Wine Science: Principles and Applications [5 ed.] 0128161183, 9780128161180

Table of contents :
Cover
Wine Science: Principles
and
Applications
Copyright
Dedication
About the Author
Preface
Acknowledgments
1 - Introduction
Grapevine and wine origins
Commercial importance of grapes and wine
Wine classification
Still table wines
Sparkling wines
Fortified wines (dessert and appetizer wines)
Wine quality
Health-related aspects of wine consumption
References
Suggested readings
2 - Grape species and varieties
Introduction
The genus Vitis
Geographic origin and distribution of Vitis and Vitis vinifera
Domestication of Vitis vinifera
Cultivar origins
Recorded cultivar development
Grapevine improvement
Standard breeding techniques
Modern approaches to vine improvement
Clonal selection
Somaclonal selection and mutation
Grapevine cultivars
Vitis vinifera cultivars
Red cultivars
White cultivars
Interspecies hybrids
American cultivars and their hybrids
French-American hybrids (direct producers)
References
Suggested reading
3 - Grapevine structure and function
Structure and function
The root system
The young root
Mycorrhizal and endophytic associations
Secondary tissue development
Root-system development
The shoot system
Buds
Shoots and shoot growth
Tissue development
Tendrils
Leaves
Photosynthesis and other light-activated processes
Transpiration and stomatal function
Reproductive structure and development
Inflorescence (flower cluster)
Induction
Inflorescence morphology and development
Flower development
Timing and duration of flowering
Pollination and fertilization
Flower type and genetic control
Berry growth and development
Berry structure
Seed morphology
Chemical changes during berry maturation
Growth regulators
Water uptake
Sugars
Acids
Potassium and other minerals
Phenolics
Pectins
Lipids
Nitrogen-containing compounds
Aromatic compounds
Cultural and climatic influences on berry maturation
Yield
Sunlight
Temperature
Inorganic nutrients
Water
References
Suggested reading
4 - Vineyard practice
Vine cycle and vineyard activity
Management of vine growth
Yield/quality ratio
Physiological effects of pruning
Pruning options
Pruning level and timing
Bearing-wood selection
Pruning procedures
Training options and systems
Bearing-wood origin
Bearing-wood length
Shoot positioning
Canopy division
Canopy height
Trunk number
Planting density and row spacing
Row orientation
Canopy management and training system development
Choice of training system
Selected training systems
Vertical shoot positioning
Scott Henry and Smart–Dyson systems
Geneva double curtain
Lyre or U system
Ruakura twin two tier
Minimal pruning
Ancient Roman example
Vigor regulation (devigoration)
Rootstocks
Vine propagation and grafting
Multiplication procedures
Grafting
Soil preparation
Vineyard planting and establishment
Irrigation
Assessing timing and need of irrigation
Water quality and salinity
Types of irrigation
Fertilization
Factors affecting nutrient supply and acquisition
Assessment of nutrient need
Nutrient requirements
Nitrogen
Phosphorus
Potassium
Calcium
Magnesium
Sulfur
Zinc
Manganese
Iron
Boron
Copper
Molybdenum
Chlorine
Organic fertilizers
Farm manure
Green manures
Compost
Biochar
Organic viticulture
Disease, pest, and weed management
Pathogen control
Chemical methods
Biological control
Environmental modification
Genetic control
Eradication and sanitation
Quarantine
Consequences of pathogenesis for fruit quality
Fungal diseases
Botrytis bunch rot
Powdery mildew (oidium)
Downy mildew (oidium)
Black rot of grapes
Eutypa dieback and other related syndromes
Esca, black measles, Petri, and black foot diseases
Ochratoxin-producing aspergilli
Bacterial diseases
Crown gall
Pierce's disease
Yellows diseases
Viruses, virus-like, and viroid diseases
Fanleaf degeneration
Leafroll
Yellow speckle
Nematode pathogens
Root-knot nematodes
Dagger nematodes
Insect and mite pests
Phylloxera
Leafhoppers (sharpshooters)
Tortricid and other moths
Mealybugs
Mites
Mammalian and bird damage
Physiological disorders
Air pollution
Ozone
Hydrogen fluoride
Elemental contaminants
Chemical spray phytotoxicity
Weed control
Tillage
Herbicides
Mulches
Cover crops
Biological control
Harvesting
Criteria for harvest timing
Sampling
Harvest mechanisms
Manual harvesting
Mechanical harvesters
Pivotal strikers
Trunk shakers (pulsators)
Striker–shaker combinations
Horizontal impactor
Robotic harvesters
Factors affecting harvester efficiency
Relative merits of mechanical harvesting
Measurement of vineyard variability
References
Suggested reading
5 - Site selection and climate
Soil influences
Geologic origin
Texture
Structure
Drainage and water availability
Soil depth
Soil fauna and flora
Nutrient content and pH
Color
Organic content
Topographic influences
Solar exposure
Wind direction
Frost and winter protection
Altitude
Drainage
Atmospheric influences
Minimum climatic requirements
Temperature
Chilling and frost injury
Minimizing frost and winter damage
Solar radiation
Physiological effects
Wind
Water
Climate change
References
Suggested reading
6 - Chemical constituents of grapes and wine
Introduction
Overview of chemical functional groups
Chemical constituents
Water
Sugars
Pectins, gums, and related polysaccharides
Alcohols
Ethanol
Methanol
Higher (fusel) alcohols
Other alcohols
Diols, polyols, and sugar alcohols
Acids
Acetic acid
Malic acid
Lactic acid
Succinic acid
Tartaric acid
Phenolics and related phenyl derivatives
Chemical groups of phenolics
Color—red wines
Color—white wines
Taste and mouthfeel
Odor
Oxidant and antioxidant action
Antimicrobial action
Clarification
Aldehydes and ketones
Aldehydes
Ketones
Acetals
Esters
Chemical nature
Origin
Lactones and other oxygen heterocycles
Terpenes and their oxygenated derivatives
Nitrogen-containing compounds
Amides
Amino acids
Other compounds
Sulfur-containing compounds
Hydrocarbons and derivatives
Macromolecules and growth factors
Carbohydrates
Lipids
Proteins
Nucleic acids
Vitamins
Dissolved gases
Carbon dioxide
Oxygen
Sulfur dioxide
Minerals
Chemical nature of varietal aromas
Appendix 6.1
Appendix 6.2
Appendix 6.3
References
Suggested reading
7 - Fermentation
Wine production
Prefermentation practices
Sorting
Prefermentative drying
Quality assessment
Stemming and crushing
Stemming
Crushing
Cryoextraction and supraextraction
Maceration (skin contact)
White wines
Rosé wines
Red wines
Dejuicing
Pressing
Vertical (basket) presses
Horizontal (moving head) presses
Pneumatic (tank, bladder, or membrane) presses
Continuous screw press
Must clarification
Adjustments to juice and must
Acidity and pH
Sugar content and alcohol potential
Reducing alcohol content
Color enhancement
Enzyme addition
Other adjustments
Blending
Decoloration and reducing browning potential
Addition of sulfur dioxide
Alcoholic fermentation
Fermentors
Batch-type fermentors
Continuous fermentation and related procedures
Fermentor size
Fermentation
Biochemistry of alcoholic fermentation
Energy balance and the synthesis of metabolic intermediates
Influence on grape constituents
Yeasts
Classification and life cycle
Yeast identification
Yeast evolution and grape flora
Succession during fermentation
Must inoculation
Spontaneous versus induced fermentation
Yeast breeding
Genetic modification
Environmental factors affecting fermentation
Carbon and energy sources
Alcohols
Nitrogenous compounds
Lipids
Phenols
Sulfur dioxide
Oxygen and aeration
Carbon dioxide and pressure
pH
Vitamins
Inorganic elements
Temperature
Pesticide residues
Stuck and sluggish fermentation
Malolactic fermentation
Lactic acid bacteria
Effects of malolactic fermentation
Acidity
Microbial stability
Flavor modification
Amine production
Origin and growth of lactic acid bacteria
Factors affecting malolactic fermentation
Physicochemical factors
pH
Temperature
Cellar practices
Chemical factors
Carbohydrates and polyols
Organic acids
Nitrogen-containing compounds
Ethanol
Other organic compounds
Fermentors
Gases
Pesticides
Biological factors
Yeast interactions
Bacterial interactions
Viral interactions
Control
Inoculation
Inhibition
Appendix 7.1
Appendix 7.2
References
Suggested reading
8 - Postfermentation treatments and related topics
Wine adjustments
Acidity and pH adjustment
Deacidification
Precipitation
Ion-exchange column
Biological deacidification
Acidification
Sweetening
Dealcoholization
Flavor enhancement
Sur lies maturation
Color adjustment
Tannin addition
Blending
Stabilization and clarification
Stabilization
Tartrate and other crystalline salt stabilization
Potassium bitartrate instability
Calcium tartrate instability
Other calcium salt instabilities
Protein haze stabilization
Polysaccharide removal and stability
Tannin removal and oxidative casse
Metal casse stabilization
Ferric (iron) casse
Copper casse
Masque
Lacquer-like bottle deposits
Microbial stabilization
Oxidation control/regulation (microoxygenation)
Fining
Activated carbon (charcoal)
Albumin
Bentonite
Kieselsol
Casein
Gelatin
Gum arabic
Isinglass
Polyvinylpolypyrrolidone
Tannin
Copper sulfate
Clarification
Racking
Centrifugation
Filtration
Depth filters
Membrane filters
Maturation in oak (and other) cooperage materials
Oak species and wood properties
Primacy of oak
Barrel production
Staves
Barrel assembly
Cooperage size
Conditioning (before use) and care (after use)
Barrel life span
Chemical composition of oak
Cell-wall constituents
Cell-lumen constituents
Compounds extracted from, and absorbed by, oak
Oxygen uptake and evaporation from barrels
In-barrel fermentation
Advantages and disadvantages of oak cooperage
Alternative sources of oak flavor
Other cooperage materials
Cork and other bottle closures
Cork
Cork and the cork oak
Culture and harvest
Cellular structure
Physicochemical properties
Stopper production
Agglomerate, technical, and hybrid cork
Cork as a potential source of wine faults
Leakage
Deposits
Taints
Alternative bottle closures
Synthetic corks
Other closure options
Closure permeability issues
Cork insertion
Leakage caused by insertion problems
Bottles and other containers
Glass bottles
Manufacture
Shape and color
Preparation for bottling
Filling
Bag-in-box containers
Aging/Shelf-Life
Effects of aging
Appearance
Taste and mouthfeel sensations
Fragrance
Loss or modification of aroma and fermentation bouquet
Origin of a bottle-aged bouquet
Additional changes
Accelerated aging
Factors affecting aging
Oxygen
Temperature
Light
Vibration
pH
Rejuvenation of old wines
Aging potential
Shelf-life
Wine spoilage
Cork-related issues
Yeast-induced spoilage
Bacterial-induced spoilage
Lactic acid bacteria
Acetic acid bacteria
Other bacterial spoilage
Sulfur off-odors
Additional spoilage problems
Infected grapes
Light exposure
Untypical (or atypical) aged flavor
Oxidation
Heat
Storage orientation
Accidental contamination
Winery waste treatment
Treatment systems
References
Suggested reading
9 - Specific and distinctive wine styles
Sweet table wines
Botrytized wines
Infection
Chemical changes associated with noble rotting
Types of botrytized wines
Tokaji aszú
German botrytized wines
French botrytized wines
Desirable varietal attributes
Induced botrytization
Nonbotrytized sweet white wine
Drying-induced concentration
Heat-induced concentration
Freeze-induced concentration
Addition of juice concentrate (sweet reserve)
Red wine styles
Dry appassimento-based wines
Production of amarone
Governo process
Carbonic maceration wines
Advantages and disadvantages
Phase I: Whole-grape (auto-)fermentation
Phase I Fermentation of released juice
Phase II: Alcoholic fermentation
Maturation/aging potential
Use with rosé and white wines
Sparkling wines
Traditional (standard, champenoise) process
Grape cultivars employed
Harvesting
Pressing
Primary fermentation
Preparation of the assemblage
Tirage
Yeasts and culture acclimation
Second fermentation (prise de mousse)
Riddling
Disgorging, dosage, and corking
Yeast enclosure
Transfer method
Bulk method
Other methods
Carbonation
Production of rosé and red sparkling wines
Effervescence and foam characteristic
Gushing
Aging
Fortified wines
Sherry and sherry-like wines
Solera system
Base wine production
Stylistic forms of Jerez sherry
Finos
Amontillado
Oloroso
Distinguishing sensory differences
Sweetening and color wines
European sherry-like wines
Non-European sherry-like wines
Solera-aged sherries
Submerged-culture sherries
Baked sherries
Porto and port-like wines
Porto
Base wine production
Maturation and blending
Sweetening and blending wines
Port-like wines
Aromatic character of ports
Madeira
Base wine production
Heat processing
Further maturation
Sweetening and blending wines
Blending
Chemical nature of the bouquet
Vermouth
Brandy
Base wine production
Distillation
Maturation
References
Suggested reading
10 - Wine laws, authentication, and geography
Appellation control laws
Basic concepts and significance
Geographic expression
France
Germany
Italy
South Africa
United States
Canada
Australia
Detection of wine misrepresentation and adulteration
Wine authentication
Validation of conformity with wine production regulations
World wine regions
Europe
Climate
Cultivars
Viticulture
Enology
Central Western Europe
France
Alsace
Bordeaux
Burgundy
Champagne
Loire
Southern France
Germany
Switzerland
Czech Republic and Slovakia
Austria
United Kingdom
Southern Europe
Italy
Northern Italy
Central Italy
Southern Italy
Spain
Rioja
Penedés
Sherry
Portugal
The Upper Douro
Vinho Verde
Setubal
Madeira
Greece
Eastern Europe
Hungary
Republics of the former Yugoslavia
Romania
Bulgaria
Russia, Moldova, Ukraine, Georgia, and other former Soviet Union states
North Africa and the Near East
Far east
China
Japan
Australia and New Zealand
Australia
New Zealand
South Africa
South America
Chile
Argentina
Brazil
Uruguay
North America
United States
California
Pacific northwest
East of the rockies
Canada
References
Suggested reading
11 - Sensory perception and wine assessment
Visual sensations
Color
Clarity
Viscosity
Spritzig (effervescence)
Tears
Oral sensations
Taste
Sweet, umami, and bitter tastes
Sour and salty tastes
Factors influencing taste perception
Mouthfeel
Astringency
Burning
Temperature
Prickling
Body (weight)
Metallic
Taste and mouthfeel sensations in wine tasting
Odor
Olfactory system
Nasal passages
Olfactory epithelium, receptor neurons, and connection with the brain
Odorants and olfactory stimulation
Sensations from the trigeminal nerve
Odor perception
Factors affecting olfactory perception
Odor assessment in wine tasting
Off-odors
Wine ranking, assessment, and sensory analysis
Conditions for sensory analysis
Tasting room
Number of wines
Presentation of samples
Glasses
Temperature
Wine identity
Breathing
Presentation sequence
Time of day
Replicates
Wine score cards
Number of tasters
Tasters
Training
Measuring tasting acuity and consistency
Tasting technique
Appearance
Clarity
Color
Viscosity
Effervescence
Tears
Orthonasal odor
In-mouth sensations
Taste and mouthfeel
Retronasal odor
Finish
Assessment of overall quality
Wine terminology
Statistical and descriptive analysis of tasting results
Simple tests
Analysis of variance
Sensory analysis
Objective wine analysis
Appendix 11.1
References
Suggested reading
12 - Wine, food, and health
Wine and food
Moderate wine consumption
Alcohol
Metabolism
Physiological actions
Potential health benefits and influences
Food value
Effects on digestion
Phenolic bioavailability
Antimicrobial action
Antioxidant effects
Cardiovascular disease
Vision
Neurodegenerative diseases
Osteoporosis
Arthritis
Diabetes
Goiter
Kidney stones
Potential health issues
Cancer
Allergies and hypersensitivity
Gout
Headaches
Dental erosion
Fetal alcohol syndrome
Toxins
Contraindications
Medication interactions
References
Suggested reading
Glossary
Index
A
B
C
D
E
F
G
H
I
J
K
L
M
N
O
P
Q
R
S
T
U
V
W
X
Y
Z
Back Cover

Citation preview

Fifth Edition

Wine Science Principles and Applications

Ronald S. Jackson, PhD

Academic Press is an imprint of Elsevier 125 London Wall, London EC2Y 5AS, United Kingdom 525 B Street, Suite 1650, San Diego, CA 92101, United States 50 Hampshire Street, 5th Floor, Cambridge, MA 02139, United States The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, United Kingdom Copyright © 2020 Elsevier Inc. All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Details on how to seek permission, further information about the Publisher’s permissions policies and our arrangements with organizations such as the Copyright Clearance Center and the Copyright Licensing Agency, can be found at our website: www.elsevier.com/ permissions. This book and the individual contributions contained in it are protected under copyright by the Publisher (other than as may be noted herein). Notices Knowledge and best practice in this field are constantly changing. As new research and experience broaden our understanding, changes in research methods, professional practices, or medical treatment may become necessary. Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using any information, methods, compounds, or experiments described herein. In using such information or methods they should be mindful of their own safety and the safety of others, including parties for whom they have a professional responsibility. To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors, assume any liability for any injury and/ or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions, or ideas contained in the material herein. Library of Congress Cataloging-in-Publication Data A catalog record for this book is available from the Library of Congress British Library Cataloguing-in-Publication Data A catalogue record for this book is available from the British Library ISBN: 978-0-12-816118-0 For information on all Academic Press publications visit our website at https://www.elsevier.com/books-and-journals

Publisher: Charlotte Cockle Acquisition Editor: Nancy Maragioglio Editorial Project Manager: Hilary Carr Production Project Manager: Prem Kumar Kaliamoorthi Cover Designer: Christian Bilbow Typeset by TNQ Technologies

This book is dedicated to the memory of Suzanne Ouellet and all those who labor, often in the background and without fanfare, to produce the wines that can so inspire us, the consumer.

About the Author The author received his bachelor’s and master’s degrees from Queen’s University, Kingston, and doctorate from the University of Toronto. His time in Vineland, Ontario, and subsequent sabbatical at Cornell University redirected his interest in Botrytis toward viticulture and enology. As part of his teaching duties at Brandon University, he developed the first wine technology course in Canada. For many years, he was a technical advisor to the Manitoba Liquor Control Commission, developing sensory tests to assess candidates for its Sensory Panel, and was a member of its External Tasting Panel. In addition, he is author of Wine Tasting: A Professional Handbook, Conserve Water Drink Wine, as well as numerous chapters and technical reviews. He is retired in Oakville, Ontario, but remains active in writing, hiking, cycling, swimming, etc. He can be reached via Elsevier, 525 B Street Suite 1800, San Diego, CA 921014495, or through the Cool Climate Viticulture and Oenology Institute, Brock University, St. Catharines, Ontario, where he is a Fellow.

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Preface

There are three pillars of wine sciencedgrape culture, wine production, and sensory analysis. Although it is traditional to cover these topics separately, a joint discussion reinforces their natural relationships. Wine’s botanical origin and microbial transformation require a discussion of the physiology and genetics of the vine, yeasts, and bacteria. These are crucial to understanding the biologic and chemical genesis of wine quality. Similarly, microclimatology and soil physicochemistry reveal the vineyard derivatives of grape quality. Equally, improved sensory procedures are permitting vineyard and winery modifications to be better evaluated, as a clearer understanding of sensory psychophysiology is revealing the basis of wine appreciation and food pairing. Finally, investigation of the health-related aspects of wine consumption is revealing its real, versus imagined, benefits and dangers. Much of the data in the text is derived from a few Western European cultivars. Thus, caution must be taken in extrapolating this information to other regions and cultivars. Conversely, challenging the established percepts has led Australian producers to sculpt exceptional wines from cultivars grown under conditions climatically distinct from their ancestral homelands. The principal theme of the work involves the various generators of wine quality. As such, the text concentrates on an understanding of the principles of grape growth and wine production. The benefits and deficits associated with the various choices at each stage are discussed, providing a background for rational selection of what may be the best options under local conditions. Even books with a regional focus find it difficult to present precise suggestions. There are too many variations even within small viticultural regions to permit a detailed formula to be recommended for producing particular wine styles in a paint-by-number approach. Science can only provide evidence-based guidance, present reasons for best practice, and enunciate the potential advantages and disadvantages of options for achieving precise goalsdno technique is without drawbacks. Because of seasonal and vineyard variability, it is rarely possible to provide choices with the clarity and certainty that grape growers and winemakers might wish. Individual experimentation is still the only means of establishing what works in a specific location, climate, and situation as well as how quality may be enhanced. Science can present a road map; winemakers decide the route. One negative side effect of our rapidly advancing knowledge is the potential for confusion. Practitioners often get annoyed with differences in opinion and changing views pontificated by experts. There is a misconception that scientists know, rather than are searchers for, the truth. Research studies are limited by time, space, funding, and analytic and statistical procedures, and if we are honest, bias at all levels. Nonetheless, lack of agreement is no reason to shun decades of dedicated research and to embrace superstition and long-disproven archaic concepts. Why would anyone genuinely wish to go back to the rigors and pain of medieval, prescientific life? Biodynamic views may simplify decisions, harkening back to some illusionary idyllic past, but at what cost? What is required is critical thinking based on reason and evidence. We need more, not less, science if affordable, high-quality wine is ever to fill the supermarket shelf. It is consumer confidence in product quality that provides employment for millions in the wine industry, maintaining farmland value and stemming the tide of developers intent on covering vineyards with asphaltinterlaced suburbia. Because knowledge is always in a state of flux, presenting where further study is needed is almost as important as outlining current opinion. While impossible to exhaustively treat all divergent views, those opinions with the greatest support, practical value, and relevance are noted. For some contentious issues, further investigation may differentiate between correlation and causation as well as veracity; for others, personal preference will always be the deciding factor. I extend my apologies to those who may feel that their views have been inadequately or inappropriately presented or for whom my expressed opinions are not to their liking. The effects of global warming on viticulture are becoming increasingly obvious and concerning. Although its rate, timing, extent, and effects are still marginally conjectural, the clear indications are dire. If even mild scenarios come to fruition, the effects will be enormous. Although famous vineyards may be underwater and grape adaptation to site

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Preface

severely dislocated, the most devastating effects will be serious disruptions to world agriculture and the political and social turmoil that will ensue. For students, I have tried to supply some periodic respites from technical detail with snips of conjecture, levity, and history. The latter demonstrates the expertise and observational acumen of our forebearers. Not only can this be humbling, but it also helps to exemplify how previous cultures have viewed a beverage so dear to our hearts. With the recent profusion of genomic and other -omic studies, new insights into the evolution and regulation of grapevine and microbial growth are being revealed. While fascinating, there is still a gap between these discoveries and their practical application. Thus, there is still ample scope for inquiry for the next generation of researchers. A list of Suggested Reading is given at the end of each chapter to guide further study. Citations in the text provide the sources of the information noted. Although most are in English, where there are none or the best sources are in foreign languages, these are provided. Several conventions have been used throughout the text. Where no common chemical name is available or preferred, IUPAC terminology is typically used. For genes and their protein transcripts, the current practice of acronym use is followed, with the first letters of the generic name used to designate the host species (e.g., Vv for Vitis vinifera). For specific genes, italicized capitals are used (e.g., VvMYBA1), whereas their protein transcripts appear nonitalicized (e.g., VvMYBA1). In writing this text, I am always reminded of what Dr. Harold Good, my most influential professor at Queen’s, once stated in reference to some ill-considered comment I made just prior to my BSc graduation: If you think you have learned a great deal during your years here, you should not graduate. What you should have learned is the extent of your ignorance.

Not what a graduating student wants to hear, but true. Over the years, I have increasingly investigated the limits of that “ignorance,” and celebrate the wonders being exposed and anticipate those to come. Our species has discovered so much that is astounding, but probably only the tip of a much more spectacular iceberg of insights.

Acknowledgments Without the astute observations of generations of winemakers and grape growers and the dedicated research of countless enologists and viticulturalists, this work would not have been possible. Thus, general acknowledgment is given to those whose work or publications have not been specifically cited. Appreciation is also given to reviewers who read chapters and provided constructive criticism for the first edition, as well as individuals who have taken their time to point out errors, most recently Jean Thurley. Credit must also go to the various editors who have helped over the years in the preparation of various editions of the text. However, special thanks go to Nancy Maragioglio. She has greatly facilitated preparation of the last three editions. Her encouragement and creativity have not only improved the product, but also added to the joy of its preparation. Appreciation also goes to all those at Elsevier whose skills have added to the quality of the final product. Gratitude is also expressed to the many researchers, companies, institutes, and publishers who have freely donated photographs, data, diagrams, or figures that illustrate the text. Finally, the work is dedicated to my late wife, Suzanne Ouellet, whose unfailing support and encouragement were essential in bringing the work to fruition.

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C H A P T E R

1 Introduction O U T L I N E Grapevine and wine origins

1

Commercial importance of grapes and wine

9

Wine classification Still table wines Sparkling wines Fortified wines (dessert and appetizer wines)

11 11 13 14

Grapevine and wine origins

14

Health-related aspects of wine consumption

15

References

17

Suggested readings

20

anthocyanin, has been found in a container associated with what appears to be a grape press (Areshian et al., 2012). Although malvidin is relatively uncommon in other fleshy fruit, it does occur in pomegranates (Punica granatum), indigenous to the region. Evidence of domesticated grapevines (Ramishvili, 1988), grape remains (Areshian et al., 2012), and residues in pottery throughout the region suggests that wine production may have been well dispersed throughout the Caucasus during the Neolithic period (P.E. McGovern, personal communication). Pyriform vessels appearing to contain wine residues have been found farther south (Godin Tepe, w3100e2900 BCE). From the distribution of the residues in the vessels, they appeared to have been laid on their side, possibly for storage (Badler, 1995). The vessels also possessed an inner slip of fine, fired clay. This could have made them comparatively nonporous. They were also apparently sealed with clay closures (Michel et al., 1993). The earliest evidence of wine residues from the Aegean comes from a Neolithic jar in Dikili Tash (northern Greece), w2300 BCE (Garnier and Valamoti, 2016), and for the Mediterranean, pottery found in a cave in Sicily, w1500 BCE (Tanasi et al., 2017). However, if all that is needed for wine’s “discovery” is a human population in a region where grapes grow indigenously, then current estimates for wine’s production may be short by several thousand years. For example, the complex of buildings in southeastern

Wine has an archeological record dating back at least 8 millennia. Residues consistent with wine (or at least grape juice) have been found in the South Caucasusdin pottery shards from Georgia, w6000e5000 BCE (McGovern et al. (2017), and a clay pot from northern Iran, w5400e5000 BCE (Fig. 1.1). The residues contained crystals of calcium tartrate, typically found in grapes and wine. Tartaric acid is rarely found in other regional fruit, excepting hawthorns. Both sites are situated along the southeastern rim of the natural habitats of wild grapevines. The relatively small, narrow-necked, ceramic vessel from Iran also contained traces of terebinth, a resin from Pistacia terebinthus. Resin was commonly used as an inner coating to waterproof pottery vessels (Serpico, 2000; Arobba et al., 2014). Amphorae so treated have been found in Egyptian tombs (Hayes, 1951; McGovern, 1997). Amphorae are a later development in pottery production used for storage and transport. They were typically two-handled and elongated, and had a capacity of about 30 L. Resin also had antimicrobial and flavorant properties. During Roman times, the resins commonly used to line amphorae were derived from pine sap (e.g., Pinus halepensis) (Columella in De Re Rustica, 12.20e14; Pliny in Historia Naturalis, 14.25). Another, possibly independent, site for wine’s origin is in neighboring Armenia (Barnard et al., 2011). Evidence for the presence of malvidin, the major grape

Wine Science, Fifth Edition https://doi.org/10.1016/B978-0-12-816118-0.00001-5

Wine quality

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2

1. Introduction

FIGURE 1.1 Pottery vessel from which residues likely to be of Neolithic wine were found in Hajji Firuz, in the northern Zagros mountains of Iran. From McGovern, P.E., Glusker, D.L., Exner, L.J., Voigt, M.M., 1996. Neolithic resinated wine. Nature 381, 480e481, by permission of Nature/Springer.

Turkey (Go¨bekli Tepe), w9000 BCE (Schmidt, 2010), must have required a large labor force. The buildings appear to have been constructed by a hunteregatherer society (predating agriculture or pottery production). If wine were produced by this society, it presumably was in limited quantities and consumed shortly after production. Older examples of fermented beverages have been discovered (McGovern et al., 2004), but they appear to have been produced from a mixture of rice, honey, and various fruits (hawthorn and/or grape). Such beverages were being produced in China as early as 7000 BCE. Although China is home to several Vitis spp., most of its alcoholic beverages have historically been rice based (Poo, 1999). As noted, the presence of calcium tartrate crystals is usually considered indicative of wine residues. Additional potential (but not exclusive) indicators are tannin residues (Garnier et al., 2003), ergosterol (produced during yeast fermentation) (Isaksson et al., 2010), waxes (from grape skins) (Isaksson, 2005), and Vitis pollen (Arobba et al., 2014). A more specific (but rare) indicator of the past presence of wine (or grapes) would be the isolation of grape DNA (Milanesi et al., 2011). Other than problems associated with differentiating between dried grape and wine residues, there is the thorny issue of what actually constitutes wine. Does spontaneously fermented grape juice qualify as wine, or should the term be restricted to juice fermented and stored in a manner capable of retaining its winelike properties? The latter would suggest purposeful intent, rather than a fortuitous happenstance. Because of low yield, seasonal availability, and marginal availability (vines growing up trees or straddling

screes), opportunities for collecting significant quantities of wild grapes would probably have been limited. Wine production, as normally defined, also demands some form of impervious, ideally sealable, container. Thus, any significant wine production probably postdates the development of agriculture and a settled lifestyle. Even accidental seed germination in rubbish piles would have taken several years before the vines produced fruit. Admittedly, all this is speculative. Thus, the timing and events that ultimately led to wine, and its spread, will always remain an enigma. Nonetheless, narrow-necked pottery vessels were being produced about 8000 years ago, and the availability of wood-derived resins (sealing the vessels) would have made wine storage possible. The use of wineskins is another option, but one for which no archaeological evidence remains. Wineskins, smeared with pitch, were used by the peasantry to store wine in Spain in the 1800s (Henderson, 1824, p. 188), possibly reflecting a technique millennia old. The presence of carbonated grape-seed remains from 3rd- and 4th-century BC Jordan suggests that viticulture, and possibly winemaking, had already spread beyond the indigenous range of wild grapevines. This suggests purposeful cultivation (McGovern et al., 1997; Zohary and Hopf, 2000). However, the first unequivocal evidence of intentional winemaking appears in representations of wine presses from the reign of Udimu (Egypt), w3000 BCE (Petrie, 1923). Wine residues have also been found in amphorae, specifically so marked, in many ancient Egyptian tombs. The earliest dates from the reign of King Semerkhet (2920e2770 BCE) (Guasch-Jane´ et al., 2004). Amphorae marked as containing white and red wine were discovered in King Tutankhamun’s tomb (1325 BCE) (Lesko, 1977). Confirmation of origin has been established by the presence of syringic acid, an alkaline breakdown product of malvidin-3-glucoside. The same technique has been used to establish the red grape origin of an ancient Egyptian drink, shedeh (Guasch-Jane´ et al., 2006). It was a filtered, heated wine (Tallet, 1995). For additional details about Egyptian wine jars, and their sealing, see Lesko (1977), Hope (1978), and McGovern et al. (1997). However, the effectiveness of reeds, covered with a clay top, in preserving wine against spoilage seems doubtful (Fig. 8.45). The amphorae are fascinating, though, in their possessing notations on the wine’s origin, the vintage, the vineyard, and occasionally the winemaker (McGovern, 1997). Equivalent indications of origin have been found on Roman amphorae in Pompeii, occasionally including indications of the owner (Jashemski, 1975). Although DNA analysis of amphora contents has begun (Milanesi et al., 2011; Foley et al., 2012), it has yet to be used to suggest varietal origin.

3

Grapevine and wine origins

Although grapes possess an extensive epiphytic yeast flora, they typically only initiate fermentation, being unable to ferment the majority of grape sugars. Only Saccharomyces cerevisiae and closely related strains are sufficiently alcohol tolerant to complete the task, producing a potentially stable wine. Surprisingly, S. cerevisiae is not a major (or typical) member of the grape-skin flora. The principal natural habitat of the likely ancestor of S. cerevisiae (S. paradoxus) appears to be oak bark, its exudate, and the surrounding soil and litter (Kowallik and Greig, 2016). The habit of grapevines, growing up trees such as oak, and the ancient harvesting of both grapes and acorns (Bohrer, 1972; Mason, 1995) could have favored the inoculation of grape juice with S. cerevisiae. The overlapping distributions of oak (Meusel et al., 1965) and wild grapes (Levadoux, 1956), the origins of Western agriculture (Bohrer, 1972; Zohary and Hopf, 2000), and advances in pottery production may have fostered winemaking (Fig. 1.2). In addition, it may not be pure coincidence that most major yeast-fermented beverages and foods have their presumptive origins in the same region. Ancient myths (Civil, 1964) suggest that grapes were the initial inoculum for beer production, a view

BLACK SEA

consistent with genomic studies (Fay et al., 2019). The first beer recipes appear on Babylonian cuneiform tablets from w4000 BCE. In addition, beer’s modern expression requires the cultivation of barley and its malting (partial germination). However, its earliest forms might have been produced from grain starch hydrolyzed by the action of lactobacilli or cooking, liberating sufficient sugars for non-Saccharomyces yeasts to ferment. Honey, if diluted, will also spontaneously ferment, producing a mildly alcoholic beverage, as could a slurry made from dates. There is no evidence that mastication (a source of salivary amylase) was used in the Near East for alcoholic beverage production, as in South and Central America (chicha and masato) or Japan (li). The earliest documented evidence for a connection between wine and Saccharomyces cerevisiae comes from lees DNA isolated from an amphora discovered in the tomb of Narmer, the Scorpion King (Egypt), w3150 BCE. The DNA showed closer similarity to modern S. cerevisiae strains than to related species (e.g., S. bayanus and S. paradoxus) (Cavalieri et al., 2003). Words clearly referring to fermentation appear in documents from w2000 BCE (Forbes, 1965). The origin, and spread, of winemaking from Caucasia to other regions is compatible with the remarkable

CA

UC

AS

US

Anatolia CASPIAN SEA

Mediterranean Sea MESOPOTAMIA

FIGURE 1.2 Comparison of the geographic distributions of Vitis vinifera f. sylvestris (shaded burgundy) and Quercus sessilis (right-slanted lines) and the origin of agriculture (left-slanted lines). Based on data from Levadoux, L., 1956. Les populations sauvages et cultive´es de Vitis vinifera L. Ann. Ame´lior. Plantes, Se´r. B 1, 59e118; Zohary, D., Hopf, M., 2000. Domestication of Plants in the Old World, third ed. Oxford University Press (Clarendon), Oxford, UK; Ducousso, A., Bordacs, S., 2003. EUFORGEN Technical Guidelines for Genetic Conservation and Use for Pedunculate and Sessile Oaks (Quercus robur and Quercus petraea). International Plant Genetic Resources Institute, Rome, Italy, p. 6; Meusel et al., 1965; and Bohrer, V.L., 1972. On the relation of harvest methods to early agriculture in the Near East. Ecom. Bot. 26, 145e155.

4

1. Introduction

similarity among words for wine in most Indo-European languages (see Table 2.1). Winemaking’s spread into Europe may have occurred simultaneous with the spread of farming cultures out of Caucasia. Studies of the mitochondrial DNA from human remains suggest a major movement of farmers into Europe, possibly via Hungary and Slovakia, beginning about 6400 BCE (Bramanti et al., 2009). This movement is also correlated with a major and rapid shift from hunting and gathering to farming. However, this is considerably earlier than known evidence suggestive of winemaking in Europe. Thus, exactly when winemaking spread into Europe remains speculative (Renfrew, 1989). These changes may have been associated with new migrants, who displaced (or intermingled with) the initial groups who brought a farm-based culture to Europe (Bramanti et al., 2009; Kristiansen et al., 2017). This may also be the origin of the predominance of Indo-European languages in Europe. Another line of evidence, suggestive of the diffusion of winemaking skills from the east, comes from Eastern Mediterranean myths. These stories consistently locate the origin of winemaking in northeastern Asia Minor (Stanislawski, 1975). For example, Bacchus, the Roman god of wine, was considered to have come from Thrace (a geographical region occupying present-day southeastern Bulgaria, northeastern Greece, and Anatolian Turkey). Unlike grain crops (e.g., wheat and barley), grapes develop an extensive yeast flora, although this rarely includes Saccharomyces cerevisiae. Piled unattended for several days, grape cells begin to self-ferment, as oxygen becomes limiting, generating small amounts of alcohol. During the process, skins weaken, facilitating berry rupture. Released juice is rapidly colonized by the skin’s yeast flora. These would continue the fermentation of grape sugars into ethanol. Without their complete fermentation, colonization by spoilage yeasts and bacteria would occur quickly (especially in the presence of oxygen). The result would be the conversion of a nascent wine into vinegar. Under most circumstances, though, even trace numbers of S. cerevisiae tend to rapidly dominate fermentation, completely metabolizing the major grape sugars, glucose and fructose. The higher alcohol content that results would donate a degree of microbial stability (if stored anaerobically). Despite the tendency of grapes to self-ferment, fermentation progresses to completion only upon crushing. Crushing releases and mixes the juice with epiphytic yeasts and those derived from the fermentation vessel. Yeast fermentation is more rapid in the presence of slight amounts of oxygen, but continued exposure favors the growth of spoilage organisms. Although vinegarization makes wine unacceptable as a human beverage, it has its own value. For example, vinegar is used to preserve (pickle) perishable foods, and

can be enjoyed as a condiment. For ancient cultures, it also expedited pottery production (as a slip, or a deflocculant in glaze formation). Of the many fruits gathered by ancient man, only grapes store carbohydrates predominantly as soluble sugars. Thus, the major caloric source in grapes is in a form readily metabolized by yeasts. Most other fleshy fruits store carbohydrates primarily as starch and pectins, nutrients unavailable to wine yeasts. The rapid and extensive production of ethanol by Saccharomyces cerevisiae quickly limits the growth of most bacteria and other yeasts. Consequently, wine yeasts generate conditions that rapidly give them almost exclusive access to grape nutrients. Subsequent microbial growth is possible, but usually after the readily fermentable sugars have been metabolizeddif oxygen is present to permit respiratory metabolism. Another unique property of grapes concerns the acids they contain. The principal acid found in mature grapes is tartaric acid. It occurs in small quantities in the vegetative parts of many plants (Stafford, 1959), but rarely in fruit. The other major grape acid, malic acid, is often metabolized as the fruit ripens. Because tartaric acid is metabolized by few microbes, wine retains sufficient acidity to limit the growth of most bacteria and fungi. In addition, acidity gives wine much of its fresh taste. The combined action of grape acidity and the accumulation of ethanol suppresses the growth and metabolism of most potential spoilage organisms. This property is enhanced in the absence of air (oxygen). The result is the transformation of a perishable, periodically available fruit into a relatively stable beverage, with novel, intoxicating properties. The latter, plus its flavor, were almost undoubtedly what initially encouraged wine productiondnot the subtleties that so appeal to the modern aficionado. The salubrious and celebratory attributes of wine have often been appreciated more than its subtleties. As Younger (1966) notes: Like medieval man, medieval wine entered birth upon a precarious existence. In a good year the natural strength of the wine may have brought it through most of its perils; in a poor year the natural strength of its drinkers may have carried them through the worst of its after-effects .

Unlike most domesticated crops, the grapevine has required little genetic modification to adapt it to cultivation. Its mineral and water requirements are low, permitting it to grow on sites ill-suited to food crops. In addition, planting on slopes, close to rivers, provided favorable conditions for fruit maturation and ready access to waterborne trade routes. The vine’s climbing nature also permitted it to grow untended up trees interspersed among field crops. This method of cultivation was used in Bronze Age Crete (Hanson, 1992), during Roman times, and in several locations up to modern times (Fregoni,

Grapevine and wine origins

1991). In addition, the vine’s immense regenerative potential allows it to withstand intense pruning. Pruning turned a trailing climber into a short, shrublike plant, suitable for monoculture. The short, stocky stature of shrubby vines minimized the need for supports, and may have decreased water stress in semiarid environments (soil shading). The regenerative powers and woody structure of the vine have also permitted it to withstand considerable winterkill and still produce commercially acceptable yields. This favored the spread of viticulture into central Europe and the subsequent selection of, or hybridization with, indigenous feral vines. Probably the major change that transformed wild vines into a domesticated crop was a return to functional bisexuality. The vast majority of wild vines are functionally unisexual, despite possessing either rudimentary anthers or a pistil (Fig. 2.2). In most cultivars, functional bisexuality has involved inactivation of a single gene. However, in some cultivars, full bisexual expression appears to have involved several mutations (Carbonneau, 1983). How, when, and where grapes were domesticated will probably never be known. However, two scenarios seem likely. Several Neolithic sites show significant collections of grape seeds (refuse piles), indicating the local importance of grape harvesting. Although these remains usually show charring, seeds escaping charring could have found conditions ideal for growth among the ashes. Had any of these progeny been self-fertile, they could have produced a crop, despite being isolated from feral vines. More likely, though, accidental selection for functional bisexuality occurred when feral vines were planted adjacent to settlements (away from endemic populations). Self-fertile revertants would have unintentionally been selected, especially if unfruitful unisexual vines were rogued. Vegetative propagation of the more fruitful (bisexual) vines, especially showing other desirable properties, could have provided a coterie of vines for viticultural improvement. Although other modifications characterize domesticated vines, those involving seed and leaf shape have no known enologic or viticultural importance. The lower acidity and higher sugar content that characterize cultivated varieties are not necessarily the exclusive attributes of domesticated vines. These properties may reflect viticultural growth conditions as much as genomic modification. The sugar and acid contents of feral grapes appear not to have been studied under cultivation (equivalent to those of domesticated cultivars). Canes, trailing along the ground, root easily, particularly when partially covered with soil. Observation of this tendency probably led to its use in vine propagation. Vegetative propagation has the advantage of retaining genetic traits. Early viticulturalists would have, sooner or later, realized the desirability of retaining traits unmodified. In arid regions, where fungal diseases tend to be minimal, vines could be left to trail on the ground. However,

5

in moister regions, vines were often planted next to trees for support. A major problem was that the fruit developed out of easy reach. Some perceptive cultivator probably discovered that vine trimming facilitated fruit gathering. Subsequent observation would have demonstrated that pruning could also favor optimal fruit maturation. The combination of easier harvesting and improved maturity probably spurred further developments in pruning and training. Combined with advances in wine production and storage, the stage was set for the evolution of the wine trade. As noted, the evolution of winemaking from a periodic, haphazard event to a dedicated agricultural activity presupposes the development of a settled lifestyle. A nomadic habit is incompatible with harvesting sufficient fruit to produce a ready supply of wine. In addition, unlike major field crops, grapevines provide significantly less yield, producing a perishable fruit (unless dried or converted into wine). A dependable supply of grapes would have become important when society began to associate wine with religion. To ensure a reliable supply, planting close to human settlements became a necessity. It takes several years for newly established vines to begin to bear a significant crop and requires several additional years to reach full productivity. Such an investment would be reasonable only if the planter resided nearby. Under such conditions, grape collection would have become compatible with predictable wine production. If, as seems reasonable, wine production is dependent on a settled agricultural existence, then significant wine production cannot predate the agricultural revolution. Because grapevines are not indigenous to the Fertile Crescent (the origin of Western agriculture), the beginnings of a dedicated viticulture probably occurred after the spread of a settled, agrarian lifestyle into the Caucasus. Grape cultivation and winemaking probably spread southward from the Caucasus into Syria, Palestine, and northern Egypt and across to Mesopotamia. From this base, wine consumption, and its socioreligious connections, has generally been viewed as central to promoting the spread of viticulture around the Mediterranean. Despite this, Stevenson (1985) and Nu´n˜ez and Walker (1989) provide archaeological and palynological evidence for an extensive system of grape culture in southern Spain about 5000 years ago. This is millennia before the Phoenicians were thought likely to have introduced winemaking to the regionda view that may be in error (Eshel et al., 2019). Nonetheless, there still is genetic evidence for an independent center of grapevine domestication in the Iberian Peninsula (Arroyo-Garcı´a et al., 2006). In addition, archeobotanical evidence for grape domestication has been found in Neolithic southern Italy (Gismondi et al., 2016). The data from Campania are particularly interesting because sufficient DNA was available to demonstrate the existence of allelic variants

6

1. Introduction

still present in Italian cultivars, but not in cultivars from sites considered ancestral to grape cultivation. Nevertheless, colonization from the eastern Mediterranean is still considered the predominant route by which grape-growing and winemaking knowledge spread into and throughout much of Europe. Evidence of major wine importation into western Europe predates Roman conquests by several centuries (Loughton, 2003). Subsequent Roman occupation expanded vineyard plantings throughout most of the empire. More recently, European colonization has spread grapevine cultivation globally. Until comparatively recently, most contemporary wine styles either did not exist or occurred in forms considerably different from their present forms. Most ancient and medieval wines were probably dry to semidry, turning vinegary by summer. The only medieval wine acknowledged to have aged well (at least remaining drinkable) was that stored in the enormous oak cooperages of Heidelberg (BadeneWu¨rttenberg), with capacities reaching 195,000e228,000 L. Generally, protection of most wines from oxidation and microbial spoilage was inadequate. The use of sulfur dioxide is not recorded until the end of the 1400s (in Germany). Thus, from the end of the Roman era until the Renaissance, prolonged aging of wine was rare, and not generally ensured for another few hundred years. Nonetheless, techniques were available to extend a wine’s “shelf-life” during Greek and Roman times. One of the most effective, burning sulfur to cleanse (disinfect) amphorae, appears not to have been one of them. However, Columella (CE 4e70 ) does describe how to clean and treat amphorae with hot pitch (De Re Rustica, 12.18). They were first heated upside down over an open flame. After uprighting, hot pitch was applied to coat their inner surfaces. A 1- to 2-mm lining waterproofed the typically porous, ceramic vessels (Pliny the Elder, Historia Naturalis 14.24, 14.27). Other treatments noted included rinsing with seawater and perfuming with myrrh. An inner wax coating was eschewed, since it was considered to turn the wine sour. For the majority of amphorae, used to transport solid goods and olive oil, this was considered unnecessary. For large fermentation vessels (dolia), hot pitch was supplied via an iron hook. Covers were often treated with pitch as well. Resins, being mildly antimicrobial, probably provided some protection against spoilage (Columella, De Re Rustica 12.24). Resinous aromatics could also have partially masked flavor modifications associated with the early stages of spoilage. Flavorants, such as saffron and defructum (grape concentrate), might also be added to the must (Pliny, 14.27). Some Egyptian amphorae may have been made somewhat impervious by hand burnishing their outsides (McGovern, 1997). However, the most effective waterproofing was with a vitreous inner lining. The technique was perfected at least by the 7th century

BCE. These amphorae, termed type A, were produced throughout Greece and elsewhere. The procedure involved adding a flux of potash to an illitic clay containing finely divided and dispersed calcium oxide. During manufacture, a rapid reductive firing (w1000 C) produced a gray, glassy inner lining (Vandiver and Koehler, 1986). Porous, type B, amphorae contain more calcium oxide and less iron and potassium oxides. The amphora’s typical outer reddish color came from the oxidation of its iron content. This develops after the introduction of air near the end of firing. Coating with beeswax or pine pitch was a less expensive, but shorter-term, means of waterproofing. Greek and Roman wine amphorae were normally sealed with cork, as evidenced by underwater archaeology (Cousteau, 1954; Frey et al., 1978). Circular caps of cork were affixed to the rim, as well as being covered with a layer of pitch (Frey et al., 1978). An overlay of pozzolana (volcanic clay) was also typical (Fig. 1.3). Amphorae closed only with a clay stopper (Koehler, 1986) were presumably used for dry goods (Foley et al., 2012). Amphorae were occasionally stored on their sides or inverted (Grace, 1979; Jashemski, 1979)da habit appropriate for keeping cork elastic enough to maintain its protective seal. Thus, the minimum requirements for extended wine aging appear to have been met. In addition, bars in Herculaneum (Pompeii) show that amphorae were binned horizontally, in semicircular shelf grooves. That successful long-term storage was achieved is suggested by the mention of aged wine in ancient literary sources. For example, Columella (De Re Rustica 3.21.10) states that wines had the property of acquiring excellence with age. Wine was generally considered better (fetched a better price) when at least 1 year old. Pliny also notes that “all the wines from beyond the sea (largely Greece and Aegean Islands) arrived at their middle state of maturity (six to seven years)” (Historia Naturalis 14.8). Varro notes that Falernian wines became more valuable the longer they were kept (De Re Rustica, 1.65). Aged wine also receives high praise from Horace, who poetically sings the glories of a 46-year-old wine. Martial also honors a friend with a 100-year-old wine: . Let the contents of this amphora, diminished by the lapse of a hundred consulships, flow forth, and let it grow brighter, turbid as it now is, strained through the purifying linen . To Flaccus, on the Return of Priscus Terentius, Epigrams 8.45

Old wine is also mentioned positively in Homer’s Odyssey (2.340) (w850 BCE). In addition, Nestor is noted to have entertained by opening 10-year-old wine (Odyssey 3.391). There is also mention of quality distinctions between vintages, specific vineyards, and different regions. Wine from Chios, Lesbos, and Mount Falernus received special praise. Tchernia (1986, p. 30) cites an amphora possessing both the date of the vintage (the Consul at the time) and its

Grapevine and wine origins

7

FIGURE 1.3 (A) Amphora (w100e90 BCE). (B and C) Neck filled with limeepozzolana paste, including cork stopper. Reproduced from Arobba, D., Bulgarelli, F., Camin, F., Caramiello, R., Larcher, R., Martinelli, L., 2014. Paleobotanical, chemical and physical investigation of the content of an ancient wine amphora from the northern Tyrrhenian sea in Italy. J. Archaeol. Sci. 45, 226e233, reproduced with permission of Elsevier.

transfer from a dolium to the amphora. Pliny specifically notes that Chian wine was served at Julius Caesar’s triumphal banquet (Historia Naturalis 14.16.97). Annotated lists of wines are supplied by Virgil (Georgics 2.89e102), Columella (De Re Rustica 3.2), and Pliny (Historia Naturalis 14.150). Comments on their sensory attributes and the specific climatic requirements of particular cultivars are also noted by Columella (De Re Rustica 3.2.7e13) and by Athenaeus (Deipnosophistae 1.32 f). With the expansion of vineyards during the Imperial period, the number of specifically distinguished wines and grape cultivars expanded from 15, noted by Virgil in Georgics (29 BCE), to 185 in Pliny’s Historia Naturalis. Regrettably, their cultivation (or at least name retention) has not survived to modern times. With the decline of the Roman Empire, a switch to barrel storage, and the loss of a sophisticated clientele, seems to have eliminated the need to differentiate between varieties, vintages, or provenance. By medieval times, varietal identification appears to have been reduced to two categories: vinum hunicum (poor quality) and vinum francicum (high quality). These terms subsequently came to

be applied to particular cultivars or grouping, designated Heunisch and Frankisch (Schumann, 1974). A few ancient authors, such as Athenaeus and Hermippos, employ wine descriptors that have a modern ring (e.g., white flowers, violets, raspberries, pineapples, roses, hyacinths, and apples) (e.g., Henderson, 1824, p. 62; Stanley, 1999). However, these may reflect the actual addition of such flavorants to their respective wines (e.g., rose petals or violets are noted as having been added by Apicius in De Re Coquinaria, Book 1, and other Roman authors). Thus, cautions must be taken in interpreting the state of wine production from literary sources (Komar, 2013). Despite the seemingly frequent addition of flavorants, there seems adequate justification in thinking that some ancient Greek and Roman wines might be rated positively by modern aficionados. What appears curious, from a modern perspective, is the frequent mention of adding water to even the finest wines (Henderson, 1824, pp. 98, 110). Those taking wine undiluted were often derided and satirized. Whether dilution was because of their marked sweetness, dehydration, or the high alcohol content is unclear.

8

1. Introduction

As noted, many ancient wines were mixed with a variety of flavorants, including herbs, spices, honey, seawater, etc. (Henderson, 1824; Tchernia, 1986; Stanley, 1999). Such additives may appear eccentric, but seem consistent with contemporary Roman cooking practice. Their recipes typically contained strong flavorants (notably garum, a sauce made from the fermented entrails of fish) and heavy doses of spices (Apicius, De Re Coquinaria). The distinguishing attributes of meals were derived more by their condiments than their main ingredients (Tannahill, 1973; Andre´, 2009). Another example of “strange” practices is noted in Theophrastus (371e287 BCE) in Concerning Odours (11.1). He mentions the apparently common habit of adding dough, kneaded with honey, to improve a wine’s flavor. Although ancient production procedures seem primitive by current standards, some modern wines still employ similar procedures. For example, Cato recommends storing amphorae of wine in the sun, to which a portion of boiled must was added (Younger, 1966, pp. 211e212). Pliny the Elder (CE 23e79) makes the same recommendation. This is probably the origin of vin santo (see Chapter 9). Several procedures for the production of sweet wine are recorded in ancient Roman texts. These include the addition of boiled-down must, or leaving the grapes to partially dry (see Stanley, 1999). More demanding were procedures involving the fermentation of juice that oozed out of grapes under their own weight, a technique used in making Priam and Saprias (Stanley, 1999, p. 111). The latter may have been made from molded (botrytized?) grapes (Stanley, 1999, p. 112)da technique used in producing high-quality Tokaji wines. Madeira may involve extended heating, but whether ancient wines, stored in a room through which smoke and heat rose, would resemble modern madeira is questionable. It was recommended by Columella to achieve early wine maturity. Although appreciated by some, Columella does note that the technique was open to abuse (De Re Rustica 1.6.20). Pliny clearly states that wine should be aged naturally, not smoked. Despite the probability that some Roman wine would please modern palates, the majority probably would not. Grape yields were often high, leading to acidic juice, low in sugar content. This is a situation about which Columella (De Re Rustica 3.3.4e7) complains bitterly. Most wines were also stored in amphorae coated with pitch. In addition, chunks of resin were not infrequently added to the wine. This undoubtedly would have masked any subtle flavors the wine might have possessed. Presumably the finest wines were stored in amphorae with a vitreous lining (not requiring a coating of pitch to make the amphora watertight). Lower-grade wines often were supplemented with must and honey, flavored with herbs or aromatic resins, such as myrrh. The product, often referred to as sapa, was prepared by reducing its volume to about a third

by slow simmering, usually in lead vessels (Eisinger, 1982). The additive reduced spoilage and provided a sweeter, more agreeable flavor. Unwittingly, this compounded the incidence of colica Pictonum (an old term for lead poisoning) (Eisinger, 1982). It was prevalent in Roman times due to their frequent use of lead plumbing. The addition of sapa is distinct from wine used to prepare a herbal solution. Such potions were used to treat not only humans but also livestock (Columella, De Re Rustica, 6.5; Estienne and Liebauit, 1572). Wine for the enslaved and plebeian classes appears to have been concocted from boiled must or pomace soaked in water, to which seawater and/or vinegar was often added. An example of a formula for preparing a vinum operarium is given by Cato (234e149 BCE): Combine 10 quadrantals of must, 2 quadrantals of sharp vinegar, 2 quadrantals of boiled must with 50 quadrantals of fresh water. Stir with a stick three times daily for five days. Add 64 sextarii of aged sea-water, seal, and let set for 10 days. The wine should last until the summer. The remainder will be excellent, sharp vinegar.

Although such concoctions seem incomprehensible today, some medieval amalgams were hardly more savory (Accum, 1820); Younger, 1966, pp. 258, 312). In addition, wine was often recommended for its reputed medical value, rather than its sensory quality. Correspondingly, herbs, honey (and later on, eau de vie) might be added, or the wine boiled (von Hirnkofen, 1478). Other additives were ostensibly used to “correct” wine faults (Best, 1976). Some were sanctioned, others notdnotably the addition of lead acetate (Hallgarten, 1986; Skovenborg, 1995). Although masking overly acidic wines, and used to rectify ropy wines, it came at the cost of human toxicity. Wine adulteration, in some form or other, was once all too common (Accum, 1820). Apparently, perpetrators went to elaborate ends to conceal their deception. Examples include staining the ends of corks red and crystalline crusts being added to bottle surfaces, to give the impression of a well-aged wine. Wines began to take on their modern expressions about the 17th century. The use of sulfur in barrel treatment started to become more widely spread in western Europe about this time (see Sulfur dioxide, Chapter 6). This would have greatly increased the likelihood of producing better wines and extending their aging potential. Stable sweet wines, able to age for decades or longer, also started to appear in the mid-1600s, commencing with the Tokaji wines of Hungary. For the commercial production of sparkling wine, bottles strong enough to withstand the pressures exerted by carbon dioxide were a prerequisite. The production of such glass began during the mid-17th century in England. The reintroduction of cork as a wine closure, combined with strong glass, set the stage for developments in sparkling wine production, as well as in wine aging.

9

Commercial importance of grapes and wine

The development of vintage port also depended on the ready availability of inexpensive, but strong bottles. This became possible with the industrial revolution. The evolution in bottle shape, from bulbous to cylindrical, permitted bottles to be laid on their sides. Correspondingly, the cork retained its moisture, elasticity, and sealing properties. Thus, wine had the opportunity to age under conditions conducive to the development of a smooth taste and complex bouquet. The development of modern port also depended on wine distillation. Distilled spirits began to be added to fermenting juice about 1720, prematurely terminating its fermentation. The practice had become common by 1754 (Henderson, 1824, p. 201). As a consequence, a microbially stable wine, both sweet and dark red, could be shipped to distant markets. Modern sherry development also depended on the addition of grape spirits. Although ancient Greek distillation techniques were subsequently perfected by the Arabs, adoption of the procedure in medieval Europe for producing a wine distillate was slow. Thus, all fortified wines are of relatively recent origin. With advancements in glass production and the widespread adoption of cork as a bottle closure in the 17th century, conditions favorable to the production of modern wine appeared. The discovery, by Pasteur in the 1860s, of the central importance of microbes to fermentation (as well as certain forms of spoilage) set in motion a chain of events that has permitted the evolution of the incredible range of wines that typify modern commerce.

Commercial importance of grapes and wine From its humble origins, grape production has developed into one of the world’s most important fresh fruit crops, estimated to be worth $44 billion in 2013 (FAOStat, 2013). Worldwide grape production in 2017 was w73  106 metric tons (73  109 kg), showing increases in the past few decades (OIV, 2018). This compares with roughly 62, 80, 70, and 136 million metric tons for oranges, bananas, apples, and tomatoes, respectively. The area planted in grapevines in 2017 was estimated to be about 7.5 million hectares. Increases have been noted principally in China, with modest increases in several wine-producing countries (e.g., Chile, Austria, Mexico, and Hungary). Decreases are reported in most European countries. Approximately 66% of world production is fermented into wine, 32% consumed fresh, and the remaining 2% dried for raisin production (OIV, 2005). Use varies widely from country to country, often depending on the climatic and politicoreligious (wine prohibition) dictates of the region. Despite their world importance, vines cover only about 0.5% of agriculture land, and their produce constitutes but 0.4% of global household expenditure (Anderson, 2004). Grape production is largely restricted to climatic regions similar to those of the indigenous range of Vitis vinifera in Europe. This zone approximates a band between the 10 C and the 20 C annual isotherms (Fig. 1.4). Grape culture is largely restricted to regions characterized by Mediterranean-type climates.

10°C 10°C 20°C

10°C 20°C 20°C

Equator

Equator

20°C 10°C

20°C 10°C

20°C

10°C

FIGURE 1.4 Association between the major viticultural regions of the world and the 10 C and 20 C annual isotherms. Drawing courtesy of H. Casteleyn, reproduced by permission.

10

1. Introduction

Extension into cooler, warmer, or wetter environs is possible when local conditions modify the climate, or viticultural practice compensates for less than ideal conditions. Commercial production can even occur in subtropical regions, where periodic severe pruning offsets the vine’s requirement for an annual dormancy period. Europe, where w40% of the world’s vineyards are located, produces about 56% of the world’s wine. Since the 1970s, wine production has ranged from about 250 to 330  106 hL (66e87 million gallons), with recent production levels averaging in the range of about 270  106 hL. Although Spain retains its position as possessing the largest wine-producing hectarage (967  106 ha in 2018), followed by France (786  106 ha) and Italy (699  106 ha), Italy and France produced the largest volumes of wine (48.5 and

46.4  109 hL). Together, France, Italy, and Spain generate w60% of wine exports. The economic importance of wine, and enhanced worldwide competition in the export market, is partially reflected in the marked increase in research conducted worldwide (Gla¨nzel and Veugelers, 2006). Statistics on wine production and export for several countries are given in Fig. 1.5. Remarkably, several major wineproducing nations, such as Argentina and the United States, export relatively small proportions of their production. In contrast, countries such as Chile and Portugal export the majority of their production. As for importation, Germany, the United Kingdom, and the United States are among the top wine-importing countries. Although Europe remains the most important wineproducing and exporting region, in terms of quantity, it

Wine Production (millions of hl)

50

40

30

20

10

Fr

Ita ly an ce U ni Sp te d ain St Ar ate ge s nt in a C Au h i l e st ra lia C G hina e So rm ut an y h Af r Po ica r R tug o u al m an R ia us H sia un ga ry N B ew r az Z e il al an Au d st r G ia re ec C an e ad a

0

Country

Wine Export (millions of hl)

25

20

15

10

5

Ita l Fr y an ce C h Au i l e s So tra lia ut h Af r G ica U erm ni te any d St at Po es N ew rtug Ze al al Ar and ge nt i M na ol do va

Sp

ai

n

0

Country

FIGURE 1.5 Wine production (top) and export statistics (bottom) (2017) for several wine-producing countries (106 hL/year). Data from OIV, 2018. Statistical Report on World Vitiviniculture 2018. http://www.oiv.int/public/medias/6371/oiv-statistical-report-on-world-vitiviniculture-2018.pdf.

11

Wine classification

is also the primary wine-consuming region. For centuries, wine has been a significant calorie source for laborers in southern Europeda bottle of wine supplying about onequarter of their daily caloric requirement. Because wine was an integral part of daily food consumption, heavy drinking did not develop the unwritten, but tacit, approval found in some countries. Alcohol abuse, especially in the United States, spawned the prohibitionist movement. Their view, that consuming any beverage containing alcohol was detrimental to human health, is in conflict with evidence supporting the healthful benefits of moderate wine consumption (see Chapter 12). The reticence of some governments to acknowledge the beneficial consequences of moderate wine consumption does injustice to the long, extensive, and efficacious use of wine in medicine (Lucia, 1963). There has been a marked reduction or stabilization in per-capita wine consumption since 1969 (Fig. 1.6). The reasons for this are complex and often region specific. Occasionally, the decline has been optimistically interpreted as a shift toward the purchase of less, but better-quality wine. Although possibly true, the decline in wine consumption in Europe appears to be partially associated with a switch to the use of distilled spirits.

Wine classification Except in the broadest sense, there is no generally accepted system of classifying wines. They may be grouped based on carbon dioxide or alcohol content, color, or stylistic, varietal or regional characteristics. Each has its advantages and disadvantages. Although sometimes arbitrary, such categorization does recognize significant differences, not only in production but also in

use. For example, classification by color supplies a rough indication of a wine’s flavor intensity. Stylistic and geographical origin often go hand in hand, at least for many European appellations, supplying additional information about the wine’s likely sensory attributes. Varietal designation may also furnish clues as to the wine’s potential flavor characteristics. Despite these potentialities, no system provides sufficient information on which to confidently base wine purchase. Although imperfect, without categorization, consumers would be even more confused than they are now. Thus, no matter how sensorially inadequate, codifying the eclectic range of wines into categories is a necessity. Of all systems, those based on geographic origin are the most commercially used, subdivided by color, carbon dioxide content, and fortification. For consumers, it permits wine selection to be based on regional/stylistic bias, giving the impression of being concrete. Regrettably, it frequently generates a false sense of security relative to flavor characteristics. The following arrangement is traditional, being based primarily on stylistic features. Wines are initially grouped based on alcohol concentration. This commonly is indicated by the terms “table” (alcohol contents usually ranging between 9% and 15% by volume) and “fortified” (alcohol contents typically varying between 17% and 22%, due to the addition of wine spirits). Table wines are subdivided into “still” and “sparkling” categories, depending on the wine’s carbon dioxide content.

Still table wines Because most wines fall into the still table wine category, it requires the largest number of subdivisions (Table 1.1). The oldest subdivision separates wines into

Per Capita Wine Consumption

140 120 100 80 60 40 20 0 Australia Argentina

France Canada

Italy Germany

Spain New Zealand

United States South Africa

Country

FIGURE 1.6

1969

1979

1989

1999

2002

2017

Changes in per-capita wine consumption (L/year) in several countries. Data from OIV, 2005. Statistical Report on World Vitiviniculture. http://www.oiv.int/oiv/info/, 2018.

12

TABLE 1.1

Classification of still wines based on stylistic differences. White

Long aging (often matured and occasionally fermented in oak cooperage)

Short aging (seldom exposed to oak)

Typically little varietal aroma

Varietal aroma commonly detectable

Typically little varietal aroma

Varietal aroma commonly detectable

Botrytized wines

Riesling

Trebbiano

Mu¨ller-Thurgau

Vernaccia di San Gimignano

Chardonnay

Muscadet

Kerner

Vin Santo

Sauvignon blanc

Folle blanche

Pinot blanc

Parellada

Chasselas

Chenin blanc

Se´millon

Aligote´

Seyval blanc

Red Short aging

Tank oak aging (many European wines, except those from France)

Barrel oak aging (most French, “new” European, and New World wines)

Little varietal aroma detectable

Varietal aroma often detectable

Tempranillo

Cabernet Sauvignon

Gamay

Dolcetto

Sangiovese

Pinot noir

Grenache

Grignolino

Nebbiolo

Syrah

Carignan

Baco noir

Garrafeira

Zinfandel

Barbera

Lambrusco

Rose´ Sweet

Dry

Mateus

Tavel

Pink Chablis

Cabernet rose´

Rosato

White zinfandel

Some blush wines

Some blush wines

1. Introduction

Long aging

13

Wine classification

white, red, and rose´ subgroups. Not only does this have the benefit of long use, it reflects distinct differences in flavor, use, and production methods. For example, red wines tend to be more flavorful, drier, and more astringent than white wines. In contrast, white wines are generally more acidic, are fragrant, and come in a range of sweetness styles. Rose´s fall in between, being lighter than red wines, but more astringent than whites, and occasionally mildly pe´tillant and sweet. Because most white wines are intended to be consumed with meals, their inherent acidic character is desirable. Combined with food proteins, the acidic aspect of the wine becomes balanced, accentuating, and harmonizing with food flavors. Most white wines are given little if any maturation in oak cooperage. Only wines with distinct varietal aromas tend to benefit from an association with oak flavors. Those with a sweet finish generally are intended to be consumed alone, as a “sipping” wine, or to replace dessert. Most botrytized, late-harvest, and ice wines fall into this subgroup. Modern red wines are almost exclusively dry. The absence of detectable sweetness is consistent with their intended use as a food beverage. The bitter and astringent compounds that characterize most red wines bind with food proteins, producing a balance they otherwise would not express. Occasionally, well-aged red wines are saved for enjoyment after the meal. Their diminished tannin content obviates the need for food to develop smoothness and balance. Also, the complex subtle bouquet of aged wines can be more fully appreciated in the absence of competing food flavors. Most red wines that age well are given the benefit of maturation in oak. Storage in small oak cooperage (w225-L barrels) usually promotes maturation and adds subtle flavors. The duration of contact and a mix of new and old barrels are frequently employed to adjust the amount and character of the oak flavor. Following inbarrel maturation, the wines typically receive further inbottle aging on the premises before release. When less oak character is desired, oak cooperage of larger (500e1000 L) capacity may be used. Alternatively, the wine may be matured in inert tanks to avoid flavor uptake. One of the more common differences between red wines depends on the intended consumer subset. Wines processed for early consumption have lighter, more fruity flavors. In contrast, those processed to enhance aging potential often do so at the expense of early enjoyment, and are initially intensely tannic. Beaujolais nouveau is a prime example of a wine designed for early consumption. In contrast, premium wines illustrate the other end of the spectrum. For these, long aging is required for the development of their finest qualities. Rose´ wines have frequently been maligned, but are experiencing a resurgence of interest and consumer

demand. To achieve the light rose´ color, juice from the crushed grapes is left in contact with the seeds and skins for only a short period. This limits not only anthocyanin extraction but also flavor uptake. In addition, rose´ wines soon lose their initial fruity character and fresh pink coloration (turning orangish). Many rose´ wines historically were finished with a slight sparkle and sweet taste. This made many aficionados view rose´s with disdain, considering them to possess the faults of both white and red wines, but none of their respective qualities. To counter the stigma formerly attached to the term “rose´,” many North American versions were designated as “blush” or “white” renderings of red cultivars. With the current upswing in the popularity of rose´ wines, this no longer seems necessary.

Sparkling wines Sparkling wines often are classified by production method (see Table 1.2). The three principal techniques are the traditional (champagne), transfer, and bulk (Charmat). They all employ yeasts to generate the effervescence that donates their distinctive attribute. Although technically precise, the system may not reflect significant sensory differences. For example, the traditional and transfer methods typically aim to produce dry to semidry wines that accentuate subtlety, limit varietal aroma, and possess a “toasty” bouquet. The bulk process is more frequently used to produce highly aromatic, sweet, sparkling wines, but not necessarily. The character of sparkling wines often differs more relative

TABLE 1.2

Classification of sparkling wines with some representative examples. Natural (without flavors added)

With added flavors, coolers (low alcohol)

Highly aromatic (sweet)

Subtly aromatic (dry or sweet)

Fruit-flavored, carbonated wines

Asti-style Muscatbased wines

Traditional style Champagne Vin Mousseux Cava Sekt Spumante Crackling/carbonated Perlwein Lambrusco Vinho Verde

14

1. Introduction

to their yeast (sur lies) contact time, and the variety of grapes used, than to their production method. Sparkling wines, produced by incorporating carbon dioxide under pressure, show an even wider range of styles. These include dry white wines, such as vinho verde (traditionally obtaining its pe´tillance from malolactic fermentation); sweet, pe´tillant red wines; most crackling rose´s; and fruit-flavored “coolers.”

Fortified wines (dessert and appetizer wines) All terms applied to this category (Table 1.3) can be somewhat misleading. For example, some subcategories achieve their elevated alcohol contents without the addition of distilled spirits (e.g., the sherry-like wines from Montilla, Spain). Thus, technically they are not fortified. The alternative designation of aperitif and dessert wines also has problems. Although most are used as aperitif or dessert wines, many table wines are used similarly. For example, dry sparkling wines are often viewed as the ultimate aperitif, whereas botrytized wines are the superlative dessert wines. TABLE 1.3

Classification of fortified wines with some representative examples.

With added flavors

Without added flavors

Vermouth

Sherry-like

Byrrh

Jerez-Xere`s-Sherry

Marsala (some)

Malaga (some)

Dubonnet

Montilla Marsala Chaˆteau-Chalon New World solera and submerged sherries Port-like Porto New World ports Madeira-like Madeira Baked New World sherries and ports Muscatel Muscat-based wines Setu´bal Samos (some) Muscat de Beaunes de Venise Communion wine

Regardless of designation, wines in this category typically are consumed in small amounts, and the bottle’s contents are seldom completely consumed upon opening. Their high alcohol contents limit microbial spoilage, and their marked flavor and resistance to oxidization often allow them to remain stable for weeks in partially empty bottles. These are definitely desirable properties for wines consumed in small amounts over days or weeks. The exceptions are fino sherries and vintage ports. They lose their distinctive properties several months after bottling or several hours after opening, respectively. Fortified wines are produced in a wide range of styles. Dry or bitter-tasting forms are normally consumed as aperitifs before meals. They stimulate the appetite and activate the release of digestive juices. Examples are fino-style sherries and dry vermouths. The latter are flavored with a variety of herbs and spices. More commonly, fortified wines are markedly sweet. Major examples are oloroso sherries, ports, madeiras, and marsalas. These wines are consumed after meals, or as a dessert substitute.

Wine quality What constitutes wine quality is often based on cultural percepts, training, and experience. Ideally, this reflects the sensory characteristics of the wine. In addition, its perception depends on the acuity of the taster and the degree of attention given the wine. Nonetheless, quality does have components independent of the individual taster. This especially applies to faults. There is less accord as to what constitutes quality. Not surprisingly, quality often is defined in diverse and abstruse ways. It may be evaluated in terms of subtlety and complexity, aging potential, stylistic purity, varietal expression, historic prestige, or expert ranking. Each has its justification and limitations. Nevertheless, the views of experts (often self-appointed) have had the greatest influence on consumers, and occasionally winemakers. Premium wine sales constitute only a small fraction of world production, but have had a profound influence on the direction of enologic and viticultural research. This has resulted in an overall improvement in wine quality. As a consequence, consumers can choose from a greater selection of quality wines than at any time in human history. Occasionally, this has been viewed as bringing “fine wine to all, on the supermarket shelf” (NOVA, 1978). Although partially true, this does not necessarily equate to appreciation. It is unlikely that simple economic availability will increase the appreciation of fine wine, any more than opera on television appears to have generated higher consumer demand. The sensory and sociological

Health-related aspects of wine consumption

features that make quality wine appealing to connoisseurs are still poorly understood. This means that for most wine producers, understanding the preferences of the majority of consumers is far more lucrative. Understanding how a target group perceives quality and value for money is particularly lucrative (Cardello, 1995; Lawless, 1995). A clear example of the importance of perception (and purchase intention) was the marked jump in red wine sales following the airing of the “French Paradox” on 60 Minutes. Its long-term influence on wine sales is less clear. For example, several studies suggest that the purported health benefits of wine have only a marginal influence on consumer wine purchases (Saliba and Moran, 2010), the effect being most noticeable in those least in needdthe healthy (Higgins and Llanos, 2015). Pretorius et al. (2006) have clearly enunciated the marketing view of quality as being defined as sustainable consumer satisfaction. However, accurately measuring this aspect is fraught with difficulty, consumer loyalty often being fickle. It is also uncertain whether purchases reflect opinions expressed on questionnaires (Jover et al., 2004; Ko¨ster, 2003). Nonetheless, perceived quality is probably the principal force influencing most connoisseur purchases. How else can one explain the continuing significance of a quality ranking generated for the 1855 World Exposition in Paris (the cru classe´ Bordeaux system), or the influence of critics such as Robert Parker? Regrettably, too few people have faith in their own sensory skills. Playing “follow the leader” seems simplerdobviating the need to do personal assessment. This also applies to food and wine combination. Studies on the physiology, neurology, and psychology of perception increasingly show how we all are, to varying degrees, “islands unto ourselves.” For the occasional wine drinker, geographic or varietal origin tends to be of secondary importance, availability, price, and previous experience being the overriding factors directing purchase. Appreciation is usually assessed on subjective, highly idiosyncratic criteria. In contrast, provenance and reputation strongly influence the purchase, and presumably appreciation, of wine aficionados. For the connoisseur, whether and how well a wine reflects expectations can be crucial to perceived quality. Historic and traditional expectations are central to the quality percepts embodied in most appellation control laws, and continue to influence consumer opinion, like a self-fulfilling prophecy. In addition to the purely subjective and historic views of quality, esthetic quality is arguably the most highly prized attribute possessed by premium wines. It is defined similar to that of artistic endeavors. This includes concepts described as balance, harmony, symmetry, development, duration, complexity, subtlety, interest, intrigue, dynamism, and uniqueness.

15

Regrettably, defining these terms precisely is no easier with wine than with artistic pursuits, owing to human variability in perception and the influences of training and cultural bias. Nevertheless, balance and harmony in wine commonly refer to a smooth taste and mouthfeel, without any aspect interfering with an overall pleasurable experience. Symmetry often applies to the perception of compatibility and balance between sapid (taste and mouth-feel) and olfactory (fragrant) sensations. Development typically refers to the dynamic temporal changes in intensity and aromatic character after pouring. When fascinating and dynamic, this feature is central to maintaining interest. Fragrance duration (lasting over the duration of the tasting) is also a critical element in the esthetic appreciation of fine wines. Complexity and subtlety are additional highly valued attributes of fragrance and flavor. The long-term impact of these factors, termed memorability (the “wow” factor), is probably the most significant determinant in the overall quality impression. It is the unexpected (when appreciated) that is most fundamental to intrigue and the embedding of memory traces in the brain.

Health-related aspects of wine consumption Until the end of the 19th century, human afflictions were often treated with wine, either directly or indirectly (as a solvent for the active ingredient) (Lucia, 1963). Early in the 20th century, well-meaning, but misguided, prohibitionists waged war against all beverages containing alcohol. They succeeded in persuading several governments to make its commercial sale illegal, and seemingly convinced many in the medical profession that its consumption was detrimental. The only exception to the general interdiction was for use in religious ceremonies. More recently, there has been a marked renewal of interest in the medical benefits of moderate wine consumption (see Chapter 12). Among the betterdocumented benefits are those relative to cardiovascular disease. In addition, wine may reduce the undesirable influences of stress, enhance sociability, lower rates of clinical depression, and improve self-esteem and appetite in the elderly (Baum-Baicker, 1985; Delin and Lee, 1992). Imperative in all such studies has been the need to minimize the potential influence of the multitude of cultural, environmental, and individual factors that could skew the results. For example, in one study, wine consumers were found to purchase more “healthy” foods than their beer-drinking counterparts (Johansen et al., 2006). Thus, the importance of studies of more than 55,000 men over a 16-year period (Mukamal et al., 2006). They accounted for comparable lifestyles when correlating alcohol consumption with coronary

16

1. Introduction

In an intriguing study by Lindman and Lang (1986), wine was the only beverage containing alcohol associated with positive social expectations. Thus, wine appears unique in its identification with happiness, contentment, and romance. Additional studies have also found that wine is associated with more socially desirable stereotypes than other alcohol-containing beverages (Delin and Lee, 1992; Duncan et al., 1995; Klein and Pittman, 1990a). Furthermore, wine consumption is rarely associated with intoxication and other alcohol-related problems (Smart and Walsh, 1999; Jensen et al., 2002) (Fig. 1.7). Finally, wine is the alcoholic beverage most associated in the mind of consumers with food consumption (Pettigrew and Charters, 2006). How and why wine came to inherit and retain its current social image is intriguing. In the ancient civilizations of the Near East, where the vine was not indigenous, wine was comparatively expensive to produce or import. Thus, its consumption was largely limited to the ruling or priestly classes. This feature donated an element of exclusivity beer did not have, beer being the drink of the proletariat. Wine’s association with religious rites also gave it an element of

62.9

heart disease. Thus, they reduced, if not eliminated, incidental factors that might influence the results. Neoprohibitionists are all too quick to point out both real and imaginary design flaws in any study that presents findings contrary to their preferred beliefs. On the other hand, many in the wine industry are all too quick to endorse results suggesting benefits. Although the vast majority of the evidence is encouraging to those who enjoy wine, the data are insufficient to justify wine’s consumption solely on health grounds. If one summed the supposed health benefits of wine consumption, eating fruits and vegetables, avoiding excessive carbohydrate and fat intake, doing physical and mental exercise, etc., we should all live to the age of Methuselah. Nonetheless, the influence of moderate wine consumption in favoring a healthy balance of low- and high-density lipoproteins in the plasma is now well established (Kinsella et al., 1993; Rimm et al., 1991; Soleas et al., 1997). Even the National Institute on Alcohol Abuse and Alcoholism (1992) was moved to record that “there is a considerable body of evidence that lower levels of drinking decrease the risk of death from coronary heart disease.”

52.9 41.2

39.9

20.1

28.8

26.0

30

29.3

31.3

35.0

40

25.2

20

Underage drinking

Beer

Fighting and rowdy behavior

Distilled spirits

Drunk driving

Wine

Alcoholism

Wine cooler

Health problems

All equally

0.4 0.3

3.8

2.2

1.6 0.1

7.0 0.8

1.4 0.1

0.8

0.1 0.2

2.3

0.5 0.1

2.2

3.1

5.5 1.2

10

8.6

13.8

Percentage of respondents

50

48.0

49.6

54.2

60

Birth defects

None

FIGURE 1.7 Comparison of the perception of adverse consequences associated with the consumption of different beverages containing alcohol. Reprinted from Klein, H., Pittman, D., 1990b. Perceived consequences of alcohol use. Intl. J. Addictions 25, 471e493, p. 481 by courtesy of Marcel Dekker, Inc.

References

divinity, occasionally associated with a particular god (Dionysus in Greece, Bacchus throughout the Roman Empire). The antibiotic properties of wine had the unrecognized benefit of partially disinfecting water and food. This was of particular value where the vine grew endemically and wine could be produced inexpensively. Wine’s ready availability is evidenced by the profusion of Roman wine bars in Ostia, Herculaneum, and Pompeii (Hermansen, 1981; Unwin, 1991). That an amphora of wine could purchase a slave in Gaul (Siculus, Bibliotheca Historica 5.26; w60e30 BCE) is a clear indication of value. It probably also suggests that wine was appreciated for reasons not altogether due to its medical/sensory benefits (Unwin, 1991). Despite wine’s ubiquity in the Roman world, Roman poetry paints a picture of wine being viewed as a prestige item. This influence continued when Christianity became the state religion, and donating vineyards to church institutions became a tradition (Rossiter, 2007). In addition, wine’s role in communion encouraged its continued production. In contrast, wine was a rarity in most northern European regions, a feature that added to its prestige for the ruling classes (see Unwin, 1991). With industrialization, and the evolution of a middle class, the desire to adopt many of the social graces of the ruling class spread wine’s appeal. In contrast, beer tended to remain the alcoholic beverage of the poor. With the development of distillation, one of the first products (brandy) was too expensive for most people. However, distilled spirits, derived from grains or sugar cane, were much less expensive to produce, and could supply the same effect, if so desired. With the resultant negative social consequences, spirits have never approached the status (with the exception of single malt whiskies) associated with wine (or brandies). Wine also has the advantage of coming in an amazing range of styles, sensory characteristics, and qualities unmatched by any other beverage. Thus, it is ideally suited to the development of connoisseurship. These elements almost demand reverence from its devotees, limiting its abuse. Finally, the finest wines have the ability to age for decades, and occasionally centuries, developing new and novel sensory dimensions. This has further increased its uniqueness and prestige among those willing and able to pay. Thus, exclusivity, and the aura this provides, even to those who cannot possess it, promotes and enhances the retention of wine’s historic perception as a beverage of high social status and, it is hoped, of moderation. In addition to revealing the potential benefits of wine consumption, researchers are also investigating the potential undesirable consequences of even moderate wine consumption. For example, the induction of headaches by red wine has been correlated with insufficient

17

production of platelet phenol sulfotransferase (Littlewood et al., 1988). Also, headache prevention has been associated with the prior use of acetylsalicylic acid (Kaufman, 1992) or other prostaglandin synthesis inhibitors. In a few medical instances, consumption of wine (and other alcoholic beverages) is counterindicated, notably gastrointestinal ulcerations and cancers. Wine may also undesirably interact with some medications (see Chapter 12). Wine consumption is also ill advised for those who have addictive tendencies, or who appreciate wine’s alcohol content more for its intoxication effects than its role in sensory appreciation. Where wine is taken to enhance the pleasures of the table, moderate wine consumption (between one and two glasses per day) has the supplemental value of being healthy (for most of us).

References Accum, F., 1820. Adulteration of wine. In: A Treatise on Adulterations of Food and Culinary Poisions, second ed. Longman, Hurst, Rees, Orme, and Brown, London, UK, pp. 92e105. Anderson, K., 2004. The World’s Wine Markets Globalization at Work. Edward Elgar Publ. Ltd., Cheltenham, UK. Andre´, J., 2009. L’Alimentation et la Cuisine a` Rome (E´tudes Anciennes Serie Latine). Les Belle Lettres, Paris, France. Areshian, G.E., Gasparyan, B., Avetisyan, P.S., Pinhasi, R., Wilkinson, K., Smith, A., et al., 2012. The chalcolithic of the Near East and south-eastern Europe: discoveries and new perspectives from the cave complex Areni-1, Amenia. Antiquity 86, 115e130. Arobba, D., Bulgarelli, F., Camin, F., Caramiello, R., Larcher, R., Martinelli, L., 2014. Paleobotanical, chemical and physical investigation of the content of an ancient wine amphora from the northern Tyrrhenian sea in Italy. J. Archaeol. Sci. 45, 226e233. Arroyo-Garcı´a, R., Ruiz-Garcı´a, L., Bolling, L., Ocete, R., Lo´pez, M.A., Arnold, C., et al., 2006. Multiple origins of cultivated grapevine (Vitis vinifera L. ssp. sativa) based on chloroplast DNA polymorphisms. Mol. Ecol. 15, 3707e3714. Badler, V.R., 1995. The archaeological evidence for winemaking, distribution and consumption at proto-historic Goden Tepe, Iran. In: McGovern, P.E., Fleming, S.J., Katz, S.H. (Eds.), The Origins and Ancient History of Wine. Gordon and Breach Publishers, Luxembourg, pp. 45e56. Barnard, H., Dooley, A.N., Areshian, G., Gsaparyan, B., Faull, K.F., 2011. Chemical evidence for wine production around 4000 BCE in the Late Chalcolithic Near Eastern highlands. J. Archaeol. Sci. 38, 977e984. Baum-Baicker, C., 1985. The psychological benefits of moderate alcohol consumption: a review of the literature. Drug Alcohol Depend. 15, 305e322. Best, M.R., 1976. The mystery of vintners. Agric. Hist. 50, 362e376. Bohrer, V.L., 1972. On the relation of harvest methods to early agriculture in the Near East. Ecom. Bot. 26, 145e155. Bramanti, B., Thomas, M.G., Haak, W., Unterlaender, M., Jores, P., Tambets, K., et al., 2009. Genetic discontinuity between local hunter-gatherers and central Europe’s first farmers. Science 326, 137e140. Carbonneau, A., 1983. Ste´rilite´s maˆle et femalle dans le genre Vitis. II. Conse´quences en ge´ne´tique et se´lection. Agronomie 3, 645e649.

18

1. Introduction

Cardello, A.V., 1995. Food quality: relativity, context and consumer expectations. Food Qual. Prefer. 6, 163e168. Cavalieri, D., McGovern, P.E., Hartl, D.L., Mortimer, R., Polsinelli, M., 2003. Evidence for S. cerevisiae fermentation in ancient wine. J. Mol. Evol. 57, S226eS232. Civil, M., 1964. A hymn to the beer goddess and a drinking song. In: Briggs, R.D., Brinkman, J.A. (Eds.), Studies Presented to A. Leo Oppenheim. Oriental Institute, Chicago, IL, pp. 67e89 (cited in Cavalieri et al., 2003). Cousteau, J.-Y., 1954. Fish men discover a 2,200-year-old Greek ship. Natl. Geogr 105 (1), 1e34. Delin, C.R., Lee, T.L., 1992. Psychological concomitant of the moderate consumption of alcohol. J. Wine Res. 3, 5e23. Ducousso, A., Bordacs, S., 2003. EUFORGEN Technical Guidelines for Genetic Conservation and Use for Pedunculate and Sessile Oaks (Quercus Robur and Quercus Petraea). International Plant Genetic Resources Institute, Rome, Italy, p. 6. Duncan, B.B., Chambless, L.E., Schmidt, M.I., Folsom, A.R., Szklo, M., 1995. Association of the waist-to-hip ratio is different with wine than with beer or hard liquor consumption. Am. J. Epidemiol. 142, 1034e1038. Eisinger, J., 1982. Lead and wine: eberhard gockel and the colica Pictonum. Med. Hist. 26, 279e302. Eshel, T., Erel, Y., Yahalom-Mack, N., Tirosh, O., Gilboa, A., 2019. Lead isotopes in silver reveal earliest Phoenician quest for metals in the west Mediterranean. Proc. Natl. Acad. Sci. https://doi.org/ 10.1073/pnas. Fay, J.C., Liu, P., Ong, G.T., Dunham, M.J., Cromie, G.A., Jeffery, E.W., et al., 2019. A polyploid admixed origin of beer yeasts derived from European and Asian wine populations. PLoS Biol. 17, e3000147. FAOStat, 2013. FAO Statistical Yearbook 2013. World Food and Agriculture. www.fao.org/publixations. Forbes, R.J., 1965. Studies in Ancient Technology, second ed., vol. 3. E. J. Brill, Leiden, The Netherlands, p. 83. note 17. Foley, B.P., Hansson, M.C., Kourkoumelis, D.P., Theodoulou, T.A., 2012. Aspects of ancient Greek trade re-evaluated with amphora DNA evidence. J. Archaeol. Sci. 39, 389e398. Fregoni, M., 1991. Origines de la Vigne et de la Viticulture. Musumeci Editeur, Quart (Vale d’Aosta), Italy. Frey, D., Hentschel, F.D., Keith, D.H., 1978. Deepwater archaeology: the capistello wreck excavation, lipari, aeolian islands. Int. J. Naut. Archaeol. Underw. Explor. 7, 279e300 (Figure 12). Garnier, N., Valamoti, S.M., 2016. Prehistoric wine-making at Dikili Tash (northern Greece): integrating residue analysis and archaeobotany. J. Archaeol. Sci. 74, 195e206. Garnier, N., Richardin, P., Cheynier, V., Regert, M., 2003. Characterization of thermally assisted hydrolysis and methylation products of polyphenols from modern and archaeological vine derivatives using gas chromatographymass spectrometry. Anal. Chim. Acta 493, 137e157. Gismondi, A., Di Marco, G., Martini, F., Sarti, L., Crespan, M., Martı´nex-Labarga, C., et al., 2016. Grapevine carpological remains revealed the existence of a Neolithic domesticate Vitis vinifera L. specimen containing ancient DNA partially preserved in modern ecotypes. J. Archaeol. Sci. 69, 75e84. Gla¨nzel, W., Veugelers, R., 2006. Science for wine: a bibliometric assessment of wine and grape research for wine-producing and consuming countries. Am. J. Enol. Vitic. 57, 23e32. Grace, V.R., 1979. Amphoras and the Ancient Wine Trade. American School Classical Studies Athens, Princeton, NJ (photo 63). Guasch-Jane´, M.R., Ibern-Go´mez, M., Andre´s-Lacueva, C., Ja´urequi, O., Lamuela-Ravento´s, R.M., 2004. Liquid chromatography with mass spectrometry in tandem mode applied for the identification of wine markers in residues from Ancient Egyptian vessels. Anal. Chem. 76, 1672e1677.

Guasch-Jane´, M.R., Andre´s-Lacueva, C., Ja´uregui, O., Lamuela Ravento´s, R.M., 2006. The origin of the ancient Egyptian drink Shedeh revealed using LC/MS/MS. J. Archaeol. Sci. 33, 98e101. Hallgarten, F.L., 1986. Wine Scandal. Weidenfeld & Nicolson, London, UK. Hanson, V.D., 1992. Practical aspects of grape-growing and the ideology of Greek viticulture. In: Wells, B. (Ed.), Agriculture in Ancient Greece: Proc. 7th Int. Symp. Swedish Institute in Athens. 1990. Paul Astroms Forlag, Stockholm, Sweden, pp. 161e166. Hayes, W.C., 1951. Inscriptions from the palace of amenhotep III. J. Near East. Stud. 10, 82e112 (pg. 96). Henderson, A., 1824. The History of Ancient and Modern Wines. Baldwin, Craddock and Joy, London, UK. Hermansen, G., 1981. Ostia: Aspects of Roman City Life. Univ. Alberta Press, Edmonton, AB (Figure 14, p. 91). Higgins, L.M., Llanos, E., 2015. A healthy indulgence? Wine consumers and the health benefits of wine. Wine Econ. Pol. 4, 3e11. Hope, C., 1978. Excavations at malkata and the birket habu 1971e1974; V. Jar sealings and amphorae of the 18th dynasty: a technological study. Egyptology Today 5 (2), 1e88. Warminster: Aris & Phillips. Isaksson, S., 2005. Wine or oil? analysis of organic residues in connection with ceramic materials from Pyrgouthi, the Berbati Valley, Argolis, Greece. In: Hjohlman, J., Penttinen, A., Wells, B. (Eds.), Pyrgouthi. A Rural Site in the Berbati Valley in the Argolid from the Early Iron Age to Late Antiquity. Skrifter Utgivna Av Svenska Institutet I Athen, vol. 40, pp. 283e298 (Stockholm). Isaksson, S., Karlsson, C., Eriksson, T., 2010. Ergosterol (5,7,22ergostatrien-3b-ol) as a potential biomarker for alcohol fermentation in lipid residues from prehistoric pottery. J. Archaeol. Sci. 37, 3263e3268. Jashemski, W.F., 1975. Vintage Pompeii. Nat. Hist. 84, 52e60. Jashemski, W.F., 1979. The Gardens of Pompeii, Herculaneum and the Villas Destroyed by Vesuvius, vols. I & II. Caratzas Brothers, New Rochelle, NY ( Plate 256). Jensen, M.K., Andersen, A.T., Sørensen, T.I.A., Becker, U., Thorsen, T., Grønbaek, M., 2002. Alcoholic beverage preference and risk of becoming a heavy drinker. Epidemiology 13, 127e132. Johansen, D., Friis, K., Skovenborg, E., Grønbaek, M., 2006. Food buying habits of people who buy wine or beer: cross sectional study. Br. Med. J. 332, 519e522. Jover, A.J.V., Montes, F.J.L., Fuentes, M.M.F., 2004. Measuring perceptions of quality in food products: the case of red wine. Food Qual. Prefer. 15, 453e469. Kaufman, H.S., 1992. The red wine headache and prostaglandin synthetase inhibitors: a blind controlled study. J. Wine Res. 3, 43e46. Kinsella, J.E., Frankel, E., German, J.B., Kanner, J., 1993. Possible mechanisms for the protective role of antioxidants in wine and plant foods. Food Technol. 47, 85e89. Klein, H., Pittman, D., 1990a. Drinker prototypes in American society. J. Subst. Abus. 2, 299e316. Klein, H., Pittman, D., 1990b. Perceived consequences of alcohol use. Int. J. Addict. 25, 471e493. Koehler, C.G., 1986. Handling of Greek transport amphoras. In: Empereur, J.-Y., Garlan, Y. (Eds.), Recherches sur les Amphores Greques E´cole franc¸aise d’Anthe`nes, Paris, France. Bull. Correspondance Helle´nique, pp. 49e67. Suppl. 13. Kowallik, V., Greig, D., 2016. A systemic forest survey showing an association of Saccharomyces paradoxus with oak leaf litter. Environ. Microbiol. Rep. 8, 833e841. Komar, P., 2013. The benefits of interdisciplinary approach e a case of studying the consumption of Greek wines in Roman Italy. In: Proceeding of the 1st Annual Interdisciplinary Conference, AIIC. 24e26 April 2013, Azores, Portugal, pp. 45e54. Ko¨ster, E.P., 2003. The psychology of food choice: some often encountered fallacies. Food Qual. Prefer. 14, 359e373.

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Poo, M.-C., 1999. The use and abuse of wine in Ancient China. J. Econ. Soc. Hist. Orient 42, 123e151. Pretorius, I.S., Bartowsky, E.J., de Barros Lopes, M., Bauer, F.F., du Toit, M., van Rensburg, P., et al., 2006. The tailoring of designer grapevines and microbial starter strains for a market-directed and quality-focussed wine industry. In: Hui, H.Y., Sherkat, F. (Eds.), Handbook of Food Science, Technology and Engineering, vol. 4. CRC Press, New York, NY, pp. 1741e1742. Ramishvili, R., 1988. Dikorastushii Vinograd Zakavkazia (Wild Grape of the South Caucasus). Ganatleba, Tbilisi, Georgia (in Russian). Renfrew, C., 1989. The origins of Indo-European languages. Sci. Am. 261 (4), 106e116. Rimm, E.B., Giovannucci, E.I., Willett, W.C., Colditz, G.A., Ascerio, A., Rosner, B., et al., 1991. Prospective study of alcohol consumption and risk of coronary disease in men. Lancet 338, 464e468. Rossiter, J.J., 2007. Winemaking after Pliny: viticulture and farming technology in late antique Italy. In: Lavan, L., Zanini, E., Sarantis, A. (Eds.), Technology in Transition: a.D. 300e650. Late Antique Archaeology, vol. 4. Koninklijk Brill NV, Leiden, The Netherlands, pp. 93e118. Saliba, A.J., Moran, C.C., 2010. The influence of perceived healthiness on wine consumption patterns. Food Qual. Prefer. 21, 692e696. Schmidt, K., 2010. Go¨bekli Tepe e the Stone Age Sanctuaries. New results of ongoing excavations with a special focus on sculptures and high reliefs. Docu. Praehist. 37, 239e256. Schumann, F., 1974. Untersuchungen an wildreben in Deutschland. Vitis 13, 198e205. Serpico, M., 2000. Resins, amber and bitumen. In: Nicholson, P.T., Shaw, I. (Eds.), Ancient Egyptian Materials and Technology. Cambridge University Press, Cambridge, England, pp. 430e474. Skovenborg, E., 1995. Lead in wine through the ages. J. Wine Res. 6, 49e64. Smart, R.G., Walsh, G., 1999. Heavy drinking and problems among wine drinkers. J. Stud. Alcohol 60, 467e471. Soleas, G.J., Diamandis, E.P., Goldberg, D.M., 1997. Wine as a biological fluid: history, production, and role in disease prevention. J. Clin. Lab. Anal. 11, 287e313. Stafford, H.A., 1959. Distribution of tartaric acid in the leaves of certain angiosperms. Am. J. Bot. 46, 347e352. Stanislawski, D., 1975. Dionysus westward: early religion and the economic geography of wine. Geogr. Rev. 65, 427e444. Stanley, P.V., 1999. Gradation and quality of wine in the Greek and Roman worlds. J. Wine Res. 10, 105e114. Stevenson, A.C., 1985. Studies in the vegetational history of S.W. Spain. II. Palynological in investigations at Laguna de los Madres, Spain. J. Biogeogr. 12, 293e314. Tallet, P., 1995. Le shedeh: e´tude d’un proce´de´ de vinification en Egypte ancienne. Bull Inst, Franc¸. Arche´ol. Orient. 95, 459e492. Tanasi, D., Greco, E., Di Tullio, V., Capitani, D., Gullı`, D., Ciliberto, E., 2017. 1H-1H NMR 2D-Tocsy, ATR FT-IR and SEM-EDX for the identification of organic residues on Sicilian prehistoric pottery. Microchem. J. 135, 140e147. Tannahill, R., 1973. Food in History. Stein and Day, New York, NY. Tchernia, A., 1986. Le Vin de l’Italie Romaine: Essai d’Histoire Economique d’apre`s les Amphores. E´cole Franc¸aise de Rome, Rome. Unwin, T., 1991. Wine and the Vine an Historical Geography of Viticulture and the Wine Trade. Routledge, London, UK. Vandiver, P., Koehler, C.G., 1986. Structure, processing, properties, and style of Corinthian amphoras. In: Kingery, W.D. (Ed.), Ceramics and Civilization, vol. 2. Technology and Style American Ceramics Society, Columbus, OH, pp. 173e215. von Hirnkofen, 1478. Von Bewahrung und Bereitung der Weine. Konrad Fyner, Esslingen, Germany (a translation of Liber de vinis, 1311

20

1. Introduction

by Arnold de Villanova and Tractatus de vino et eius proprietate by Gottfried vin Francon). An English translation appears in Sigerist, H.E., 1943. The Earliest Printed Book on Wine. Schuman, New York, NY. Younger, W., 1966. Gods, Men and Wine. George Rainbird, London, UK. Zohary, D., Hopf, M., 2000. Domestication of Plants in the Old World, third ed. Oxford University Press (Clarendon), Oxford, UK.

Suggested readings Wine history Denecker, E., Vandorpe, K., 2007. Sealed amphora stoppers and tradesmen in Greco-Roman Egypt: archaeological, papyrological and inscriptional evidence. BABesch 82, 115e128. Forni, G., 2012. The origin of “Old World” viticulture. In: Maghradze, D., Rustioni, L., Turok, J., Scienza, A., Failla, O. (Eds.), Caucasus and Northern Black Sea Region Ampelography, Vitis Special Issue, pp. 27e38.

Johnson, H., 1989. Vintage: The Story of Wine. Simon & Schuster, New York, NY. Lesko, L.H., 1977. King Tut’s Wine Cellar. Albany Press, Albany. McGovern, P.E., 2003. Ancient Wine: The Search for the Origins of Viniculture. Princeton University Press, Princeton, N.J. McGovern, P.E., Fleming, S.J., Katz, S.H. (Eds.), 1995. The Origins and Ancient History of Wine. Gordon and Breach Publishers, Luxembourg. Thurmond, D.L., 2016. From Vines to Wines in Classical Rome: A Handbook of Viticulture and Oenology in Rome and the Roman West. Brill Academic Publ., Leiden, Netherlands. Unwin, T., 1991. Wine and the Vine. An Historical Geography of Viticulture and the Wine Trade. Routledge, London, UK.

Wine and culture Burton, B.J., Jacobsen, J.P., 2001. The rate of return on investment in wine. Econ. Inq. 39, 337e350. Charters, S., 2006. Wine and Society: The Cultural and Social Context of a Drink. Elsevier, Butterworth-Heinemann, Burlington, MA. Oczkowski, E., 2001. Hedonic wine price function and measurement of error. Econ. Rec. 77, 374e382.

C H A P T E R

2 Grape species and varieties O U T L I N E Introduction

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The genus Vitis

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Geographic origin and distribution of Vitis and Vitis vinifera

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Domestication of Vitis vinifera

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Cultivar origins

Standard breeding techniques Modern approaches to vine improvement Clonal selection Somaclonal selection and mutation

48 52 53 60

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Grapevine cultivars Vitis vinifera cultivars Interspecies hybrids

60 60 65

Recorded cultivar development

42

References

68

Grapevine improvement

46

Suggested reading

75

Introduction

Cyphostemma, Nothocissus, Parthenocissus, Pterisanthes, Pterocissus, Rhoicissus, Tetrastigma, and Yua.

Grapevines are classified in the genus Vitis, within the Vitaceae. Other well-known members of the family are the Boston ivy (Parthenocissus tricuspidata) and Virginia creeper (P. quinquefolia). Members of the Vitaceae are typically woody, show a climbing habit, have leaves that develop alternately on shoots (Fig. 2.1), and possess swollen or jointed nodes. These may generate tendrils or flower clusters opposite the leaves. The flowers are minute, are uni- or bisexual, and occur in large clusters. Most flower parts appear in groups of fours or fives, with the stamens developing opposite the petals. The ovary consists of two carpels, partially enclosed by a receptacle that develops into a two-compartmented berry. The fruit contains up to four seeds. The Vitaceae is predominantly a tropical to subtropical family, containing about 900 species, divided among some 14 genera (Galet, 1988; Wen, 2007). In contrast, Vitis is primarily a temperate-zone genus, occurring indigenously only in the Northern Hemisphere. Related genera include Acareosperma, Ampelocissus, Ampelopsis, Cayratia, Cissus, Clematicissus,

Wine Science, Fifth Edition https://doi.org/10.1016/B978-0-12-816118-0.00002-7

The genus Vitis Grapevines are distinguished from related genera primarily on floral characteristics. The flowers are typically functionally unisexual, being either male (possessing erect, functional anthers, and lacking a fully developed pistil) or female (containing a functional pistil, and either producing recurved stamens and sterile pollen or lacking anthers) (Fig. 2.2). The petals are fused, forming a calyptra or cap. The petals remain connected at the apex, splitting along the base only at maturity, when the calyptra is shed (see Plate 3.6). Occasionally, though, the petals may separate at the top, while remaining attached at the base (Plate 2.1). These “star” flowers possess an appearance resembling typical flowers. This feature characterizes some members of the Vitaceae, for example, Cissus. In some cultivars, star flower production is induced by cool temperatures, whereas in others it is a constitutional property (Longbottom et al., 2008). The trait is not genetically

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2. Grape species and varieties

FIGURE 2.1 Vitis vinifera shoot, showing the arrangement of leaves, clusters (Cl), and tendrils (T); Ax B, axillary buds; Bl, blade; I, internode; P, petiole; Sh T, shoot tip; Stip, stipule. After von Babo, A.F., Mach, E., 1923. Handbuch Weinbaues und der Kellerwirtschaft, fourth ed., vol. 1. Parey, Berlin, Germany, Part 1. From Pratt, C., 1988. Grapevine structure and growth stages. In: Pearson, R.C., Goheen, A.C. (Eds.), Compendium of Grape Diseases. APS Press, St. Paul, Minnesota, pp. 3e7, reproduced by permission.

transmissible in Vitis as the fruit is seedless (Chardonnay) or no fruit is produced (Shiraz). Swollen nectaries occur at the base of the ovary (see Fig. 3.25C). Although they produce no nectar, they generate a mild fragrance that can attract pollinating insects. The sepals of the calyx form only as vestiges and degenerate early in flower development. The fruit is juicy and acidic. The genus has typically been divided into two subgenera, Vitis and Muscadinia. Vitis (bunch grapes) is the larger of the two subgenera, containing all species except V. rotundifolia and V. popenoei. The latter are placed in the subgenus Muscadinia (muscadine grapes). The two subgenera are sufficiently distinct to have induced some taxonomists to raise Muscadinia to the genus level. Members of the subgenus Vitis are characterized by having shredding bark, nonprominent lenticels, a pith interrupted at nodes by woody tissue (the diaphragm), tangentially positioned phloem fibers, branched tendrils, elongated flower clusters, berries that adhere to the fruit stalk at maturity, and pear-shaped seeds possessing a prominent beak and smooth chalaza. The chalaza is a pronounced, circular, depressed region on the dorsal (back) side of the seed (Fig. 2.3C). In contrast, species in the subgenus Muscadinia possess a tight, nonshredding bark, prominent lenticels, no diaphragm

FIGURE 2.2 Diagrammatic representation of the variety of male, female, and bisexual flowers produced by Vitis vinifera. After Levadoux, L., 1946. E´tude de la fleur et de la sexualite´ chez la vigne. Ann. E´c. Natl. Agric. Montpellier 27, 1e90, reproduced by permission.

interrupting the pith at nodes, radially arranged phloem fibers, unbranched tendrils, small floral clusters, berries that separate individually from the cluster at maturity, and boat-shaped seeds with a wrinkled chalaza. Some of these characteristics are diagrammatically illustrated in Fig. 2.3. Plate 2.2 illustrates the appearance of Muscadinia grapes and leaves. The two subgenera also differ in their chromosomal composition. Vitis species contain 38 chromosomes (2n ¼ 6x ¼ 38), whereas Muscadinia species possess 40 chromosomes (2n ¼ 6x ¼ 40). The symbol n refers to the number of chromosome pairs formed during meiosis, and x refers to the number of chromosome complements (genomes that were involved in their evolution). Successful crosses can be experimentally produced between species of the two subgenera, primarily when V. rotundifolia is used as the pollen source. When V. vinifera is used as the male plant, the pollen germinates, but does not effectively penetrate the style of the V. rotundifolia flower (Lu and Lamikanra, 1996). This may result from the synthesis of inhibitors, such as quercetin glycosides in the pistil (Okamoto et al., 1995). Although generally showing vigorous growth, the progeny frequently are infertile. This probably results from imprecise pairing of the unequal number of chromosomes

The genus Vitis

23

PLATE 2.1 (A) Typical Chardonnay star flowers: early in development when the ovary is small (a) and later in development when the ovary has enlarged (b). Note the red/pink pigmentation at the tips of the petals. (B) Chardonnay flowers: (a) normal; (b) star flower, dissected longitudinally just prior to opening. Note the differences in shape of the flower buds, the relatively small size of the ovules, and the pale-colored anthers of the star flower. The anther in the foreground of (b) has been positioned deliberately to more clearly expose the inner organs of the flower. The bar represents 1 mm. From Longbottom, M.L., Dry, P.R., Sedgley, M., 2008. Observations on the morphology and development of ‘star’ flowers of Vitis vinifera L. cvs Chardonnay and Shiraz. Aust. J. Grape Wine Res. 14, 203e210, Figs. 3 and 4, p. 206, reproduced by permission.

(19 þ 20) and the consequential unbalanced separation of the chromosomes during meiosis. The genetic instability so produced disrupts pollen growth, resulting in infertility. The evolution of the Vitaceae appears to have involved hybridization, associated with chromosome doublingda feature common in many plants (Soltis and Soltis, 2009). Based on DNA genomics, three separate whole-genome duplication events are suspected (Jaillon et al., 2007; Velasco et al., 2007). Their proposals differ basically only in the timing of the duplicationsd whether the last duplication occurred before or after the progenitors of the Vitaceae split off from other Rosid dicotyledons. Although involving hexaploidy, similar to that proposed by Patel and Olmo (1955), they differ markedly in the nature and timing of the hybridizations.

Using cytogenetic evidence, Patel and Olmo (1955) envisioned two duplication eventsdthe first involving hybridization between progenitors with six and seven chromosome pairs (6 þ 7 ¼ 13, doubling to 26) and a later hybridization occurring between the tetraploid and a diploid, giving rise to hexaploid grapevines. A cross between a diploid parent with six chromosome pairs gave rise to bunch (Vitis) grapes (13 þ 6 ¼ 19, doubling to 38), whereas a diploid with seven chromosome pairs gave rise to muscadine grapes (13 þ 7 ¼ 20, doubling to 40) (Fig. 2.4). Variations in the chromosome numbers of other genera can be viewed as derived from tetraploid or hexaploid vines, followed by chromosome loss, fusion, translocation, and/or doubling. Examples of other generic chromosome

24

2. Grape species and varieties

FIGURE 2.3 Properties of the Vitis (1) and Muscadinia (2) subgenera of Vitis. (A) Internal cane morphology; (B) tendrils; (C) front and back seed morphology; (D) bark shredding. (A, B, and D) From Bailey, L.H., 1933. The species of grapes peculiar to North America. Gentes Herb. 3, 150e244. (C) From Rives, M., 1975. Les origines de la vigne. Recherche 53, 120e129, reproduced by permission.

complements include Cyphostemma (22), Tetrastigma (22, 44), Cissus (24, 22, or 26), Cayratia (32, 72, or 98), and Ampelocissus and Ampelopsis (40). Older cytogenetic evidence, consistent with polyploidy, involves the apparent association of four nucleoli in species possessing 24 and 26 chromosomes, and six nucleoli-related chromosomes in species possessing 38 or 40 chromosomes. Typically, diploid species possess only two nucleoli-related chromosomes. Chromosome structure itself provides little information concerning evolutionary events in the Vitaceae, due to their minute size (0.8e2.0 mm) and similar morphology (Fig. 2.5). Modern genomic studies (Malacarne et al., 2012) envision an autotetraploid (2n ¼ 2x ¼ 24) hybridizing with a related but distinct diploid (2n ¼ 12), followed by chromosome doubling. The resulting paleohexaploid would have

possessed six sets of chromosomes (2n ¼ 2x ¼ 36). The other pair of chromosomes in the subgenus Vitis (2n ¼ 38), and two pairs for the Muscadinia (2n ¼ 40) are unexplained. The scheme also differs in when the ancestral hexaploid arose. Genomic analyses (Salse, 2016) and estimates based on evolutionary clocks (Magallo´n and Castillo, 2009) place the Vitales near the origin of dicotyledonous plants, about 100e110 million years ago. The earliest known, clearly identifiable, fossilized fruit and seed of the Vitaceae come from the late Cretaceous in India. However, these features may have evolved subsequent to the origin of the progenitors. As the fossil finds are up to 10 million years earlier than similar finds in Europe and North America, Manchester et al. (2013) suggest an “out-of-India” genesis. Such an Asian origin is consistent with phylogenetic data provided by Pe´ros et al. (2011).

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The genus Vitis

(A)

(B)

PLATE 2.2 Vitis rotundifolia. (A) fruit; (B) leaf. (A) Photo courtesy of Chris Evans, The University of Georgia, www.insectimages.org. (B) Photo courtesy of Carl Hunter © USDA-NRCS PLANTS Database/USDA SCS. 1991. Southern wetland flora: field office guide to plant species. South National Technical Center, Fort Worth, TX, respectively.

Regardless of the details, all schemes propose diploidization (Soltis and Soltis, 1999; Cui et al., 2006). Diploidization typically involves significant genomic reorganization (usually involving transposons) and the loss (or inactivation) of many duplicated genes. This may explain why only two nucleoli appear prominently in the cells of Vitis spp. (Haas et al., 1994), in contrast to the six that would otherwise be expected. However, it appears all chromosome complements are still existent. This would permit some homologous genes to mutate without detriment, potentially taking on new or modified roles. This could explain the abnormally large number of genes affecting wine attributes. Examples are the multiple gene clusters associated with terpene and stilbene synthesis. Terpenes are important not only to many varietal aromas (Mateo and Jimenez, 2000), but also to planteenvironment interactions. Of the 32 stilbene synthetase genes (Parage et al., 2012) some are involved in disease and pest resistance. The increased potential for environmental adaptability seems one of the principal evolutionary features favoring hybridization and chromosomal doubling (Fawcett et al., 2009). Alternatively, gene duplicates may become silenced, generating nonfunctional (null) pseudogenes. Both changes convert polyploids into functional diploids. An important secondary consequence of these modifications is preventing (or limiting) multivalent crossovers, unbalanced chromosome separation, and aneuploidy development during meiosis. Unequal chromosome complements in ovules and pollen often lead to partial or complete seed sterility. In contrast to the relative genetic isolation (infertility), imposed by the differing chromosome complements between the two grapevine subgenera, crossing among species within subgenera can produce fertile

First stage Parents:

Diploid (2x = 14)

X

Diploid (2x = 12) n=6

Gametes:

n=7

Progeny:

Diploid (2x = 13)

Chromosome doubling:

Tetraploid (4x = 26)

Second stage Parents:

Diploid X Tetraploid (4x = 26) (2x = 12)

Gametes:

n = 13

n=6

Tetraploid X Diploid (4x = 26) (2x = 14) n = 13

n=7

Progeny:

Triploid (3x = 19)

Triploid (3x = 20)

Chromosome doubling:

Hexaploid (6x = 38)

Hexaploid (6x = 40)

Progenitors of:

Euvitis

Muscadinia

FIGURE 2.4

Hypothesized evolution of the Vitis and Muscadinia subgenera of Vitis, involving sequential hybridization and chromosome doubling of the progeny. Based on the work of Patel, G.I., Olmo, H.P., 1955. Cytogenetics of vitis. I. The hybrid V. vinifera  V. rotundifolia. Am. J. Bot. 42, 141e159.

progeny. Although facilitating the incorporation of traits from one species into another, the ease with which interspecies crossing occurs complicates

26

2. Grape species and varieties

FIGURE 2.5 Karyotypes and idiograms of Vitis vinifera (1) and V. rotundifolia (2). From Patil, S.G., Patil, V.P., 1992. Karyomorphology of Vitis vinifera and V. rotundifolia and their hybrid. Cytologia 57, 91e95, reproduced by permission.

delineating species. This is further compounded by many species being sympatric (Fig. 2.6) (facilitating hybridization) and the scarcity of distinctive morphological features. Quantitative differences that distinguish species, such as shoot and leaf hairiness, are often influenced by environmental conditions. This is often interpreted as evidence that speciation is still active within the genus, and evolution into distinct species remains incompletedlocal populations often being viewed more as ecospecies or ecotypes than biological species. These complications may be partially resolved with genomic analysis (Tro¨ndle et al., 2010). The most recent classification of eastern North American species of Vitis is given by Moore (1991). A summary of the classification of Chinese species is given in Wan et al. (2008). Data derived from molecular (DNA) markers generally support relationships based on traditional morphologic features, but also highlight differences (Aradhya et al., 2008). Their data suggest considerable gene flow among species.

Geographic origin and distribution of Vitis and Vitis vinifera Current distribution includes northern South America (the Andean highlands of Colombia and Venezuela), Central and North America, Asia, and Europe. This would correlate with the ancestry of at least the original polyploidy having occurred while North America and Europe were still part of Pangea. In contrast, species in the subgenus Muscadinia are restricted to the southeastern United States and northeastern Mexico, possibly indicating its origin having occurred close to or after North America had split from Pangea (during the Cretaceous). This correlates to when flowering plants began to replace gymnosperms as the predominate land plants. The North American distribution of Vitis species is shown in Fig. 2.6.

In the 19th century, many extinct species of Vitis were proposed, based on fossil leaf impressions (see Jongmans and Junk, 1939). The validity of these designations is in question, due to the dubious nature of the evidence (Kirchheimer, 1938). Not only do several unrelated plants possess similarly shaped leaves, but individual grapevines show remarkable variation in leaf shape, lobbing, and dentation (Zapriagaeva, 1964). Of greater value in species delineation is seed morphology (Chen and Manchester, 2011), even though significant interspecies variation also exists (Fig. 2.7). On the basis of seed morphology, two groups of fossilized grapes have been distinguished, Vitis ludwigii and V. teutonica. Seeds of the V. ludwigii type, resembling those of muscadine grapes, have been found in Europe from the Pliocene (2e10 million years BP). Those of the V. teutonica type, resembling those of bunch grapes, have been discovered as far back as the Eocene (34e55 million years BP). However, these identifications are based on comparatively few specimens, and, thus, any conclusions remain tenuous. In addition, related genera, such as Ampelocissus and Tetrastigma, produce seed similar to those of Vitis. This would fit with the suspected much earlier evolution of these genera. In contrast, species radiation in the genus Vitis is estimated to have begun about 6e6.6 million years BP (Zecca et al., 2012). Although most grape fossils have been found in Europe, this may reflect more the distribution of appropriate sedimentary deposits (or paleobotanical interest and investigation) than Vitis distribution. Baranov (in Zukovskij, 1950) suggests that the progenitors of Vitis were bushy and inhabited sunny locations. As forests expanded during the more humid Eocene, the development of a climbing habit would have permitted them to retain their preference for sunny conditions. This may have involved mutations modifying some floral clusters into tendrils, thus improving clinging ability. This hypothesis is not unreasonable, based on the differentiation of buds into flower clusters or tendrils, reflecting the ratio of gibberellin to cytokinin

27

Geographic origin and distribution of Vitis and Vitis vinifera

(A)

(B) 50°

50°

40°

40°

30°

30°

120°

100°

80°

(C)

120°

100°

80°

100°

80°

100°

80°

(D) 50°

50°

40°

40°

30°

30°

120°

100°

120°

80°

(E)

(F) 50°

50°

40°

40°

30°

30°

120°

100°

80°

(G)

120°

(H)

50°

50°

40°

40°

30°

30°

120°

100°

80°

120°

100°

80°

FIGURE 2.6 Geographic distribution of Vitis species in North America. Subgenus Muscadinia: (A) Vitis rotundifolia var. rotundifolia (downsloping to right), and V. rotundifolia var. munsoniana (downsloping to left). Subgenus Vitis: (B) series Ripariae, V. riparia (downsloping to left), V. rupestris (downsloping to right), and V. acerifolia (horizontal); (C) series Labruscae, V. labrusca (downsloping to left), V. shuttleworthii (downsloping to right), and V. mustangensis (horizontal); (D) series Cordifoliae, V. vulpina (downsloping to left), V. monticola (downsloping to right), and V. palmata (horizontal); (E) series Cinerescentes, V. cinerea var. cinerea (downsloping to left), V. cinerea var. floridana (downsloping to right), V. cinerea var. helleri (horizontal), and V. cinerea var. baileyana (vertical); (F) series Aestivalis, V. aestivalis var. aestivalis (downsloping to left), V. aestivalis var. bicolor (downsloping to right), and V. aestivalis var. lincecumii (horizontal); (G) series Occidentales, V. californica (downsloping to left), V. girdiana (downsloping to right), and V. arizonica (horizontal); and (H) hybrids, V.  novae-angliae (downsloping to left), V.  champinii (downsloping to right), and V.  doaniana (horizontal). Data supplied by Moore, M., Botany Department, University of Georgia, Athens, GA.

28

2. Grape species and varieties

FIGURE 2.7 Lateral (front) and dorsal (back) chalaza views of grape seeds. (A) Vitis vinifera; (B) V. labrusca; (C) V. vulpina; (D) V. cinerea var. helleri; (E) V. cinerea var. baileyana; (F) V. illex; (G) V. vulpina var. praecox; (H) V. rotundifolia; (I) V. rotundifolia var. munsoniana. From Bailey, L.H., 1934. The species of grapes peculiar to North America. Gentes Herb. 3, 150e244, reproduced by permission.

(Srinivasan and Mullins, 1981; Martinez and Mantilla, 1993). Regardless of the manner and geographic origin of Vitis, it established its present range by the end of the last major glacial period (w8000 BCE). It is believed that periodic advances and retreats during the last glacial period markedly affected the evolution of Vitis and its survival in Eurasia. The alignment of the major mountain ranges in the Americas, vs. Eurasia, appears to have had an important bearing on the respective survival (and/or evolution) of the genus in the Northern Hemisphere. In the Americas and eastern China, the mountain ranges run predominantly northesouth, whereas in Europe and western Asia they run principally eastewest. This would have permitted North American and eastern Chinese species to relocate, south or north, relative to the movement of glacial ice sheets. In contrast, the southward movement of grapevine species in Europe and western Asia would have been largely restricted by the predominant eastewest alignment of the Pyrenees, Alps, Caucasus, and Himalayas. This may explain the existence of but one species of Vitis (V. vinifera) in Europe and western Asia, in contrast to the more expanded species presence in North and Central America (about 34 endemic species) (Rogers and Rogers, 1978) and China (with about 30 indigenous species) (Fengqin et al., 1990). A few naturalized, foreign Vitis spp. are occasionally found in Europe. They are probably “escaped” rootstocks, imported for grafting, subsequent to the phylloxera epidemic in the late 1800s (Arrigo and Arnold, 2007), or were cultivated as a fruit crop (Cangi et al., 2006). Some of these have also yielded hybrids with indigenous wild Vitis vinifera (Bodor et al., 2010). Although glaciation destroyed most of the favorable habitats in the Northern Hemisphere, major southward displacement was not the only option open for survival. In certain areas, isolated but favorable sites (refuges) permitted continued existence throughout the glacial period. In Europe, refuges occurred around the Mediterranean basin and south of the Black and Caspian seas

FIGURE 2.8 Distribution of wild Vitis vinifera vines about 1850 (dots), superimposed on forest refuges in the Mediterranean and Caucasian regions during the last ice age (within lined/dashed areas). After Levadoux, L., 1956. Les populations sauvages et cultive´es de Vitis vinifera L. Ann. Ame´lior. Plant. Se´r. B 1, 59e118, reproduced by permission.

(Fig. 2.8). For example, grape seeds have been found associated with anthropogenic remains in caves in southern Greece (Renfrew, 1995) and southern France (Vaquer et al., 1985), both near the end of the last glacial advance. These refuges may have played a role in the evolution of the various varietal groups of Vitis vinifera (see later). Periodically displaced during the various Quaternary glacial periods (Fig. 2.9), V. vinifera was inhabiting the southern regions of France and northern Greece some 10,000 years ago (Planchais and Vergara, 1984; Wijistra, 1969). For the next several thousand years, the climate slowly improved to an isotherm about 2e3 C warmer than currently (Dorf, 1960). Refuge habitats of wild V. vinifera were mild, humid forests south of the Caspian and Black seas and adjacent Transcaucasia, along the fringes of the cooler mesic forests of the northern Mediterranean, and into the heartland of Europe, along the banks and hillsides of the Danube, Rhine, and Rhoˆne rivers. These sites vary in altitude, from sea level to 1000 m.

Domestication of Vitis vinifera

29 FIGURE 2.9 Fossil grape remains in eastern Europe during the Quaternary. Pollen data refer to all but the Wu¨rm period (occurring only around the Mediterranean basin) and macrofossils for the MindeleRiss interglacial. Planchais, N., 1972e1973. Apports de l’analyse pollinique a` la connaissance de l’extension de la vigne au quaternaire. Nat. Monspel. Ser. Bot. 23e24, 211e223, reproduced with permission.

0

500 Km

P – Lower and middle Quaternary

P – Post-glacial

P–

Mindel – Riss Interglacial

P – Human influences

M–

From Frenzel, 1968

(P = pollen, M = Macrofossils)

P – Riss – Würn Interglacial

Although remnant populations are in risk of extinction, current interest in discovering them, and their preservation, may stabilize the decline. A general account of the tenuous situation is provided by Arnold et al. (1998).

Domestication of Vitis vinifera Grapevine cultivars show few of the standard signs of plant domestication (Meyer and Purugganan, 2013). These characteristics can be summarized as follows: conversion from cross- to self-fertilization, release from the need for seed and bud vernalization (cold treatment), elimination of photoperiod phenologic regulation, inactivation of seed dehiscence or fruit separation upon maturation, development of parthenocarpy (fruit production independent of seed development), increase in shoot-to-root ratio, enhanced fruit (or seed) size, augmentation in crop yield, reduction in phytotoxin

production, a shift from a bi- or perennial habit to an annual habit, and a reduction in genetic diversity. Of these changes, only conversion to self-fertility is clearly expressed in domesticated grapevines. Other modifications associated with domestication are less noticeable. There is a slight reduction in photoperiod sensitivity and need for seed vernalization, easier fruit separation from the cluster, increased fruit size, seedlessness in some table and raisin cultivars, and increased sugar and reduced acid contents. Other features that differentiate domesticated from wild grapevines include a shift from small, round berries to larger, elongated fruit; more fruit per cluster; bark separating in wider, corecoherent strips (vs. bark separating in long thin strips); larger, elongated seeds (vs. smaller, rounded seeds); and large leaves with entire or shallow sinuses (vs. smaller, usually deep, three-lobed leaves) (Olmo, 1976). Some of these features are illustrated in Fig. 2.10. Plate 2.3 also illustrates the characteristic features of wild (sylvestris)

30

2. Grape species and varieties

FIGURE 2.10 Morphological differences between the leaves (L), flowers (F), fruit clusters (B), and seeds (S) of cultivated (sativa) and wild (sylvestris) grapevines. From This, P., Lacombe, T., Thomas, M.R., 2006. Historical origins and genetic diversity of wine grapes. Trends Genet. 22, 511e519, reproduced by permission.

grape clusters, including the dominant (wild-type) bluish fruit coloration. Even the population of endophytic bacteria in feral vines is more varied and extensive than those of domesticated vines (Campisano et al., 2015). Grapevines also illustrate features typical of other perennial crop domestications: prolonged juvenile stages,

maintenance of considerable ancestral genetic diversity, limited sexual recombination, and clonal propagation. Due to the last, as well as possible backcrossing with wild strains, domestication tends to be slow and progressive, probably occurring recurrently in different locations. Whole-genome sequencing has demonstrated

Domestication of Vitis vinifera

PLATE 2.3 Cluster of grapes of Vitis vinifera f. silvestris in southern Spain. Photo courtesy of Nun˜ez, D., Universidat de Murica.

both a separation between domesticated and wild vines and a separation between wine and table grapes (Zhou et al., 2017). Domestication is particularly evident in shifts in loci associated with sugar accumulation and bisexuality. Their data suggest that domestication may have begun considerably before a settled (agricultural) lifestyle developed. Additional data are available in Wales et al. (2016), providing a discussion of the relative value of nuclear, mitochondrial, and plastid DNA analyses. Color variation is another feature often associated with domestication (Plate 2.4), but not readily preserved in the archaeological record. This diversity results from a

PLATE 2.4 Grapevine heterogeneity as illustrated by the diversity in morphology, structure, and coloration in grapes and grape clusters. Photo courtesy of Ku¨hn-Institute, Institute for Grapevine Breeding, Geilweilerhof, Siebeldingen, Germany.

31

combination of factors, notably mutation in anthocyanin genes (Fournier-Level et al., 2009), and how they respond to activation. Although pale or white cultivars are more typical of table grapes, one of the genetic factors involved in the loss of anthocyanin synthesis appears to have occurred millennia before domestication (Mitani et al., 2009). The principal physical indicator of domestication, detectable in archaeological remains, is a shift in seed indexdthe ratio of seed width to length. Although of no known selective advantage, the shift appears to correlate with a change from cross- to self-fertilization. This may reflect some unknown and unsuspected pleiotropic effects. Seeds from wild (sylvestris) vines are rounder, possess a nonprominent beak, show a well-developed chalaza, and demonstrate an average seed index of about 0.64 (ranging from 0.54 to 0.82). In contrast, seeds from domesticated (sativa) vines are more elongated, possess a prominent beak, and have a seed index averaging about 0.55 (often ranging from 0.44 to 0.75) (Renfrew, 1973, Fig. 2.11). However, evidence based on carbonized seed remains may be of uncertain value. Charring appears to increase the relative seed length, decreasing the seed index (Margaritis and Jones, 2006). It does, though, enhance survival in sites. In addition, considerable variation in seed size and shape (Zapriagaeva, 1964) makes conclusions based on small sample sizes unreliable. A more detailed and sophisticated method for differentiating between cultivated and wild grape seeds has confirmed the differences established by earlier studies and extended them to identifying cultivar subgroups (Terral et al., 2010). Seed index data have been used to suggest the slow domestication of grapevines in northeastern Iran and Anatolia (Miller, 1991). Some of the earliest archaeological findings have been found in Shulaveri, Georgia (w6000 BCE) (Ramishvili, 1988). Remains become increasing prevalent during the middle to late Bronze age (w3000 BCE). Clear evidence of cultivation appears in Jericho about 3200 BCE (Hopf, 1983), and in Macedonia and Greece after 2800 BCE (Renfrew, 1995). This is considerably later than archaeological evidence suggestive of wine production in northern Iran (5400e5000 BCE) (McGovern et al., 1996) and Georgia (6000e5800 BCE) (McGovern et al., 2017). However, the time frame is consistent with the predicted spread of agriculture out of the Fertile Crescent into northern Iran and Anatolia (Ammerman and Cavalli-Sporza, 1971). Thus, dedicated wine production may have developed concurrent with the establishment of an agrarian culture, but prior to the preservation of morphological evidence of grapevine domestication (Zohary, 1995). In the Mediterranean, the earliest presumptive evidence of winemaking comes from the northern Aegean (Valamoti et al., 2007), from about the middle of the 5th millennium BCE. From seed characteristics, it involved

32

2. Grape species and varieties

20

18 Sativa Silvestris 16

14

Frequency

12

10

8

6

4

2

0 0.44

0.48

0.52

0.56

0.60 0.64 Ratio

0.68

0.72

0.76

0.80

FIGURE 2.11 Variation in the width/length index of seeds of wild (sylvestris) and cultivated (sativa) subspecies of Vitis vinifera. From Stummer, A., 1911. Zur Urgeschichte der Rebe und des Weinbaues. Mitt. Anthropol. Ges. Wien 41, 283e296, reproduced by permission.

wild grapes. In western Europe, evidence of wild grapevine use has been found in a Neolithic village near Paris (about 4000 BCE) (Dietsch, 1996). Residues, indicative of wine production, have been isolated from a vessel in Crete dating to the end of the 3rd millennium BCE (Martlew, 1999), and in Sicily, about the Middle Bronze Age (1560e1250 BCE) (Tanasi et al., 2017). Semidomesticated grape seed remains (2700 BCE) have been discovered in England (Jones and Legge, 1987), and in several Neolithic and Bronze Age lake dwellings in northern Italy, Switzerland, and the former Yugoslavia (see Renfrew, 1973). Data from southern France suggest a slow progression from a mix of seed morphotypes (w50 BCE) to predominantly domesticated forms (CE w500) (Bouby et al., 2013). In addition, grape seed and pollen remains have been identified from Neolithic sites in southern Sweden and Denmark (Rausing, 1990). At that time, the climate was warmer than today. Evidence of grape culture (Stevenson, 1985) and domestication (Nu´n˜ez and Walker, 1989) has been found in southwestern Spain (w2500e3000 BCE). The existence of a Spanish center of grapevine domestication has received molecular support (Arroyo-Garcı´a et al., 2006; Laucou et al., 2018). Differences in floral sexual expression and leaf shape occur between wild and domesticated

grapevines (Levadoux, 1956). For example, male vines have larger inflorescences and flower earlier and for a longer period than bisexual flowers (Negi and Olmo, 1971). Male flowers also show poorly developed nectaries. These features are, however, not preserved or recognizable in vine remains. Additional differences have been noted by Patel and Olmo (1955), but most of these features are readily influenced by cultivation. Another potentially useful property, preserved in archaeological remains, involves changes in the fruit pedicel (see Fig. 3.23 for terminology). In domesticated Vitis vinifera, the pedicel frequently breaks at the main stem (rachis) when berries detach. In contrast, this seldom occurs with wild vines, due to their stouter stems. Thus, the relative frequency of fruit remains attached to the pedicel may be an additional indicator of domestication in archaeological remains. Nonetheless, of all fossil evidence, the greatest confidence can be placed in pollen data. Pollen found in sedimentary deposits is sufficiently distinct and well preserved to establish the presence of a species or subspecies at a prehistoric site (Fig. 2.9). The outer wall (exine) of pollen consists primarily of sporopollenin, a material particularly resistant to decay. Pollen frequency is often used as an indicator of the relative abundance of

Domestication of Vitis vinifera

wind-pollinated species in a region. Correspondingly, grapevine pollen frequency has been used as an indicator of ancient viticulture (Turner and Brown, 2004). For example, a pronounced increase in pollen counts was associated with increased human activity and presumptive viticulture in northern Greece after CE 1500 (Bottema, 1982). Of particular importance is the difference between fertile and sterile pollen (produced by male/bisexual flowers vs. female flowers). Fertile pollen is tricolporate (containing three distinct ridges) and produces germ pores, whereas sterile pollen is generally acolporate (possessing no ridges) and possesses no germ pores (Fig. 2.12). In addition, sterile pollen is often associated with disrupted chromosome separation during nuclear division and possesses inner-wall abnormalities. Although some of these features could be retained in pollen samples, they appear to have been little used in assessing the relative frequency of dioecious (wild) and bisexual (cultivated) vines at prehistoric sites (see Planchais, 1972e1973). This may result from the poorer release of pollen from bisexual flowers and their corresponding restricted presence in sedimentary deposits. As noted, it is generally believed that domestication began in or around Transcaucasia, or neighboring Anatolia, about 4000 BCE. About 8000 years ago, the climate started to become warmer and generally more humid than currently. In particular, rainfall increased considerably in eastern Anatolia. This was associated with an advance of a Quercus-dominated steppeeforest into what had been a semiarid environment. The forest reached its maximum extension about 4200 BCE (Wick et al., 2003). Thus, growth of V. vinifera was probably more extensive in regions associated with the earliest known archaeological evidence of wine production (northwestern Zagros mountains) than today. This is also the region in which domestication of wheat is thought to have occurred (Heun et al., 1997). Subsequent

33

climatic drying, beginning about 2000 BCE, slowly shifted the vegetation to match the current semiarid state in the region, resulting in a shrinkage of the Vitis habitat. The recent, dramatic shrinkage of feral V. vinifera populations has resulted from habitat destruction, disease (phylloxera and mildew), and competition from American and Asian Vitis escapes (Arrigo and Arnold, 2007). There are current attempts to retain existing populations of wild vines as a reserve of genetic diversity (e.g., anthocyanin diversity) (Revilla et al., 2010). The likely feral distribution, prior to the phylloxera epidemic, is illustrated in Fig. 2.8. Despite human migration and its consequences relative to the cultivar movement, changes in seed morphology and pollen shape indicate grape domestication long before the agricultural revolution spread into southern and central Europe. Traits such as selffertilization and large fruit size have often been attributed to varieties imported by colonists, but this is not requisite. Fruit size often increases under cultivation, but could have resulted independent of flb gene mutation (or related genes) (Fernandez et al., 2006). In addition, the occurrence of self-fertilization in wild (sylvestris) grapevines (Schumann, 1997; Failla et al., 1992; Anzani et al., 1990) need not presuppose the introduction of domesticated vines. Functional bisexuality has been found (albeit rarely) in a range of Vitis species (Dorsey, 1912; Fengqin et al., 1990). Locally derived cultivars, possibly associated with hybridization with introduced vines, would have more likely been adapted to prevailing soil and climatic conditions. This is consistent with data suggesting that most cultivars fall into ecogeographic groups (Bourquin et al., 1993). The presence of numerous traits (such as rounder seeds) reminiscent of V. vinifera spp. sylvestris occurs in cultivars such as Traminer, Pinot noir, and Riesling. This has been construed as evidence of indigenous origin. Comparison of the genetic traits of long-established Italian varieties with indigenous feral vines is FIGURE 2.12 Diagrammatic representations of pollen of Vitis vinifera. (A) Fertile pollen: (1) swollen grain; (2) dry grain, top view; (3) swollen grain in water; (4) dry grain, side view. (B) Infertile pollen: (1e6) dry grains; (7) swollen grain in water. After Levadoux, L., 1946. E´tude de la fleur et de la sexualite´ chez la vigne. Ann. E´c. Natl. Agric. Montpellier 27, 1e90, reproduced by permission.

34

2. Grape species and varieties

consistent with some (Grassi et al., 2003), but not all, cultivars (Scienza et al., 1994; Bacilieri et al., 2013) being of local origin. This also applies to Iberian cultivars (Arroyo-Garcı´a et al., 2006). Data from a range of studies suggest that European cultivars have originated from a mix of importation, introgression, and de novo domestication. Domestication probably occurred progressively, with the slow accumulation of agronomically valuable mutations. The early association of wine with religious rites in the Near East (Stanislawski, 1975) could have provided the incentive for cultivar selection, and the beginnings of concerted viticulture. Initially, cultivation would have required the planting of both fruitbearing (female) and non-fruit-bearing (male) vines. Propagating only fruit-bearing cuttings would have resulted in a reduction or cessation of productivity, by separating female vines from pollen-bearing vines (growing wild in potentially distant, open, moist

TABLE 2.1

woodlands or riparian habitats). So doing would have highlighted the productivity of any self-fertile revertants, and presumably led to their selection. Viticulture also would have favored the selection of vines showing increased fruit size, as well as improved visual, taste, and aroma attributes. An obvious example might be the augmented terpene production in Muscat cultivars. Additional support for the connection between winemaking and grapevine domestication comes from the remarkable similarity between the words for wine and vine in European languages (Table 2.1). In contrast, little resemblance exists between words for grape. The persistence of local terms for grape in European languages suggests that grapes were used long before the introduction of winemaking. The spread of related terms (wine and vine) may have been associated with the demic diffusion of Neolithic farmers (Chicki et al., 2002; Bramanti et al., 2009) and the associated dispersion of Indo-European languages into Europe (Renfrew, 1989).

Comparison of synonyms for “wine,” “vine,” and “grape” in the principal Indo-European languages.

Language

Wine

Vine

Grape

Greek

oῐvoz

ᾶmpεloz

_ bosryz

Latin

vı¯num

vı¯tis

 va u

Italian

vino

vite, vigne (pl.)

uva

French

vin

vigne

raisin

Spanish

vino

vid

uva

Rumanian

vin

vita

strugure

Irish

fı¯n

finemain

fin

Breton

gwin

gwinienn

rezinenn

Gothic

wein

weinatriu

weinabasi

Danish

vin

vinranke (-stok)

drue

Swedish

vin

vinranka (-stock)

druva

English

wine

vine

grape

Dutch

wijn

wijnstok

druif

German

wein

weinstock, rebe

traube, weinbeere

Lithuanian

vynas

vynmedis

vynuog_e

Latvian (Lettish)

vı¯ns

vı¯nogulajs

vı¯noga

Serbo-Croatian

vino

losa

grozd

Czech

vı´no

re´va, vinny´ ker

hrozen

Polish

wino

winorosl

winogrono

Russian

vino

vinograd

vinograd

Sanskrit

draks xarasa

draks xa-

draks xa-

Information from Buck, C.D., 1949. A Dictionary of Selected Synonyms in the Principal Indo-European Languages. University of Chicago Press, Chicago, IL.

35

Cultivar origins

FIGURE 2.13 Geographical distribution of the genetically differentiated grape cultivars based on 10,000 genome-wide single-nucleotide polymorphisms. Clusters (C) are positioned according to the main geographic origin. Their contributions of cultivars in other geographic zones are indicated by lines and dots. Seven geographic regions were considered: the Iberian peninsula in light yellow, western and central Europe in blue, the Balkans in light green, the Italian peninsula in purple, the Eastern Mediterranean and Caucasus in orange, the Middle and Far East in brown, Russia and the Ukraine in green, and northern Africa in dark yellow. Cluster C1, C2, and C3 includes mainly wine cultivars; C4 and C5 consist of mainly table cultivars; C6 consists of eastern wine cultivars, many Georgian; C7 consists of mainly Balkan wine cultivars; C8 consists mainly of Iberian cultivars. From Laucou, V., Launay, A., Bacilieri, R., Lacombe, T., Adan-Blondon, A.-F., Be´rard, A., et al., 2018. Extended diversity analysis of cultivated grapevine Vitis vinifera with 10K genome-wide SNPs. PLoS One 13 (2), e0192540, reproduced with permission.

This is probably associated with the spread of viticulture and winemaking into Early Bronze Age Macedonia and Crete during the 3rd millennium BCE (Hamilakis, 1999; Valamoti et al., 2007), in Italy (Marvelli et al., 2013). This dispersion was accentuated with the spread of the Roman Empire, but preceded by the implantation of Greek colonies in Italy and southern France, beginning in the 8th and 7th centuries BCE. Current genomic data suggest several centers of cultivar development and focal points of diffusion (e.g., Fig. 2.13). In contrast to European languages, words for grape, wine, and grapevines all have the same root derivative in Near Eastern languages (Forbes, 1965). This similarity is consistent with the likelihood that wine preceded, or coincided with, the appearance of the grapevine and viticulture in Semitic cultures. Gamkrelidze and Ivanov (1990) believe their related terms all derive from what may have been the archetypal word for wine, woi-no. Although grapevine domestication appears to have occurred independently in several locations (Aradhya et al., 2003; Arroyo-Garcı´a et al., 2006), the oldest is in the southern Caucasus. This is supported by the advanced state of cultivar variation in that region. Local varieties possess many recessive mutants, such as smooth leaves, large branched grape clusters, and medium-sized juicy fruit. The number of recessive traits is often considered an indicator of cultivar age. Varieties showing these particular traits were classified by Negrul (1938) as members of the proles orientalis grouping. Also included in this group are the genetically distinct V. vinifera cultivars of China and Japan (Goto-Yamamoto et al., 2006). Evidence from Jiang et al. (2009) suggests that the first tentative cultivation of V. vinifera began in China about 300 BCE. Cultivars in the northern Mediterranean and in central

Europe were viewed by Negrul as being of relatively recent origin (middle to late 1st millennium BCE). This view was based on their possessing few recessive traits and their close resemblance to wild vines. They were placed in the proles occidentalis grouping. Varieties found in contiguous regions (e.g., Georgia and the Balkans) show properties intermediate between proles orientalis and proles occidentalis. These Negrul designated as proles pontica. Characteristics of the three groupings are given in Table 2.2. Data giving support to the genetic relationship of these groupings are now accumulating (De Lorenzis et al., 2015). Additional studies along these lines were conducted by Levadoux (1956). Extensions of Negrul’s classification system can be found in Tsertsvadze (1986) and Gramotenko and Troshin (1988).

Cultivar origins Except for cultivars of recent origin, for which there is a documented record, the origin of most cultivars is shrouded in mystery. Archaeological finds are insufficiently informative, while ancient writings do not discuss the issue. Although many authors have attempted to correlate Roman cultivar names with current varieties, even Roman authors disagreed on their appropriate use. For example, Virgil (Georg 2.106), possibly with poetic license, compared their number to the sand grains in Libya. Even more recently, connecting existing cultivars with the names used by Abbe´ Rosier in Chaptal et al. (1801) is dubious. Without voucher specimens, speculation on current equivalents is often problematic. Occasionally, vines thought to be

36

2. Grape species and varieties

TABLE 2.2 Classification of varieties of Vitis vinifera according to Negrul (1938). Proles orientalis

Proles pontica

Proles occidentalis

Georgia, Asia Minor, Greece, Bulgaria, Hungary, Romania

France, Germany, Spain, Portugal

Buds glabrous, shiny

Buds velvety, ash-gray to white

Buds weakly velvety

Lower leaf surfaces glabrous to setaceous pubescent

Lower leaf surface with mixed pubescence (webbed and setaceous)

Lower leaf surfaces with webbed pubescence

Leaf edges recurved toward the tip

Leaf edges variously recurved

Leaf edges recurved toward the base

Grape clusters large, loose, often branching

Grape clusters medium size, compact, rarely loose

Grape clusters generally very large, compact

Fruit generally oval, ovoid, or elongated; medium to large; pulpy

Fruit typically round, medium to small, juicy

Fruit often round, more rarely oval; small to medium; juicy

Varieties mostly white with about 30% rose´s

About equal numbers of white, rose´, and red varieties

Varieties commonly white or red

Seeds medium to large with an elongated beak

Seeds small, medium, or large (table grapes)

Seeds small with a marked beak

Many varieties partially seedless, some seedless

Many varieties partially seedless, some completely so

Seedless varieties rare

Varieties produce few, lowyielding fruiting shoots

Varieties often produce several, highly productive fruiting shoots

Varieties typically produce several, highly productive fruiting shoots

Varieties short-day plants with long growing periods, not cold hardy

Varieties relatively cold hardy

Varieties long-day plants with short growing periods, cold hardy

Most varieties are table grapes, few possess good winemaking properties

Many varieties are good winemaking cultivars, a few are table grapes

Most varieties possessing good winemaking properties

Grapes low in acidity (0.3% e0.6%), sugar content commonly 18%e20%

Grapes acidic (0.6%e1.0%), sugar content commonly 18%e20%

Grapes acidic (0.6%e1%), sugar content commonly 18%e20%

Self-crossed seedlings of certain varieties possessing simple leaves

Self-crossed seedlings of certain varieties with dwarfed shoots and rounded form

Self-crossed seedlings of certain varieties having mottled colored leaves

Regions Central Asia, Afghanistan, Iran, Armenia, Azerbaijan Vine properties

Fruiting properties

After Levadoux, L., 1956. Les populations sauvages et cultive´es de Vitis vinifera L. Ann. Ame´lior. Plant. Se´r. B 1, 59e118, reproduced by permission.

representatives of a single cultivar have been shown by DNA analysis to be separate cultivars. Seed collection has been the principal means by which annual crops have been propagated. By contrast, vegetative propagation has been the principal means of propagating perennial crops (such as grapevines). Vine propagation by layering was quicker and ensured cultivar consistency. Even before the genetic origin of variation was known, the variability generated by seed propagation was known (Anonymous, 1852, pp.

17e20). Nonetheless, the prevalent view at the time was of environmentally acquired (Lamarckian) inheritance (Chaptal et al., 1801, pp. 146e8). During Roman times, the benefits of breeding were well known, at least for animals. However, documentation of intentional grapevine breeding is absent. If and when it occurred, crossing probably was accidental, being favored by the common intermixture of cultivars. This habit could have had the advantage of buffering against the vagaries of climate and soil conditions.

Cultivar origins

Although the fortuitous germination and growth of seedlings to maturity in vineyards may have been uncommon, both events might have occurred more frequently in small peasant plantings or next to dwellings. Because fruit as well as leaves often shows marked phenotypic variability, recognizing new character combinations is more likely in small plantings. The most likely sites for any breeding would have been abbeys and noble estates. Monks, being literate, would have been able to read ancient texts, as well as having the time and writing skills to observe and collect data on vine performance. This may be the origin of the linked parentage for several Italian and French cultivars (Fig. 2.14). Even before cultivar origins could be established by molecular means, many cultivars were considered ancient. Examples of current cultivars likened to those named by Pliny are Greco with vitis aminea gemina, Fiano with vitis apiano, and Sciascinoso with vitis oleagina (see Thomson, 2004). During the Middle Ages, varietal designation was largely abandoned, beginning to reappear in the 14th and 15th centuriesdexamples being Traminer (1349), Rula¨nder (1375), and Riesling (1435) (Ambrosi et al., 1994). Name derivation has occasionally been used to suggest local origin, for example, Se´millon from semis (seed), and Sauvignon from sauvage (wild) (Levadoux, 1956). However, the existence of multiple and unrelated synonyms for many European cultivars does not lend credence to name derivation as an indicator of origin. Far better evidence is now based on DNA studies. Until the development of DNA sequencing, the best evidence for varietal origin came from morphological (ampelographic) comparisons. Such data were

Parent-offspring Full-siblings 2 relatives

Mondeuse Blanche

Pinot

Dureza

Syrah

Teroldego

Marzemino

?

Lagrein

FIGURE 2.14 Most likely pedigree reconstruction from an analysis of 60 microsatellite markers of cultivars from northern Italy and southern France. Reprinted by permission from Macmillan Publishers Ltd. Vouillamoz, J.F., Grando, M.S., 2006. Genealogy of wine grape cultivars: ‘Pinot’ is related to ‘Syrah’. Heredity 97, 102e110. Allele 1 Allele 2 Allele 3 Allele 4

37

particularly useful when cultivars had diverged from a common ancestor via somatic mutation. Examples are the color mutants of Pinot noirdPinot gris and Pinot blanc. Vegetative propagation maintains the traits of the progenitor, except where modified by somatic mutations. In contrast, seed propagation in highly heterozygous plants, such as grapevines, produces progeny that can possess markedly different phenotypic characteristics from their parents (Bronner and Oliveira, 1990). This tends to blur morphologic traits, making leaf characteristics suspect as indicators of varietal origin. Another line of evidence has used chemical indicators. Isozyme and phenolic distributions are examples. However, the growing numbers of DNA fingerprinting techniques, such as microsatellite allele (simple sequence repeat) (SSR) and single-nucleotide polymorphism (SNP) analysis, are far more powerful and universally applicable. They circumvent some of the difficulties associated with interpreting restriction fragment-length polymorphism patterns and standardization with the random amplified polymorphic DNA procedure. In addition, because microsatellite markers are seldom located in functional genomic regions, they are neither selected for nor selected against evolutionarily. This permits the rapid (in terms of centuries) accumulation of variations, permitting varietal (and occasionally clonal) differentiation. Markers may contain one or multiple nucleotide repeats, as noted in the accompanying illustration (Illustration 2.1). These genomic markers also have the advantage of high polymorphism and being inherited codominantly (valuable in parentage studies). For example, crossing vines possessing VVMD5 genotypes with base pair lengths 228/238 and 236/236 could yield offspring only with 228/236 or 238/236 genotypes (as well as the original combinations, if self-crossing occurred). When comparing the SSR or SNP genotypes of varieties, it is possible to identify the synonymy of cultivar names, as well as supporting or refuting presumed parentage (e.g., Table 2.3). In one example of 381 crosses, the pedigrees of 126 were discovered to be different from those indicated by their breeders (Lacombe et al., 2013). The ability of genomic analysis to study grapevine domestication and uncover unsuspected cultivar relationships is impressive. Automation of genomic analysis, combined with the functional diploidy, heterozygosity, and small genome

GATTACCGACCTACACACACACACACACACACACACACACACACATGACATTACTCATCT GATTACCGACCTACACACACACACACACACA TGACATTACTCATCT TGACATTACTCATCT GATTACCGACCTACACACACACA TGACATTACTCATCT GATTACCGACCTACACA

ILLUSTRATION 2.1 Hypothetical example of a simple sequence repeat marker with allelic forms with 2, 5, 9, and 16 CA tandem repeats.

38

Partial data indicating the presumptive parentage of Madeleine Royale as a cross between Pinot and Schiava Grossa.

Marker

Pinot

Madeleine Royale

Schiava Grossa

Chasselas

VVMD5

238e228

236e228

238e236

236e228

VVMD6

205e205

214e205

214e211

212e205

VVMD7

243e239

247e243

247e247

247e239

VVMD8

143e141

157e143

167e157

143e143

VVMD17

221e212

222e212

222e222

212e212

VVMD21

251e249

249e249

249e249

266e249

VVMD24

218e216

216e214

214e210

214e210

VVMD25

253e243

253e245

259e245

259e245

VVMD26

255e249

255e249

251e249

251e249

VVMD27

189e185

189e181

185e181

189e185

VVMD32

273e241

273e253

273e253

241e241

The relationship between Madeleine Royale and Chasselas is ruled out by discrepancies (e.g. 241e241 pair, shown in boldface) of the Chasselas genotype. Data from Vouillamoz, J.F., Arnold, C., 2010. Microsatellite pedigree reconstruction provides evidence that ‘Muller-Thurgau’ is a grandson of ‘Pinot’ and ‘Schiava Grossa’. Vitis 49, 63e65.

size (475e500 Mb), has made SSR analysis the preferred tool in parentage and synonymy studies. It has also been useful in identifying cultivars found in old, traditional vineyards (Jung and Maul, 2004). These have proven to be “treasure troves” of old and near-extinct varieties. Their preservation acts as a reserve of the existent genetic diversity. Genomic analysis is also critical in producing a genetic map of the vine’s 19 linkage groups (Vezzulli et al., 2008). Additional markers from chloroplast DNA are of particular interest in determining maternal inheritanced chloroplast DNA (as well as mitochondrial DNA) is derived only from egg cells (Strefeler et al., 1992; Arroyo-Garcı´a et al., 2002). This has permitted the identification of Gouais blanc (syn. Heunisch Weiss) as the maternal parent of cultivars such as Chardonnay, Gamay noir, and Aligote´ (Hunt et al., 2010) and Sauvignon blanc as the female parent of Cabernet Sauvignon. This determination is of more than just academic interest. Chloroplast DNA can influence properties such as chilling injury (Chung et al., 2007) and fungal toxin tolerance (Avni et al. 1992). In addition, it has attributes superior to nuclear DNA in discerning evolutionary trends, due to its low mutation rate and the absence of genetic recombination (typical of nuclear DNA). These attributes have been used to identify two regions (central Italy and the Caucasus) as being ancient genomic refuges of Vitis vinifera spp. sylvestris from the last European glacial period (Grassi et al., 2006), demonstrating the extensive

genetic diversity of Georgian cultivars (Schaal et al., 2010) and its being the likely center of diversity for many European cultivars (Imazio et al., 2006). Wild vines also show extensive morphological diversity in the Caucasus region (Akhalkatsi et al., 2012), as well as possessing (along with central Italy) all five known grapevine chloroplast haplotypes (Grassi et al., 2006). Genomic studies have also been used to show the importance of introgression in Portuguese (Lopes et al., 2009) and Spanish (De Andre´s et al., 2012) cultivars, but its lack in Sardinian cultivars (Zecca et al., 2010). Mainland Italian varieties seem to be a mix of introduced cultivars and introgression with sympatric wild vines and those from western and central Europe (Sefc et al., 2003). Their data also show that, for the 164 cultivars studied, genetic divergence was roughly correlated with geographic separation (Fig. 2.15). However, data from a larger set of feral vines in Italy appear to contradict the involvement of local vines in the origin of many Italian cultivars (Imazio et al., 2015). Data from the eastern Adriatic region also suggest a separation of cultivars and wild accessions (Zdunic et al., 2017). Nonetheless, Veronese cultivars appear related to local feral vines (Vantini et al., 2003) and Georgian cultivars to sympatric feral vines (Ekhvaia et al., 2010). For example, Rka´tsiteli showed 90% similarity with wild vines growing in the Lekjura Gorge region. In contrast, other studies support the significance of cultivar importation. Data implicative of Georgian cultivars being involved in the origin of several western cultivars are found in Vouillamoz et al. (2006). These include Muscat, Chardonnay, Cabernet Sauvignon, 0.5

0.4 Nei’s genetic distance

TABLE 2.3

2. Grape species and varieties

0.3

0.2

0.1

0 0

500

1000

1500

2000

2500

3000

3500

Geographic distance (km)

FIGURE 2.15

Scatterplot of geographic vs. genetic distance between grapevine gene pools of 164 European cultivars. This suggests the relatively minor role played by introduced cultivars from Greek or Phoenician sources. From Sefc, K.M., Steinkellner, H., Lefort, F., Botta, R., da Caˆmara Machado, A., Borrego, J., et al., 2003. Evaluation of the genetic contribution of local wild vines to European grapevine cultivars. Am. J. Enol. Vitic. 54, 15e21, reproduced by permission.

39

Cultivar origins

(Silvaner  Blaue Zimmettraube) (Maul et al., 2016), Sil¨ sterreichisch weiss) (Sefc vaner (probably Traminer  O et al., 1998), Sangiovese (Ciliegiolo  Calabrese de MontenuovodCalabrian cultivars) (Vouillamoz et al., 2007), Syrah (Dureza  Mondeuse blanchedsouthern French cultivars) (Bowers et al., 2000), and Tempranillo (Albillo Mayor  Benedicto) (Iba´n˜ez et al., 2012). Other suggestions, such as Pinot noir (Traminer  Meunier) (Regner et al., 2000), are possible, but have yet to be confirmed. However, Meunier is often viewed simply as a named clonal variant of Pinot noir. Amazingly, many classic cultivars appear to have a common parent (Fig. 2.16). Traminer appears to be one of the parents of cultivars such as Riesling, Sauvignon blanc, and Chenin blanc. Pinot noir also appears to have participated in multiple offspring. Surprisingly, a cultivar of little apparent enologic significance, Gouais blanc (Heunisch Weiss), is the presumptive parent, along with Pinot, of multiple French cultivars, notably Chardonnay, Gamay noir, Aligote´, and Melon

Syrah, Pinot noir, Nebbiolo, and Chasselas. Introgression with eastern cultivars is also suggested by data presented in Myles et al. (2011). Introduction vs. local domestication also seems likely for Bulgarian and Tunisian table grapes (Dzhambazova et al., 2009; Snoussi et al., 2004), as well as North African cultivars (Riahi et al., 2012). Thus, there appears to be no common denominator to the origin of existent cultivars, some appearing to be selections from local wild vines, others being the offspring of imported and sympatric feral vines, and still others being transplanted from elsewhere, possessing no direct genetic relationship to indigenous vines. Where presumptive parents are known, it is unknown whether the crosses occurred spontaneously or purposefully. Examples are Chardonnay (Pinot noir  Gouais blanc) (Bowers et al., 1999), Cabernet Sauvignon (Cabernet franc  Sauvignon blanc) (Bowers and Meredith, 1997), Merlot (Cabernet franc  Magdeleine Noire des Charentes) (Boursiquot et al. 2009), Blauer Portugieser

Muscat Blanc

Reichensteiner

Gamay

Schönburger

Gelber Ortlieber

Chardonnay

Ortega Ehrenfelser Müller-Thurgau Rotberger

Petite Bouschet

Pinot Noir Siegerrebe

Optima Trollinger

Alicante Bouschet

Wittberger Riesling

Teinturier du Cher Petit Manseng Osteiner

Perle

Aramon

Kerner Verdelho

Muscat Hamburg Perlriesling

Sylvaner

Rotgipfler

Roter Veltliner

Donzillinho Traminer

Taminga Muscat of Alexandria Perle de Csaba Tinta Madeira Flora

Bequignol Grüner Veltliner

Semillon Fer Servadou

Pé agudo

Sauvignon Blanc Trousseau Chenin Blanc

Royalty

Sibling or equivalent Parent-offspring

Cabernet Franc Cabernet Sauvignon

Trincadeiro Merlot

Ruby Cabernet Meslier-Saint-François Carignan Colombard

FIGURE 2.16 Network of first-degree relationships among common grape cultivars. Solid lines represent likely parenteoffspring relationships. Dotted lines represent sibling relationships or equivalent. Arrows point from parents to offspring. Myles, S., Boyko, A.R., Owens, C.L., Brown, P.J., Grassi, F., Aradhya, M.K., et al., 2011. Genetic structure and domestication history of the grape. Proc. Natl. Acad. Sci. U.S.A. 108, 3530e3535, reproduced by permission.

40

2. Grape species and varieties

Although the actual origins of many cultivars may never be known, due to one or both parents being no longer extant, DNA studies can suggest probable ancestry. The inclusion of multiple SSR markers greatly reduces the probability of predicting incorrect pedigrees. Twenty-five markers are often considered a minimum. While certainty of parentage is difficult, DNA fingerprinting techniques can easily establish what is essentially impossible. For example, it has shown that Mu¨ller-Thurgau is the result of neither a Riesling selfcross nor a Riesling  Silvaner cross (Bu¨scher et al., 1994). The latest detailed analysis suggests that Riesling and Madeleine Royale are the progenitors of Mu¨ller-Thurgau (Vouillamoz and Arnold, 2010). Microsatellite analysis has also shown the presumed parentage of some recent Ukrainian cultivars to be in error (Goryslavets et al., 2010). The technique has even revealed unsuspected aspects of the diversity and geographic distribution of Cabernet Sauvignon (Moncada et al., 2006) and Merlot (Herrera et al., 2002) clones. In the latter instance, the clone of Merlot grown in Chile is distinctly different from that typically grown in France. One of the limitations of all such studies is that they must be based on existent cultivars. What potential lineages might be suggested, were extinct cultivars or feral strains available for sampling, will never be known. Although most cultivars thus far investigated appear to be monozygous (derived from a single seed), this

(Bowers et al., 1999). It is also speculated to be one of the parents of Riesling (Regner et al., 1998). A fascinating recount of the history, characteristics, and color variants of Heunisch Weiss is given by Maul et al. (2015). Examples of other cultivars, now rare and/or of little viticultural interest, involved in the origin of important cultivars are Benedicto, Blaue Zimmettraube, and Magdeleine Noire des Charentes. These could all be viticultural examples of a phenomenon called hybrid vigor (heterosis)dwhere the quality of the progeny seems out of all proportion with that of one or both parents. Although extremely useful in breeding, the origin of heterosis still remains a mystery, but may relate to partial dominance or overdominance of particular alleles or linked genes. The importance of Gouais blanc in several French cultivars is partially reflected in the importance of Sangiovese and Garganega in the origins of Italian cultivars (Fig. 2.17; Di Vecchi Staraz et al., 2007) and Lista´n Prieto in South American varieties (Tapia et al., 2007). The latter, a little-known Spanish cultivar, crossed with Muscat of Alexandria, gave rise to two Torronte´s varieties, and with Jaen B and Negra Mole, to produce other local cultivars. Lista´n Prieto itself was important in the early establishment of vineyards in the Americas, being variously called the Mission grape (California), Pais (Chile), and Criolla chica (Mexico). Criolla is used to cover a group cultivars in South America (Martı´nez et al. 2006).

Ciliegiolo

?

Calabrese di Montenuovo

Sangiovese

Tuccanese di Turi

Foglia tonda Morellino del Casentino Morellino del valdarno

Susumaniello

Vernaccia nera del valdarno

Gaglioppo

Nerello mascalese

Frappato Mantonicone

Tuscany

Sicily and Calabria

Apulia

FIGURE 2.17 Schematic representation of the presumptive origin of ‘Sangiovese’ and its involvement in the origin of other Italian cultivars. From Crespan, M., Calo`, A., Giannetto, S., Sparacio, A., Storchi, P., Costacurta, A., 2008. ‘Sangiovese’ and ‘Garganega’ are two key varieties of the Italian grapevine assortment evolution. Vitis 47, 97e104, reproduced by permission.

41

Cultivar origins

does not necessarily imply genetic identity. Mutations subsequent to origin can progressively generate distinct clones, showing allelic polymorphy. Examples of clearly polyclonal varieties are Cabernet franc, Cabernet Sauvignon, Traminer, Riesling, Pinot noir, Chenin blanc, and Grolleau (Pelsy et al., 2010); Meunier (Franks et al., 2002; Stenkamp et al., 2009); Pinot gris and Pinot blanc (Hocquigny et al., 2004); and Pinot noir and Chardonnay (Riaz et al., 2002). In addition, clonal divergence may also be derived polyzygotically (that is, siblings are so morphologically similar as to be considered members of the same variety). A suspected example of a polyzygotic cultivar involves two lines of Fortana (Silvestroni et al., 1997). However, siblings are usually distinctly different, as indicated by the variety of cultivars derived from Gouais blanc  Pinot (Fig. 2.18). Additional sources of variation may arise if distinct clones are involved in multiple crosses (e.g., Pinot noir vs. Pinot blanc). Furthermore, differences can arise depending on which parent acted as the femaledit being the sole origin of traits associated with chloroplast and mitochondrial genes. Surprisingly, some mitochondrial genes are of chloroplast origin (Goremykin et al. 2009), indicating the translocation of genes between organelles. Some

nuclear genes are also thought to have been translocated from one of their organelles (Leister, 2005). The designation of a variant as a variety or a clone is partially subjective, often depending on precedent (Boursiquot and This, 1999). An example of a variant given a specific name is the Musque´ clone of Chardonnay. Alternatively, it may be distinguished only by accession number, for example, Riesling clones #239, #239-20, #239-12, and #110-1. However, if the variants make distinctly different wines, they usually go under separate, but related, names (e.g., Pinot gris and Pinot blanc, clones of Pinot noir). These are particularly interesting due to the distinct fragrances of their respective wines. The chemical origins of these differences are unknown, but may arise from increased production of volatile phenols or terpenoidsdconsequences of a chemical backup due to disrupted anthocyanin synthesis (Rinaldo, 2014). Other clonal variants are termed biotypes (e.g., many Sangiovese clones) (Calo` et al., 1995). In other instances, clones have been grouped relative to their vegetative characteristics. Examples with Pinot noir include those termed Pinot findtrailing, low-yielding vines, with small tight clusters; Pinot droitdhigher-yielding vines with upright shoots; Pinot fructiferdhigh-yielding strains; and

Pinot × Gouais blanc

Sacy Aligote

Roublot Aubin vert

Auxerrois

Romorantin

Bachet noir

Peurion

Beaunoir

Melon Chardonnay

Knipperle

Dameron

Gamay noir Franc noir de la Haute Saoöne

Gamay blanc Gloriod

FIGURE 2.18 Schematic representation of the original French cultivars thought to be progeny of a crossing of ‘Pinot’ clones and ‘Gouais blanc.’ Data from Bowers, J., Boursiquot, J.-M., This, P., Chu, K., Johansson, H., Meredith, C., 1999. Historical genetics: the parentage of Chardonnay, Gamay, and other wine grapes of Northeastern France. Science 285, 1562e1565.

42

2. Grape species and varieties

Mariafeld-type strainsdloose clustered, moderateyielding vines (Wolpert, 1995). If the number of allelic variants can be used as a measure of cultivar age, then data from Pelsy et al. (2010) would suggest that Pinot noir and Traminer are ancient cultivars. This fits morphological attributes that give the impression that both are ancient, probably selections of wild or slightly domesticated vines. Their association as one of the parents of multiple other cultivars is also consistent with their presumptive age. Nonetheless, based on the few accessions studied, the limited somatic diversity within Riesling clones (Pelsy et al., 2010) would be at variance with its presumptive ancient origin. However, the number of detected genotypes could easily vary, based on the number and appropriateness of the clones assessed. In an investigation of 24 Traminer accessions (Imazio et al., 2002), all were found to be genetically identicalda finding distinctly different from the observations of Pelsy et al. (2010). In addition, there is no evidence that the rate at which allelic SSR differences develop is constant, unlike the rate of point mutationd which tends to be relatively constant over long periods (Drake, 1991). SSR differences appear to depend primarily on polymerase slippage, or the activity of transposons, not nucleotide substitutions induced by tautomeric shifts. Another significant application of genomic analysis has involved unraveling cultivar synonymy. For example, Zinfandel in the United States is Primativo in Italy, and Crljenak kastelanski in Croatia (Maletiae et al., 2004). Molecular analysis has also shown the synonymy between most Californian Petite Sirah and Durif (Meredith et al., 1999). Durif is the progeny of a crossing between Syrah and Peloursin. Sequence analysis has also permitted clarification of the interconnections among Muscat cultivars (Fig. 2.19). Initially, sequencing techniques were thought to be independent of the tissues useddin contrast to isozymes and pigments, both of which are produced only in certain tissues or at particular developmental stages. x Trebbiano toscano x Sangiovese

However, tissue-specific DNA banding has now been identified (Donini et al., 1997). This is not overly surprising, due to the possible selective amplification or silencing, as well as other possible epigenetic factors and/or mutations of genes during tissue development. In addition, clonal chimeric differences, and their distribution within plant tissues, may be used to distinguish them. Conversely, chimeric differences may generate some of the data divergence among research labs. DNA fingerprinting has also been applied to DNA isolated from seeds (Manen et al., 2003) and other archaeological remains. Amplification has generated useful microsatellite markers from both waterlogged and charred seeds (600 BCE to CE 300). For example, DNA has been successfully isolated from seeds dating to the Late Neolithic in Sicily (w4000 BCE) (Gismondi et al., 2016). Regrettably, most genomic analysis is based on markers of no or unknown genetic significance. For example, SSR markers appear to be located in sections of the genome other than the 4% that codes for functional (protein sequencing) genes (Lodhi and Reisch, 1995). The remaining DNA consists primarily of variously repeated segments of unknown function, transposon copies, gene regulator sections, or nonfunctional (null or pseudo-) genes. Thus, SSR relationships are likely to have no phenotypically (selective) value. It is a moot point whether it would be preferable for relationships to be based on evolutionarily (ecologically) significant properties (structural genes), somewhat similar to taxonomic studies, vs. changes in selectively neutral segments of the genome (see Jones and Brown, 2000).

Recorded cultivar development Until little more than a century ago, the selection of new cultivars was limited, or at least mostly unrecorded. Some of the earliest examples seem to be accidental

Garofana Ciliegiolo

Moscato violetto Duraguzza x Moscato bianco x Axina de tres bias Schiava grossa x Muscat of Alexandria Muscat of Hamburg x Bicane x Regina dei Vigneti x Sultanina x Raboso Veronese

x Cataratto bianco x Bombino bianco x Chasselas dorée

Grillo Moscatello selvatico Mathiasz 210

Italia Early Muscat Queen Manzoni Moscato

FIGURE 2.19 Pedigree of the Muscat group of cultivars based on first-degree parentage. Moscato bianco (syn. Muscat blanc a` petits grains) seems to be the most ancient cultivar and appears in boldface. Reprinted by permission from Springer Nature, from Cipriani, G., Spadotto, A., Jurman, I., Di Gaspero, G., Crespan, M., Meneghetti, S., et al., 2010. The SSR-based molecular profile of 1005 grapevine (Vitis vinifera L.) accessions uncovers new synonymy and parentages, and reveals a large admixture amongst varieties of different geographic origin. Theor. Appl. Genet. 121, 1569e1585.

Recorded cultivar development

‘Sémillon’

43

Wild species #1 FFw + R+? red or black

HFk + w/w white

‘Catawba’

Wild species #2 FFw + B+? black

HFw + R/w red

‘Concord’ HFw + B/w black

FIGURE 2.20 The pedigree of Concord based on phenotypic and genotypic data, based on berry color simple sequence repeat markers, a flower sexelocus marker, and genome-wide genetic analysis concerning wild (dark burgundy) and Vitis vinifera (light burgundy) portions. B, black berry color allele, R, red berry color allele, w, white berry color allele; H, hermaphrodite allele, Fk, female allele, Fw, “wild female allele”. \, female flowers; ,hermaphrodite flowers. From Huber, F., Ro¨ckel, F., Schwander, F., Maul, E., Eibach, R., Cousins, P., To¨pfer, R. 2016. A view into American grapevine history: Vitis vinifera cv. ‘Se´millon’ is an ancestor of ‘Catawba’ and ‘Concord’. Vitis 55, 53e56, reproduced by permission.

crosses between indigenous Vitis spp. and European varieties in New England. For example, Catawba is probably the progeny of a Se´millon  indigenous Vitis crossing (Huber et al., 2016). Concord was originally viewed as a wild V. labrusca accession (Hedrick, 1908), but now recognized as the progeny of a cross between a wild Vitis sp. and Catawba (Fig. 2.20). The seed is reported to have been planted by E.W. Bull in Concord, Massachusetts, in 1843. Its interspecies origin is supported by the bisexual nature of its flowers (a property rare in wild grapevines), the presence of a typical vinifera Gret1 retrotransposon (Mitani et al., 2009), its seed shape, and the pinkish coloration and vinous flavor of the fruit. Other likely examples of interspecific crosses are Dutchess (V. labrusca  unknown V. vinifera), Delaware (V. vinifera  V. aestivalis  V. labrusca), Noah and Clinton (V. riparia  V. labrusca), Herbemont and Lenoir (V. aestivalis  V. cinerea  V. vinifera), and Cynthiana and Norton (V. vinifera  V. aestivalis) (Stover et al., 2009). Most of these cultivars have fruit sizes within the range of vinifera cultivars. Other American cultivars, such as Ives and Isabella, are often considered to be direct V. labrusca selections. These early cultivars are termed American hybrids, to distinguish them from hybrids produced in France between V. vinifera and one or more of V. rupestris, V. riparia, and V. aestivalis var. lincecumii. The last three are variously termed French-American hybrids, French hybrids, or direct producers. The last designation comes

FIGURE 2.21 Estimated Vitis vinifera content in 64 commercial French-American grape cultivars used in wine production. (A) Distribution of V. vinifera ancestry estimates in hybrids. (B) V. vinifera ancestry estimates for each cultivar. Bars are colored if a hybrid

44

2. Grape species and varieties

=

cultivar’s ancestry estimate falls within the 95% confidence interval of an F1, F1  indigenous North American Vitis spp., or F1  V. vinifera cross, based on simulated values. Dotted lines indicate mean values for the wild Vitis spp. and V. vinifera samples. From Migicovsky, Z., Sawler, J., Money, D., Eibach, R., Miller, A.J., Luby, J.J., et al., 2016. Genomic ancestry estimation quantifies use of wild species in grape breeding. BMC Genomics 17, 478, reproduced by permission.

from their ability to grow ungrafted in phylloxerainfested soils. It was once thought that backcrossing to Vitis vinifera had been extensive (to enhance the expression of vinifera-like winemaking qualities). However,

TABLE 2.4

about one-third of French-American hybrids have genomes consistent with F1 hybrids, with the remaining possessing genomes ranging from 11% to 76% vinifera parentage (Fig. 2.21). Additional details on the relatedness of many of these cultivars can be found in Pollefeys and Bousquet (2003). In contrast, modern hybrids, such as Regent, are often crosses between V. vinifera and V. rotundifolia. The progeny are subsequently backcrossed with the vinifera parent. Selection for desirable vinifera traits is combined with retaining rotundifolia disease resistance (Eibach et al., 2007). The original aim of most French breeders was to develop cultivars containing the wine-producing

Overview of disease susceptibility, vigor, and hardiness/cold tolerance of 41 fungus-resistant varieties. Susceptibility to diseasesa,b

Variety

DM

PM

BOT

BR

+

++

+

+++

AT

DA

CG

EUT

Vigor

Hardiness/Cold tolerance ( F)

+

+++

++

high

10 to 20

moderate

20 to 30

moderate/high

5 to15

moderate

10 to 20

reds Baco noir Baltica

+

+

+

Cabernet Carbon

/+

++

/+

Cabernet Jura



/+

/+

Chambourcin

+

+

++

+++

Chancellor

+++

++

+

+

Chelois

+

+++

+

+

+++

Concord

+

++

+

+++

+++

+

+++

high

15 to 25

De Chaunac

++

++

+

+

+

++

++

+++

moderate/high

15 to25

Frontenac

/+

++

++

++

+

++

high

20 to 30

Le´on Millot

+

++

+

+

high

15 to 25

Lucie Kulhmann

+

++

Mare´chal Foch

+

++

+

++

Marquette



+

+

+

Petite Perle

+

+

+

+

Regent

+

+

+

Sabrevois

+

+

+

+

++

+++

++

+ +

+

10 to 20

++

++

++

+

moderate moderate

15 to 25

+++

high

20 to 30

+

moderate

20 to 30

+

+++

+++

5 to15

+ +

+

moderate

20 to 30

+

+

+

++

moderate

20 to 30

++

++

++

+

+

moderate/high

20 to 30

Aurore

++

++

++

+++

high

10 to 20

Bianca

+

+

/+

Bronner

/+

+

+

Cabernet blanc

++

++

/+

Frontenac blanc

/+

+

Skandia St. Croix whites

+

++

+++

5 to15

++

5 to15 ++

++

high

20 to 30

45

Recorded cultivar development

TABLE 2.4

Overview of disease susceptibility, vigor, and hardiness/cold tolerance of 41 fungus-resistant varieties.dcont’d Susceptibility to diseasesa,b

Variety

DM

PM

BOT

Frontenac gris

/+

+

Geisenheim

+

+++

+

Helios

++

+

+

Hibernal

++

+

+

Johanniter

++

/+

++

Kunlea´ny

++

+++



La Crescent

+

+

Louise Swenson

+

+

Osceola Muscat

++

Seyval

Vigor

Hardiness/Cold tolerance ( F)

+

high

20 to 30

+

moderate

BR

AT

++

+

DA

CG

EUT

+

moderate

++

++

high

20 to 30

+

+

++

low

20 to 30

++

+

+

+

high

15 to 25

+

+++

++

++

+

moderate

10 to 20

Solaris

++

/+

++

Soleil blanc

+

/+

++

St. Pepin

+

+

+

Traminette

+

+

+

VandalCliche

+

+

+

Vidal

+

++

/+

Villard blanc



+

+

++

10 to 20

+ +

+++

++

++

+++ +

+

+

+++

+

moderate

20 to 30

moderate/high

10 to 20

moderate/high

15 to 25

moderate

5 to15

+

From Pedneault, K., Provost, C., 2016. Fungus resistant grape varieties as a suitable alternative for organic wine production: benefits, limits, and challenges. Sci. Hortic. 208, 57e77, reproduced by permission from Elsevier.

attributes of V. vinifera and the phylloxera resistance of their American parent. It was hoped that this would avoid the expense and problems associated with grafting existing V. vinifera cultivars to American rootstocks. Grafting was the only effective control against the devastation being wrought by phylloxera (Daktulosphaira vitifoliae). It also was hoped that breeding would incorporate resistance to other American pathogens, notably powdery and downy mildews. Table 2.4 provides an overview of the relative disease resistance, vigor, and cold tolerance of a large selection of FrenchAmerican and other hybrid cultivars. More by accident than design, several hybrids were more productive than pure vinifera cultivars. They became so popular that by 1955 about one-third of French vineyards were planted with French-American hybrids. Their productivity aggravated a growing problem, termed the “wine lake.” Even in recent times, an excess of 20e40  106 hL of wine has had to be distilled into industrial alcohol (Eurostat, 2013). In addition, their different aromatic character came to be viewed as a threat to the established reputation of viticultural regions. Political pressure culminated in a series of laws designed to restrict their use in AOC (appellation

d’origine controˆle´e) wines, as well as cultivation in France. Although still permitted in some European wines, or as a fresh fruit crop, hybrid cultivation is currently minimal. Similar European Union legislation, commencing in the 1970s (Meloni and Swinnen, 2013), has drastically reduced their planting across Europe. Largely rejected in the land of their origin, FrenchAmerican hybrids have found broad acceptance in many northeastern regions of North America. They initially helped foster wine production in much of the continent, outside of California, and generated regionally distinctive wines. They are still used predominantly in regions where the survival of V. vinifera cultivars is tenuous. French-American hybrids are also grown in some other parts of the world, notably Brazil and Japan. In the coastal plains of the southeastern United States, commercial viticulture and wine production is based primarily on muscadine cultivars (Vitis rotundifolia) (Morris and Brady, 2007). Scuppernong is the most well known muscadine cultivar, but has the disadvantage of being unisexual. Newer cultivars such as Noble, Magnolia, and Carlos are bisexual, and so avoid the necessity of interplanting male vines to achieve adequate fruit set. Another complication

46

2. Grape species and varieties

with muscadine cultivars is the tendency of berries to separate (shatter) from the cluster as they ripen. Newer cultivars, such as Fry and Pride, show less tendency to shatter. In addition, applying ethephon (2-chloroethyl phosphonic acid) can promote more uniform ripening. The resistance of muscadine grapes to most indigenous diseases and pests in the southern United States has permitted a local wine industry to develop, where the commercial cultivation of V. vinifera cultivars is difficult to impossible. Although Pierce’s disease has limited the cultivation of most non-muscadine cultivars in the region, the varieties Herbemont, Lenoir, and Conquistador are exceptions. One of the important cultivars developed for northern wine-growing regions is Betada Concord  Vitis riparia progeny. Although grafting prevented the demise of grape growing and winemaking in Europe during the late 1800s, early rootstock cultivars created their own problems. Most of the initial rootstocks were simple selections from feral V. riparia or V. rupestris. As most were poorly adapted to the high-calcium soils found in many European regions, the incidence of lime-induced chlorosis increased markedly. Nonetheless, the roots of both species grafted well, and were relatively phylloxera tolerant. Vitis riparia rootstocks also provided some cold hardiness, resistance to coulure (unusually poor fruit set), and restricted vigor on deep rich soils and favored early fruit maturity; whereas V. rupestris selections limited lime-induced chlorosis and tended to root deeply. The latter property provided drought tolerance in deep soils, but was unacceptable on shallow soils. The problems associated with early rootstock selections, such as Gloire de Montpellier and St. George, led to the breeding of new hybrid rootstocks (Fig. 2.22). These incorporated desirable properties from several Vitis species. For example, Vitis berlandieri (V. cinerea var. helleri) can supply resistance to lime-induced chlorosis, V.  champinii can donate tolerance to root-knot nematodes, V. vulpina (V. cordifolia) provides drought tolerance under shallow soil conditions, and V. mustangensis (V. candicans) is a source of root-knot nematode resistance. However, these species have themselves major drawbacks as rootstocks. For example, V. berlandieri and V. vulpina root with difficulty, V. mustangensis has only moderate resistance to phylloxera, and both V. mustangensis and V. vulpina are susceptible to limeinduced chlorosis. Important cultural properties of some of the more widely planted rootstocks are given in Tables 4.6 and 4.7.

Grapevine improvement Grapevine cultivars number in the thousands, but only a few dominate commercial cultivation. About

Species and bihybrid cultivars V. mustangensis SO 4 8B 34 EM 5C 420 A 161-49 C

V. cinerea var. helleri

99 R 110 R 140 RU 1103 P ‘Paulsen’

‘Solonis’ V. riparia (“Gloire de Montpellier”)

143 B

3306 C 3309 C 101-14 Mgt ‘Schwarzmann’

V. vinifera

V. rupestris (‘St. George’)

1202 C 93-5 C AxR1

41 B 333 EM ‘Fercal’

Multiple-species cultivars 196-17 Ca

V. riparia x (V. rupestris x V. vinifera)

44-53, 106-8

V. riparia x (V. rupestris x V. cordifolia)

1616 C

V. rupestris x ‘Solonis’

216-3 Ca

‘Solonis’ x ‘Othello’ (V. labrusca x V. riparia x V. vinifera)

‘Harmony,’ ‘Freedom’

‘Dog Ridge’ x 1613 C

FIGURE 2.22 Parentage of some important hybrid rootstock varieties. ( ), selection from a single species; ,, interspecies crossings. The Champin varieties, ‘Dog Ridge’ and ‘Salt Creek’ (Ramsey), are considered to be either natural Vitis mustangensis  Vitis rupestris hybrids or strains of V.  champinii. ‘Solonis’ is considered by some authorities to be a selection of Vitis acerifolia (Vitis longii), rather than a Vitis riparia  V. mustangensis hybrid. On this basis, some multiplespecies hybrids noted above are bihybrids. Modified from Pongra´cz, D.P., 1983. Rootstocks for Grapevines. David Philip, Cape Town, South Africa, reproduced by permission.

three-quarters of these are closely related (Migicovsky et al., 2016), indicating limited genetic diversity. This is of considerable concern, relative to current pathological and developing climatic challenges, even with the new possibilities opened with CRISPR (clustered regularly interspaced short palindromic repeats)-based genetics (Dolgin, 2017). As with other perennial plants, and especially due to the small size and minor morphologic differences between Vitis chromosomes (see Fig. 2.5), few data on its genetics have accumulated using cytogenetic techniques. This deficiency is now being filled by genomic (DNA) analysis (Carrier et al., 2012; Laucou et al., 2018). Most of this is based on an analysis of polymorphisms based on SNP and nucleotide insertions and deletions (indels). Fig. 2.23 represents the Vitis genome, noting the chromosomal location of many genes. It is estimated that the Vitis genome contains some 30,000 protein-coding genes. Preliminary data suggest that as many as 13% of the genes are heterozygous (Velasco et al., 2007). In addition,

47

Grapevine improvement

(B)

(A)

LG2

LG1

LG3

LG8

LG9

LG7 LG4

LG5

LG6

LG14

LG10

LG11

LG19

LG12 LG13

LG18

LG15

LG16

LG17

FIGURE 2.23 Chromosomal organization of disease-resistance genes of Vitis vinifera. (A) Phylogenetic analysis of NBS-LRR protein sequences of V. vinifera present in Pinot noir. The phylogeny of these genes is based on a distance-matrix neighbor-joining analysis (Clustal X; bootstrap of 1000) after alignment of sequences by TCoffee (version 5.05). The phylogenetic clades, in general, correspond to the classification based on protein domains (however, see text). (B) Genes assigned to LGs are represented by dots. Their gene number is specified in LG-specific insets. NBS clades (see (A)) contain mainly genes of the following classes: (1) TIR-NBS-LRR in blue; (2) CC-NBS-LRRa in green; (3) CC-NBS-LRRb in yellow; (4) NBS-LRR in cyan; (5) CC-NBSLRR in red. Other resistance genes belonging to NBS and TIR-NBS groups are represented by the open and filled dots, respectively. Resistance-related genes different from NBS ¼ nucleotide binding site; LRR ¼ leucine-rich repeat; TIR ¼ Toll-inerleukin1 receptor; LG ¼ linkage group; CC ¼ coiled coil genes are shown in black. The size of each LG is given in megabases (on the right), and http://genomics.research.iasm. From Velasco et al. (2007), reproduced by permission.

48

2. Grape species and varieties

grapevines possess an abnormally high number of isogenes (closely related genes). For example, there are 43 stilbene synthase, 89 functional terpene synthase, and 2 geranyl diphosphate synthase genes (more than twice as many as most other plants) (Jaillon et al., 2007). These genes are thought to be particularly important to grapevine health and varietal flavor distinctiveness. For example, stilbene synthases are involved in the production of resveratrol and other antimicrobial phytoalexins, terpene synthases are responsible for the production of aromatic terpenes, and geranyl diphosphate synthases are critical in the production of terpene progenitors. Knowledge of the location, and the ability to detect a gene’s presence early (circumventing dominance phenotypic masking), is being used to facilitate and speed varietal improvement. This often involves marker-assisted breeding (MAS) (Mackay et al., 2009). In grapevines, MAS is being used to incorporate disease-resistance loci (or inactivate pathogen effector sites). This has been valuable in incorporating Pierce’s disease resistance into Vitis vinifera cultivars (Riaz et al., 2009). The technique has also been used to combine (pyramid) several genes from V. rotundifolia, V. amurensis, V. rupestris, and V. aestivalis that are resistant to powdery and downy mildews (Schneider et al., 2014). Other resistance enhancements have augmented stilbene (phytoalexin) synthesis with an allelic form of the MYB14 transcription factor from Vitis sylvestris (Duan et al., 2016). MYB15 is another transcription factor involved in activating stilbene synthesis. Other interesting opportunities could involve enhanced activity of anthocyanin acylation genes (in cultivars characterized by pale colors, e.g., Pinot noir, which has a dysfunctional acyltransfer gene), inactivation of anthocyanin diglucosidation genes in non-vinifera cultivars (monoglucosidic anthocyanins generate more intense colors), and the incorporation of cold-resistance factors (Zhang et al., 2012). Additional understanding of grapevine genetics is likely to arise from comparison of the known functions of structurally related gene clusters in other plants. Such cross-comparison should greatly accelerate the speed with which functions of unknown regions of the grape genome can be interpreted. Understanding the correlation between genes and their regulation of biochemical pathways in vines is essential to applying many of the latest genetic techniques, notably CRISPR, TALENs (transcription activator-like effector nucleases), and ZFNs (zinc-finger nucleases), with MAS.

Standard breeding techniques The focus of most grape breeding has changed little since the early work of breeders such as F. Baco, A. Seibel, and B. Seyve (Neagu, 1968). Standard goals include improving the agronomic properties of rootstocks (e.g.,

ease of grafting, disease, pest and environmental adaptation) and enhancing the scion attributes (superior grape quality, optimal yield, and pest, disease, and environmental adaptation). The major changes that have occurred have entailed the availability of improved analytical instrumentation and a better understanding of genetics. This has dramatically increased a breeder’s ability to design crosses with enhanced chances of achieving the desired goals. However, the duration of any grapevine breeding program remains longdfor scions, it is in the range of 20 years. The small, bisexual nature of Vitis flowers, enclosed in a calyptra, adds additional challenges in selecting the adequate timing for emasculation and cross-pollination (Eibach and To¨pfer, 2015). Of breeding programs, those developing new rootstocks are potentially the simplest. Improvement often aims at enhanced lime, drought, disease, and/or pest tolerance/ resistance. When one or a few genes are involved, incorporation of these properties may require only the crossing of a desirable variety with the source of the new trait(s), followed by selection of offspring possessing the trait(s) desired (Fig. 2.24A). Backcross breeding (Fig. 2.24B) has been the principal means of integrating one or a few linked, dominant traits into an existing cultivar. Developing new scion (fruit-bearing) varieties is more complex. The repeated backcrossing, traditionally used to reestablish expression of desirable traits, can result in diminished seed viability and vine vigor (termed inbreeding depression) (Charlesworth and Willis, 2009). It occasionally can be countered if more than one cultivar can serve as a recurrent parent, or with embryo rescue. Embryo rescue involves the isolation and cultivation of seedling embryos on culture media, which otherwise would abort during seed development (Spiegel-Roy et al., 1985; Agu¨ero et al., 1995). Alternatively, the effects of inbreeding depression may be limited by treating germinating seeds with a demethylating agent (Pennisi, 2011). Gene inactivation is often associated with DNA methylation. Newer techniques that enhance heterosis (the opposite of inbreeding depression) may also be applicable (McKeown et al., 2013). However, an even more intractable problem in breeding new cultivars is the highly heterozygous nature of most desirable traits (Plate 2.4). As a consequence, breeding tends to disrupt the complex balance that donates a variety’s desirable attributes. In addition, with few exceptions, most properties associated with commercial success are quantitative, depending on the accumulative action of multiple genes, often located on separate chromosomes. Thus, disruption of desirable combinations is particularly likely during recombination and the random assortment of chromosomes during meiosis. Gene reassortment can also complicate the selection process, by blurring the distinction between environmentally and genetically induced variation.

49

Grapevine improvement

(B)

(A) Simple cross

Backcross Cultivar × Source

Parents:

Progeny:

C 1,

C2,

C3,

Cultivar × Source

C4, … C n

Selection for several generations:

C1 ,

C 2,

C3 ,

C 4, … C n

First backcross

BC1, BC2, BC3, BC4, … BCn

Second backcross

BC1, BC2, BC3, BC4, … BCn

Third backcross

BC1, BC2, BC3, BC4, … BCn

Selection for several generations:

New variety:

FIGURE 2.24

Genetic strategies for improving rootstocks. (A) Simple cross-breeding program with vegetative propagation following the initial cross. (B) Backcross-breeding program involving a cross followed by several backcrosses (BC) to a common (parental variety) to reestablish the desirable properties of the parental variety.

These problems may be partially diminished if the genetic basis of the trait(s) is known. For example, selection for cold hardiness might be facilitated if the nature, location, and relative importance of the regulatory genetic loci were known. Individual aspects of this particular complex property involve diverse features, such as the control of cellular osmotic potential (timing and degree of starch/sugar interconversion), the unsaturated fatty acid content of cellular membranes and its changes, and the population of ice-nucleating bacteria on leaf surfaces. This knowledge would permit each aspect to be individually assessed and selected for during breeding. Breeding for disease resistance is also far from simple, due to the frequently complex nature of disease and pest resistance and the practicalities of measuring the degree of resistance. Plant resistance is often categorized into innate resistance (mechanisms against biotic as well as abiotic agents) and resistance that is specific against particular pathogens or strains (see Genetic Control in Chapter 4). Chemical indicators and other easily detected genetically controlled features are especially useful in aiding the early selection of suitable offspring. The efficient and

early elimination of undesirable progeny ensures that precious time and resources are spent only on potentially useful offspring. For example, the presence of methyl anthranilate and other volatile esters was used in the early elimination of progeny possessing a labrusca fragrance (Fuleki, 1982). Improved knowledge of the factors associated with color stability in red wines might be useful in the early selection of offspring possessing better color retention attributes. In addition, selection for disease resistance would be both sped up and facilitated were the property assessable in seedlings by a simple chemical test. Regrettably, phytoalexin production and toxin resistance are often stage specific and not necessarily adequately expressed in seedlings. Finally, laboratory trials frequently do not adequately simulate field conditions. Another factor that can facilitate breeding is combining ability. This somewhat nebulous term refers to a strain’s relative success in breeding programs. Regrettably, at the time of this writing, combining ability is impossible to assess in advance. Its genetic basis is unknown. Nonetheless, it is suspected to be partially associated with the genetic difference between the parents. A

50

2. Grape species and varieties

possible example of combining ability may involve Gouais blanc (Heunisch Weiss) and Pinot noir. Many important cultivars have them as their parents (see Fig. 2.18). Gouais blanc is suspected to be an ancient Croatian cultivar, whereas Pinot noir is often viewed as a strain selected from wild vines, or an introgressed strain retaining many feral characteristics. Adding to the complexities of grapevine breeding is the time required for adequately assessing winemaking attributes. Vines begin to bear sufficient fruit for testing only in their third year. Also, microvinification does not necessarily, or adequately, replicate commercial conditions. Increasing the population of a potentially new cultivar to a point at which features such as winemaking potential can be adequately evaluated can take decades. Ideally, the length of a breeding program can be shortened by inducing precocious flowering (Mullins and Rajasekaran, 1981; Baby et al., 2014). This is especially useful in studying properties assessable with small fruit samples. Flowering can be induced within 4 weeks of seed germination, by the application of synthetic cytokinins such as benzyladenine or 6-(benzylamino)-9-(2tetrahydropyranyl-9H). It induces tendrils to develop into flower clusters (Fig. 2.25). This shortens the interval between crosses. Generation cycling can be further condensed by exposing seed to peroxide and gibberellin, prior to chilling (Ellis et al., 1983). The treatment shortens the normally prolonged cold treatment required for seed germination. In theory, the combination of precocious flowering and shortened seed dormancy could reduce the generation time from 3 to 4 years to about 8 months. Inducing dormant cuttings to flower is another means of accelerating the breeding process. With a greenhouse, crossing is not limited by seasonal conditions and could be conducted year-round. An alternative route for shortening the life cycle involves the mutant Vvgai1 allele. It confers a dwarf stature, diminished generation cycle, and continuous flowering (Chaı¨b et al., 2010, Fig. 2.26). The microvines grow well under 100% LED (light-emitting diode) illumination, permitting year-round growth, while reducing growth chamber energy and conditioning costs. Although not directly applicable to developing new varieties, the technique has potential in studying the genetic nature of particular traits. Clear indicators of gene expression are highly desirable; regrettably few agronomically important genetic traits have this property. Assessing properties, such as winemaking quality, can easily extend beyond the career of an individual breeder. The long gestation period for generating new scion varieties is further complicated by consumer conservatism and regulatory intransigence. In Europe, most appellations rigidly limit cultivar use, effectively legislating against innovation. This automatically places new cultivars under legal and

FIGURE 2.25 Induction of precocious flowering and fruiting in grapevine seedlings. (A) Inflorescence formation by a tendril of a 4week-old seedling of Cabernet Sauvignon. The seeding was treated daily for 7 days with 6-(benzylamino)-9-(2-tetrahydropyranyl)-9Hpurine (BPA). (B) Flower formation by four tendrils of a 6-week-old seedling of Muscat Hamburg after treatment with BPA. (C) Inflorescence formation in a 6-month-old seedling of Muscat Hamburg when sprayed with BPA and chlormequat when 3 months old. (D) Two bunches of ripe grape produced by an 8-month-old seedling of Muscat Hamburgdbunches developed from tendrils of a lateral shoot. From Mullins, M.G., 1982. Growth regulators and the genetic improvement of grapevines. In: Grape Wine Centennial Symposium Proceedings 1981. University of California, Davis, CA, pp. 143e147, reproduced by permission.

marketing constraints. Restrictions are even more severe when involving interspecies crosses, despite many hybrids possessing vinifera-like aromas (Becker, 1985). Correspondingly, incorporating new sources of disease and pest resistance into existing V. vinifera cultivars is essentially precluded using standard techniques. Although species other than V. vinifera are the primary sources of disease and pest resistance, untapped resistance may still exist within existing vinifera cultivars. For example, the offspring of a crossing between two Riesling clones (Arnsburger) showed resistance to Botrytis not apparent in either clonal progenitor (Becker and Konrad, 1990).

51

Grapevine improvement

Microvine

Normal grapevine

A

1-2 months

B

D 3-4 months

A

1 month

≥3 years

B

D 3-4 months

4-5 months

C

C

Total time of a generative cycle ≥3.5 years

1 month

1-2 months

Total time of a generative cycle < 1 years

FIGURE 2.26

Comparison of the duration of each stage within the reproductive cycle in a normal (nondwarf) grapevine and a microvine. AeB, from seed to seedling; BeC, from seedling to flowering; CeD, from flowering to berry ripening; DeA, treatment for breaking seed dormancy. From Chatbanyong, R., Torregrosa, L., 2015. A highly efficient embryo rescue protocol to recover a progeny from the microvine. Vitis 54, 41e46, reproduced with permission.

Despite North American Vitis species having been historically used as the primary source for new traits in grapevine breeding, Asian species also possess desirable traits. For example, V. amurensis is a potential source of mildew and Botrytis resistance (along with V. armata in the latter case). In addition, V. amurensis and V. piasezkii are valuable sources of cold hardiness, early maturity, and resistance to coulure. For cultivation in subtropical and tropical regions, sources of disease and environmental stress resistance include V. aestivalis and V. shuttleworthii (southeastern United States and Mexico), V. caribaea (Central America) (Jimenez and Ingalls, 1990), and V. davidii and V. pseudoreticulata (southern China) (Fengqin et al., 1990). Most interspecies hybridization has involved crossings within the subgenus Vitis. However, the production of partially fertile progeny from V. vinifera  V. rotundifolia is encouraging. Success is limited both by the inability of Vitis pollen to penetrate the style of muscadine grapes (Lu and Lamikanra, 1996) and by the aneuploidy caused by unbalanced chromosome pairing during meiosis (Viljoen and Spies, 1995). Backcrossing to one of the parental species (usually the pollen source) has been used in several crops to restore fertility to interspecies hybrids. Although crossing bunch with muscadine grapes is far from simple, V. rotundifolia is the principal source of Pierce’s disease resistance. It is also a source of resistance to downy and powdery mildew, anthracnose, black rot, and phylloxera; tolerance to root-knot and dagger nematodes; and enhanced heat insensitivity. Advances in vegetatively propagating V. vinifera  V. rotundifolia hybrids may enhance their commercial availability (Torregrosa and Bouquet, 1995) and adoption. Similar breeding

programs could also introduce V. vinifera-like winemaking properties into muscadine cultivars. Although most interspecies hybrids possess aromas distinct from those characteristic of V. vinifera cultivars, their aroma is not necessarily less enjoyable. Their distinctiveness could form the basis of regional wine styles. It was the regional distinctiveness of certain European wines that historically provided them with much of their appeal. There is no rational reason this could or should not apply to North America or elsewhere. Regrettably, tradition-bound critics and many connoisseurs are inordinately opposed to changedvinous neophobia. Acceptable flavors seem to have become petrified in their connection with a few “classic” cultivars. This situation severely limits the experience of new, and initially unbiased, wine consumers. It also restricts the spectrum of flavors available to all. The associated boredom may be one of the root causes of stagnated or diminished wine sales in some countries. Reticence to new sensory traits restricts the options associated with most grape-breeding programs. However, this appears to be based on experience, not genetics, meaning that alternative experiences have the potential to mitigate the existing Eurocentric bias (Perry et al., 2019). In addition, traditional views demand that progeny be given a new cultivar name. Thus, they lose the marketing advantage of their parents’ name(s). Most consumers are conservative, viewing wines with unknown names, or from unfamiliar countries, as suspect, if not inferior. This may also stem from new cultivars being relegated to regions where classic varieties do not grow profitably, or where increased yield or reduced production costs offset the loss of varietal name recognition.

52

2. Grape species and varieties

The increasing popularity of organically grown wines may enhance the acceptance and cultivation of new varieties. These cultivars have often been bred to require less pesticide and fertilizer use than existing cultivars.

Modern approaches to vine improvement Although standard breeding techniques can be successful, they usually involve decades of work, much effort, and considerable expense. There is also the issue of disrupting the allelic combination that gives each cultivar’s unique traits. In contrast, modern genetic procedures have the potential of introducing, upregulating, or inactivating selected genes, without disrupting other genetic traits. Consequently, the central commercial attributes of the cultivar remain unchanged. Many desirable traits, such as those regulating some aspects of disease resistance or aromatic synthesis, may be controlled by one or a few dominant traits. Examples are the R-type resistance genes, Run1, Ren1, and PdR1. They donate resistance to powdery mildew in Vitis rotundifolia and V. vinifera and to Pierce’s disease in V. arizonica/candicans, respectively. However, other traits, such as yield/quality ratio, cold hardiness, and overall flavor, are more likely to be under multigenic control and/or be recessive. Thus, they are neither easily identified nor localized, features requisite to facilitate transfer or modification. Advances in molecular genomics are facilitating gene sequencing, as well as expediting the identification of gene function (often by their inactivation). The location of gene linkage groups and correlation with function are also aided by comparing the Vitis genome map with those of other plants, where function has already been established. For example, whole chromosome segments may be densely populated with related genes. In addition, SSR and SNP segments are assisting MAS (Mackay et al., 2009). Their presence can also facilitate the identification of successfully transformed embryos or seedlings. Correspondingly, only potentially desirable progeny are propagated and studied, saving time, effort, and greenhouse space. The procedure can also streamline traditional breeding programs, by clearly identifying plants likely to possess the desired traits for subsequent crossing (Frisch and Melchinger, 2005). By directly assessing a gene’s presence (without having to detect it by its phenotypic expression), MAS avoids difficulties due to the masking of desirable, but recessive, traits by the presence of a dominant, but undesirable, allele. What may be missed, though, are epistatic or other allelic interactions. These may be crucial in gene expression, or present unsuspected and unsuitable pleiotropic influences. These may be partially predicted with techniques such as metabolomics.

Metabolomics embraces advances in analytical chemistry that permit the simultaneous assessment of multiple metabolites (Fernie and Schauer, 2009). It may associate phenotypic traits, notably those that affect berry chemistry, with the genetic characteristics of specific cultivars, the progeny of designed crosses, or changes induced by genetic transduction. The breeder can obtain a broad image of the effects of particular genes and their interaction within a specific genetic and environmental background. This is particularly valuable when attempting to understand the complexities of secondary metabolites and the flavor chemistry that affects a wine’s varietal aroma. Metabolomics is also useful in breeding, by enhancing the understanding of metabolic pathways leading to varietal uniqueness. More restricted in application is chlorophyll fluorescence imaging. It can measure the photosynthetic efficiency of individual plants (Baker and Rosenqvist, 2004). This, in turn, can be used as an objective measure of vine health (e.g., assessing resistance/tolerance to various biotic and abiotic stress factors). Additional technical advances have involved the isolation, amplification, and insertion of genes (Vivier and Pretorius, 2000; Pretorius and Høj, 2005). Insertion has typically used either the vector Agrobacterium (Rhizobium) or biolistics (particle bombardment) (ColovaTsolova et al., 2009). Viticultural examples have included incorporation of the GNA gene from Galanthus nivalis (snowdrop) and a protein-coat gene of the grapevine fanleaf virus (Mauro et al., 1995). These can enhance resistance to fanleaf degeneration, by either reducing feeding by viral-transmitting nematodes or preventing infection directly. Alternatively, the addition of a superoxide dismutase gene from Arabidopsis thaliana has been experimentally incorporated into scion varieties to improve cold resistance. Protocols for genetic transformation using Agrobacterium (Rhizobium) as the gene carrier are described in Agu¨ero et al. (2006) and Bouquet et al. (2006). Despite some success, the technique is imprecise (that is, as to where it inserts the genetic material), expensive, and time consuming, and often requires the incorporation of additional genes to permit efficient isolation of the rare transformants. Biolistics is another method used for gene insertion, whereby nano-sized particles of heavy metals (e.g., gold) are coated with DNA and injected into cells with a “gene gun.” In either case, essentially only large agricultural companies can afford the process, thereby limiting its use to crops that are highly remunerative. These (GMO) techniques also suffer from the perception that incorporating foreign DNA is inherently dangerous. Newer developments in genetics should help to negate some, if not all, of the former concerns about genetic engineering. The newer techniques no longer rely

Grapevine improvement

on transforming agents. In addition, they often do not involve the insertion of “foreign” DNA. They are also highly precise, and far less expensive to use (therefore applicable to crops such as grapevines). For example, markers associated with the CRISPR RNA segment can potentially locate a specific site for editing (Fig. 2.27). An associated DNA endonuclease (Cas9) produces double-stranded breaks in the DNA segment recognized by CRISPR. Subsequent DNA repair may involve processes such as nonhomologous knitting (end joining) or homologous recombination. Such repair mechanisms

FIGURE 2.27 RNA-guided DNA cleavage by Cas9. (A) In the native system, the Cas9 protein (light blue) is guided by a structure formed by a CRISPR RNA (crRNA, in black), which contains a 20-nt segment determining target specificity, and a trans-activating CRISPR RNA (tracrRNA, in red), which stabilizes the structure and activates Cas9 to cleave the target DNA (protospacer). The presence of a protospacer-adjacent motif (PAM, in yellow), i.e., an NGG (or less frequently NAG) sequence directly downstream of the target DNA, is a prerequisite for DNA cleavage by Cas9. Among the 20 RNA nucleotides determining target specificity, the so-called seed sequence of approximately 12 nt (in orange) upstream of the PAM is thought to be particularly important for the pairing between RNA and target DNA. (B) Cas9 can be reprogrammed to cleave DNA by a single guide RNA molecule (sgRNA, in green), a chimera generated by fusing the 30 end of the crRNA to the 50 end of the tracrRNA. From Bortesi, L., Fischer, R., 2015. The CRISPR/Cas9 system for plant genome editing and beyond. Biotechnol. Adv. 33, 41e52, reproduced with permission from Elsevier.

53

(editing) may result in the excision of a gene segment (eliminating or changing the gene’s function), facilitate the insertion of a donor DNA segment (adding new functionality), or upregulate (increase) the gene’s transcription rate (see Liu et al., 2016). Correspondingly, the procedure can be particularly valuable in MAS and the potential pyramiding (inclusion) of several useful attributes (e.g., donating stable disease resistance). Other related procedures include ZFNs and TALENs. While each has its advantages, these two are more time consuming and expensive to use. As none otherwise modifies the cultivar’s traits, there should be no need to change the cultivar name, other than to note it as a new clonal variant. Despite the potential of these procedures, their application to grapevine breeding will be partially dependent on solving issues associated with tissue culture (Saporta et al., 2016). Their use requires the use of cell culture, followed by plant regeneration from callus tissue (Vidal et al., 2009). For many cultivars this remains challenging. Nonetheless, targeted mutagenesis using the CRISPR/Cas9 procedure has been successfully demonstrated (e.g., Ren et al., 2016; Wang et al., 2018). Several stages in plantlet generation from callus tissue are shown in Plate 2.5. Another issue, often associated with vines propagated from tissue culture, is phenotypic variability. Embryonic development within callus tissue appears to reset the biological age back to a juvenile state. Such atypical phenotypic expression typically diminishes as the vine ages, or with repeated vegetative propagation (Mullins, 1990). Juvenility is reduced when terminal buds, derived from long canes (40th node onward) are grafted onto a desirable rootstock (Grenan, 1994). Why juvenile or other phenotypic modifications are expressed in vines derived from tissue culture is unknown. However, it is suspected to be the result of epigenetic modifications induced in parenchyma during callus dedifferentiation and subsequent embryonic development (Schellenbaum et al., 2008).

Clonal selection Although modern genetic approaches possess the greatest potential for significant vine improvement, clonal selection is an older, less precise alternative, without modifying vital varietal characteristics. However, improvements are limited largely to those that already exist. This usually involves gene mutations (or epigenetic modifications) that have accumulated since the cultivar’s origin. The most visually obvious are those that affect grape pigmentation and vine morphology. Nevertheless, most clonal mutations have more subtle effects, such as those affecting aromatic character or that influence yield, disease resistance, or climatic sensitivity (Fig. 2.28).

54

2. Grape species and varieties

(A)

(B)

(C)

(D)

(E)

(G)

(F)

(H)

(I)

PLATE 2.5 Embryogenesis and plant regeneration. (A and B) Effect of activated charcoal on embryogenesis: callus cultured on ½ MS (A) and on ½ MSAC (B). (C and D) Calli cultured on ½ MSAC, at different stages of development of somatic embryos. (E) Very soft nonembryogenic calli (type II). (F) Different stages of germination of the somatic embryos on a medium containing IAA, GA, and AC. (G) Plant developed on halfstrength MA in test tubes. (H and I) Acclimatization and culture under greenhouse conditions. AC ¼ activated charcoal; GA3 ¼ gibberellic acid; IAA ¼ indole-3-acetic acid; MS ¼ Murashige and Skoog medium; MSAC ¼ Murashige and Skoog supplemented with 0.25% activated charcoal. From Lo´pez-Pe´rez, A.J., Carren˜o, J., Martı´nez-Cutillas, A., Dabauza, M., 2005. High embryogenic ability and plant regeneration of table grapevine cultivars (Vitis vinifera L.) induced by activated charcoal. Vitis 44, 79e85, reproduced by permission.

55

Grapevine improvement

22.0 Total soluble solids (°Brix)

21.8 21.6 21.4 21.2 21.0 20.8 20.6 20.4 60

70

80

90

100

110 120 130 Yield (kg/a)

140

150

160

170

180

30 Compact bunches Upright growing shoots Loose clusters Small berries Clone mariafeld

Bunch rot (%)

25 20 15 10 5 0 9.0

9.5

10.0

10.5

11.0 11.5 12.0 Titratable acidity (g/L)

12.5

13.0

13.5

14.0

FIGURE 2.28 Correlation between yield and total soluble solids (top) and between titratable acidity and Botrytis bunch rot (bottom) of 44 Pinot noir clones. Data are means of the 1993e98 harvests (n þ 6). From Forneck, A., Benjak, A., Ru¨hl, E., 2009. Grapevine (Vitis spp.): example of clonal reproduction in agricultural important plants. In: Scho¨n, I., Martens, K., Van Dijk, P. (Eds.), Lost Sex: The Evolutionary Biology of Parthenogenesis, pp. 581e598. Reproduced with the kind permission Springer ScienceþBusiness Media B.V., Dordrecht, The Netherlands.

Another source of genetic variation, usually undesirable, results from infection by endogenous pathogens. One of the principal functions of clonal selection is the elimination of virus and viroid agents. They typically limit the potential of the cultivar (Credi and Babini, 1997). In contrast, some fungal or bacterial endophytes (essentially symbiotic inhabitants) may be beneficial (e.g., salt tolerance) (Rodriguez and Redman, 2008). Clonal selection refers to a series of procedures designed to identify, isolate, propagate, and provide premium vinestock to grape growers. Selection usually goes through several steps (Fig. 2.29), where cuttings are cleared of systemic pathogens, multiplied, and repeatedly selected for their viti- and vinicultural traits. Because clonal selection depends on genetic differences inherent within the cultivar, it is particularly useful with older varieties. Depending on the age and genetic stability

of the cultivar, it may possess few to many tissue-specific mutations. This is especially true with monozygotic cultivars (being derived from a single seed). Polyclonal varieties (derived from a collection of related individuals) are usually more variable, as they possess not only mutations that have accumulated in the individual lines, but also genetic differences inherent from the origins of the various lines. Because viruses and other systemic pathogenic agents invade most somatic tissues, selection alone may be unable to eliminate these agents. Thermotherapy has been particularly valuable in this regard. One version involves exposing dormant cuttings to high temperatures (w38 C) for several weeks. Alternatively, excision and micropropagation of vines from bud tissue, unfertilized ovules, stigmas, styles, or anthers (Carimi et al., 2005) may eliminate viruses that do not invade meristematic tissue. Nevertheless, isolating clones free

56

2. Grape species and varieties

FIGURE 2.29 Schedule of clonal selection at Geisenheim. From Schmid, J., Ries, R., Ru¨hl, E.H., 1995. Aims and achievements of clonal selection at Geisenheim. In: Rantz, J.M. (Ed.), Proceedings of the International Symposium on Clonal Selection. American Society of Enology and Viticulture, Davis, CA, pp. 70e73, reproduced by permission.

of all known systemic agents (bacterial, phytoplasmal, viral, and viroidal) can be onerous. In addition, reinfection often occurs where the agent is well established in a

region. Another, more intractable, problem is that some systemic agents may not provoke readily recognizable disease symptoms. Thus, their presence may go

57

Grapevine improvement

expressed as genetic differences between the vine’s outer and inner tissues (Fig. 2.30). The outer (TI) tunica gives rise to the epidermis and divides only perpendicularly to the surface. The inner tissues give rise to a more or less defined inner tunica (TII) and corpus (C) tissues. Somatic mutations generally occur and remain in the layer in which they originate, generating a periclinal chimera (see Table 2.5 for examples). As the bud grows and differentiates into the various tissues and organs of the vine, these genetic differences are propagated throughout the shoot system. Chimeric variations presumably also occur in the root system, but are little studied (being predominantly out of sight). Because adventitious roots from canes originate from the interfascicular cambium, epidermal chimeric traits are not found in the roots. In addition, epidermal chimeric traits are not transmitted sexually (via seed). Only internal tissues are so positioned as to potentially generate gametes. This situation can change, though, if the normal (tissue layer) distinction is disrupted, as when tertiary buds develop due to death of the primary and secondary buds (e.g., Marcotrigiano, 2000). Tissue layer disruption also results when cells are propagated in liquid culture. On embryogenesis, former epidermal cells may be located internally and potentially give rise to gametes.

unnoticed and the vine remain a reservoir of infection for sensitive cultivars. Detection may be possible only with immunological or nucleic acid analysis. For rootstock varieties, clonal selection is often limited to certifying them as disease free. Due to recent origin, insufficient time has elapsed for the accumulation of useful somatic mutations. In addition to basic traits, genetic stability and phenotypic consistency are essential. Correspondingly, elimination of phenotypic variability within a clone is an important goal. Regrettably, detection of such instability is not necessarily simple, expression often depending on local environmental conditions. For example, the Muscat character of some Chardonnay clones can vary markedly from year to year (Versini et al., 1992). To compensate for this, clonal selection trials usually occur at a range of sites over many years. Nonetheless, prudent vineyard owners conduct their own, onsite, comparative assessments prior to making major planting commitments. Growers also need to be aware that clonal traits may require adjustment in viticultural practice to achieve the benefits desired. Clonal instability is occasionally due to chimeric mutationsdgenetic modifications found in only a particular meristematic layer (Franks et al., 2002). This may be

(A)

TI

a

b

c

TII

C b

(B) a

c

FIGURE 2.30 Chimeric structures and their origins in the stem apex. (A). Apical meristems is composed of an outer tunica (TI) layer and an inner tunica (TII) and corpus (C) layers. The outer tunica divides perpendicular to the surface (anticlinal plane), whereas the inner tissues may divide anticlinally, or in both anticlinal and periclinal (parallel) planes. (B) Somatic mutations (shown in darker coloration) arising in the outer tunica typically remain in the epidermis (a), whereas mutations arising in the inner layers may give rise to both anticlinal (b) and periclinal (c) chimeras. Swellings in the apex are primordial leaves.

58

2. Grape species and varieties

TABLE 2.5

Microsatellite alleles detected in leaf, wood, and root tissue. Alleles

Grape type

Clone

Marker

Leaf

Wood

Root

Layer in which mutation occurs

Pinot noir

CTPS 115

VMCNg2fl2

a, b, X

a, b

a, b

TI

CTPS 386

VMCNg2fl2

a, b, Y

a, b

a, b

TI

VMClg3.2

a, b, X

b, X

b, X

TII

VMCle8

a, b, X

b, X

b, X

TII

16

VMC2fl2

a, b, X

a, b

a, b

TI

S17

VMCNglh7

a, b, X

a, b

a, b

TI

CTPS 96

VMC5g7

a, b, X

a, b

a, b

TI

VMC6c 10

b, X

b, X

b, X

TI, TII

Chardonnay

a and b indicate the two standard alleles, whereas X and Y are variant alleles. From Riaz, S., Tenscher, A.C., Graziani, R., Krivanek, A.F., Rammine, D.W., Walker, M.A. 2009. Using marker-assisted selection to breed Pierce’s disease-resistant grapes. Am. J. Enol. Vitic. 60, 199e207, reproduced by permission.

Although chimeras can induce clonal instability, most are stable. Examples are the color variants of Pinot noir (Pinot blanc and Pinot gris) (Pelsy et al., 2015), the bronze and white variants of Cabernet Sauvignon

(Malian and Shalistin) (Plate 2.6), and the hairy leaf phenotype of Pinot Meunier (Boss and Thomas, 2002). In pale-colored chimeric clones, anthocyanin production tends to be limited to the epidermis (e.g., Carbonell-

PLATE 2.6 Photographs of the colored sports of Cabernet Sauvignon. (A) Grape clusters of the clones Malian and Shalistan. (B) The original mutated cane, bearing bronze berries on the parental vine. (C) Malian vine with white bunch. (D) White sector on a Malian berry. From Walker, A.R., Lee, E., Robinson, S.P., 2006. Two new grape cultivars, bud sports of Cabernet Sauvignon bearing pale-coloured berries, are the result of deletion of two regulatory genes of the berry colour locus. Plant Mol. Biol. 62, 623e635, reproduced by permission.

59

Grapevine improvement

Bejerano et al., 2017). White chimeric clones produce anthocyanins in neither the epidermis nor the hypodermis. Plate 2.7 illustrates an example of an unstable epidermal chimera. The origin of this instability is unknown, but may be due to unstable gene methylation. In addition to eliminating systemic pathogens and phenotypic stability, improved crop yield and grape quality are central to all clonal selection procedures. Improved yield, if associated with increased vegetative vigor, can result in reduced grape quality. This undesirable correlation, if present, may potentially be avoided by adjusting viticultural practice (e.g., a more open canopy, basal leaf removal, increased planting density). However, increased yield is not a priori correlated with reduced quality (e.g., sugar content) (Fig. 2.31). In a study of Riesling clones, the clone that showed both the highest yield and the highest  Brix values was low in vegetative vigor (Scho¨fflinger and Stellmach, 1996). Such clones have the potential for combining the economic and environmental benefits of reduced fertilizer input with maximized yield and quality. In clonal selection, grape quality is always paramount. Regrettably, there are no universally accepted benchmarks. Brix and acidity values are standard indicators, but are by themselves insufficient. Measures of aroma potential are still insufficiently precise (depending on the cultivar and wine style desired), and anthocyanin assessment is complex and inadequate as an indicator of wine color or stability. There is also no clear or simple correlation between different quality indices. Typically,

PLATE 2.7

Grapes showing partial to complete chimeras in their outer pigmented tissues. Photo courtesy of Reynonds, A., Brock University.

Sugar content 470 383 Sugar content average 174

300 471 525

524

585

381 99 100

Yield average

Yield

FIGURE 2.31 Results of 10 years of comparative experiments on the behavior of clones of Syrah. Each clone position for yield (abscissa) and for sugar content (ordinate) is indicated in comparison with the average of all clones. From Boidron, R., 1995. Clonal selection in France. Methods, organization, and use. In: Rantz, J.M. (Ed.), Proceedings of the International Symposium on Clonal Selection. American Society of Enology and Viticulture Davis, CA, pp. 1e7, reproduced by permission.

microvinification tests must be conducted over several years from vines grown at different locations. In addition, the wines need to be aged for various periods, and assessed by an experienced panel of wine judges (McCarthy and Ewart, 1988). Regrettably, “quality” as perceived by wine judges and winemakers does not necessarily equate with that as perceived by consumers. These demands make the establishment of significant differences between clones long, complex, and costly. Nonetheless, done adequately, the exercise can be worth the effort. Clones can differ in yield by as much as 60%. Other features frequently integrated into clonal selection involve factors such as unique varietal aromas (e.g., Chardonnay clones 77 and 809 with a Muscat-like nuance) (Boidron, 1995); differences in winemaking potential (e.g., Gamay 222 vs. 509, used respectively for nouveau vs. cru beaujolais) (Boidron, 1995), wine style (e.g., Pinot fin for red burgundy vs. Pinot droit for champagne) (Bernard and Leguay, 1985), and growth habit (e.g., erect for mechanical harvesting); preferences in berry shape and size (affecting flavor and color potential) (Watson et al., 1988); and factors such as berry cluster morphology or epidermal wax features (affecting resistance to pathogens or physiologic disorders). One of the great successes in clonal selection, in addition to the elimination of endemic disease-causing agents, has been providing growers with an increasing palate of subtle flavor differences. Clonal uniformity also enhances a grower’s potential to produce fruit of more consistent ripeness. The benefits of uniformity are based on the belief that wine quality is enhanced by grape homogeneity. Although generally viewed as desirable, uniformity could limit a wine’s aromatic complexity, or exaggerate environmental sensitivity. Thus, the phenotypic plasticity associated with planting several clones may be a judicious form of crop insurance (Huba´ckova and Huba´cek,

60

2. Grape species and varieties

1984). Boidron (1995) recommends at least two clones for small vineyards, and up to five or six for large vineyards. In some situations, where soil-borne viral diseases (transmitted by parasitic nematodes) are a problem, preplanting soil fumigation may be necessary. Although clonal selection has many benefits, varietal variation must not be eroded in the process. Correspondingly, less desirable lines are often retained as a library of diversity, providing a future source of potentially useful variation.

Somaclonal selection and mutation The elimination of viral infection, in association with clonal selection, has made significant improvements in the planting material available to grape growers. However, further significant advances will probably depend on the generation or incorporation of new genetic variations by genetic modification procedures such as CRISPR. It is likely to replace somaclonal selection, a technique that involves placing tissue culture under conditions that can be adjusted to favor or permit only the growth of cell lines with desired attributes. For example, lines possessing tolerance for salinity or fungal toxins (Soulie et al., 1993) may be isolated by exposing cells to these conditions in culture media. Regrettably, the tolerance selected for under culture conditions may not be expressed in regenerated whole plants (Lebrun et al., 1985). This can result when selection involves only cells that have temporarily adapted to the culture conditions used, but have not been genetically modified. Selective culture techniques have also been used to isolate rare transformed cells from the majority of nontransformed cells (Mauro et al., 1995; Kikkert et al., 1996). The liquid tissue culture technique has also been used to disrupt the distinction between TI and TII meristematic cells (Skene and Barlass, 1983). As a result, callus tissue can be derived from either cell line and potentially generate embryos. This permits the isolation of plantlets that possess, or lack, somatic mutations found in one or the other of the cell lines. For example, it was used to isolate a dwarf strain of Meunier (Boss and Thomas, 2002). It also permits chimeric traits located only in the TI layer to be transmitted sexually (gametes are derived only from cells beneath the epidermis). Conversely, somaclonal selection could be used to eliminate undesirable chimeric traits.

Grapevine cultivars With named cultivars approaching 15,000 (including synonyms), a comprehensive system of cultivar classification would be useful. Regrettably, no such system exists. In some countries, there have been attempts to rationalize local cultivars into related groups (Bisson,

1989). Most ecogeographic associations have been based on ampelographic attributes. Because of the localized distribution of each group, they may be related. This view has received support from microsatellite DNA (SSR) analysis (see above). For example, most French cultivars appear to be distinct and partially related to wild accessions. As well, Armenian, Georgian, and Turkish varieties appear to be isolated genetically (Vouillamoz et al., 2006). Nevertheless, the same study found evidence that some western European cultivars (Chasselas, Nebbiolo, Pinot noir, and Syrah), or their ancestors, appear to possess traits characteristic of Georgian cultivars. Cultivar nomenclature is further complicated by the absence of an international agreement on cultivar designation. Cultivars considered identical ampelographically may be distinct genomically. In addition, some ampelographic attributes are more stable than others (e.g., vein angles vs. vein length) (see Bodor et al. 2013). The situation might be put on a more solid foundation if cultivars were associated with type specimens, as are most species names. In the future, both cultivar and clonal identification may be tied to their genomic (cultivar) and epigenomic (clonal) signatures (Meneghetti et al., 2012). For the present, however, ampelographic procedures (e.g., Galet, 1979) are likely to remain the primary means by which cultivars are identified, despite the plasticity of these traits. Ampelography is based on vegetative characteristics, such as leaf shape, veinal branching patterns, and pubescence (hairiness). It is hoped that the development of a computer-aided digitizing system for determining ampelographic measurements will simplify grapevine identification for the nonampelographer (Alessandri et al., 1996; Bodor et al., 2012). On a broader scale, cultivars are grouped according to their species or interspecific hybrid origin. Most commercial varieties are pure V. vinifera accessions. FrenchAmerican hybrids constitute the next largest group. They were derived from crosses between V. vinifera and one or more of the following: V. riparia, V. rupestris, and V. aestivalis. Early American cultivars are either selections from indigenous grapevines or hybrids between them and V. vinifera. Interspecific cultivars generally refer to modern (20th century and later) crosses between V. vinifera and species such as V. amurensis, V. riparia, V. armata, and V. rotundifolia. In most instances, their parentage is known.

Vitis vinifera cultivars Because of the huge number of named cultivars, only a few of the better-known or regionally significant are briefly noted here. Although often world famous, they may not constitute the principal varieties grown, even in the country of origin. Productivity is usually lower

Grapevine cultivars

and cultivation more demanding. Their reputation comes from their wines possessing distinctive varietal aromas, frequently combined with long-aging potential. In favorable locations, their winemaking properties can command prices that more than compensate for their reduced yield and increased production costs. Due to shifts in consumer interest/demand, proportional plantings have changed considerably (Fig. 2.32). Many of the varieties listed below are French cultivars. This presumably is a geographic or historic accident, with many worthy cultivars being little recognized outside their homelands. The cool climate of the best vineyards in France favored the retention of subtle fragrances, complete fermentation, and longaging potential. The former position of France as a major political and cultural power, and its proximity to rich connoisseur-conscious countries, encouraged the selection and development of the best local cultivars and wines. The long and frequent contacts between England and France, and the global expansion of British influence, fostered a preference for, and the dispersal of, French and a few German cultivars throughout much of the world. There is no doubt that these cultivars produce excellent wines. Regrettably, fine cultivars from southern and eastern Europe did not, and still do not, receive the recognition they probably deserve and correspondingly the geographic dispersal. Examples of several important regional, but less well known, varieties with distinctive aromas are Arinto (white, w), Ramisco (red, r), and Touriga Nacional (r) from Portugal; Corvina (r), Dolcetto (r), Negroamaro (r), Fiano (w), Garganega (w), and Torbato (w) from Italy; Rhoditis (w or rose´) from Greece; Furmint (w) from Hungary; and Malvasia (w), Parellada (w), Viura (w), and Graciano (r) from Spain. Red cultivars Barbera is the most widely cultivated variety in Piedmont. It is also important in the rest of Italy, being the third most cultivated variety, after Sangiovese and Trebbiano Toscano. Outside Italy, it is principally planted in California and Argentina. It is moderately high yielding, producing fruit with an intense color, high in acidity, but with moderate tannin content. The clusters possess long green stalks, making mechanical and manual picking easy. Cultivation is also uncomplicated, due to its adaptability to different soils. Barbera can make a varietally distinctive, fruity wine, but is commonly blended with other cultivars to add acidity and a fruity character to wines of high pH. Regarding disease susceptibility, Barbera is sensitive to grapevine leafroll. Some clones may be severely affected by bunch rot. Cabernet franc has fame primarily as one of the parents of its more illustrious offspring, Cabernet Sauvignon

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and Merlot. It may be the principal source of the generally undesirable bell pepper (methoxypyrazine) flavors of both progenies. Only rarely is it appreciated as a varietal wine. This may result from its comparative tolerance of cooler climates than its offspring. Cabernet Sauvignon is one of the best-known red cultivars, due to both its association with one of Europe’s bestknown red wines (bordeaux) and its ability to produce excellent wines in many parts of the world. Under optimal conditions, it produces a fragrant wine possessing a black currant (or violet) fragrance. Under less favorable conditions, it expresses a bell pepper aroma. The berries are small, acidic, and seedy and possess a darkly pigmented, tough skin. The cultivar is frequently cane pruned to accentuate production and provide better sun exposure for its upright shoots. The easy separation of the berries from the cluster facilitates mechanical harvesting. The cultivar is highly susceptible to several fungal diseases, notably Eutypa and esca wood decays, powdery mildew, and phomopsis. DNA fingerprinting techniques indicate that Cabernet Sauvignon is probably the progeny of a Cabernet franc  Sauvignon blanc cross (Bowers and Meredith, 1997). In Bordeaux, and increasingly in other regions, wines made from Cabernet Sauvignon are blended with wines produced from other, related, cultivars, notably Cabernet franc and Merlot. The latter moderates the tannin content and accelerates maturation. Ruby Cabernet is a Davis cross between Carignan and Cabernet Sauvignon. It possesses a Cabernet aroma, but grows better in hot climates than Cabernet Sauvignon. Dolcetto is an Italian cultivar almost exclusively grown in Piedmont. Nevertheless, it still ranks eighth in overall hectarage in Italy. It has rarely been tried outside Italy. In growth, Dolcetto possesses comparatively weak vigor, producing small- to medium-sized clusters, with small rounded berries. It produces a wine usually light, but bright, in color, medium bodied, and with a mild distinctive aroma. Gamay noir a` jus blanc is the primary (white-juiced) Gamay cultivar. Its notoriety rests on the popularity of Beaujolais wines, with half its hectarage occurring in Beaujolais. It is little cultivated outside France. Crushed and fermented by standard procedures, Gamay produces a light red wine with few distinctive attributes. This is often accredited to its high productivity. When processed by carbonic maceration, though, it yields a distinctly fruity wine. Most of these features come from the grape fermentative process that precedes alcoholic fermentation (see Chapter 9). Gamay produces medium-sized fruit, with a tough skin. It is sensitive to most fungal grapevine diseases. The Gamay Beaujolais and Napa Gamay grown in California are not directly related to Gamay noir. They are considered clones of Pinot noir and Valdiguie´, respectively.

2000

1990 2000

0

Muscat of Alexandria

1990

Colombard Muscat Blanc a Petits.. Cereza

Airen Garnacha Tinta Rkatsiteli Sultaniye Trebbiano Toscano Mazuelo Merlot Cabernet Sauvignon Monastrell Bobal Sangiovese Catarratto Bianco Chardonnay Criolla Grande Barbera Cayetana Blanca Muscat of Alexandria Cinsaut Chenin Blanc Aligote Riesling Palomino Fino Pedro Ximenez Tempranillo Sauvignon Blanc Macabeo Cereza Malvasia Bianca di Candia Tribidrag Pinot Noir Negroamaro Cabernet Franc Muller Thurgau Colombard Syrah

350000

Macabeo Cot Cayetana Blanca Alicante Henri Bouschet Aligote Cinsaut Chenin Blanc Montepulciano Catarratto Bianco Tribidrag Gamay Noir Isabella

Cabernet Sauvignon Merlot Airen Tempranillo Chardonnay Syrah Garnacha Tinta Sauvignon Blanc Trebbiano Toscano Pinot Noir Mazuelo Bobal Sangiovese Monastrell Grasevina Rkatsiteli Cabernet Franc Riesling Pinot Gris

62 2. Grape species and varieties

500000

(A) Top varieties in 1990, compared with 2000 and 2010

450000

400000

300000 2010

250000

200000

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(B) Top varieties in 2010, compared with 1990 and 2000

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FIGURE 2.32 World’s top 35 grape varieties in 1990, 2000, and 2010 (in hectares). (A) Top varieties in 1990, compared with 2000 and 2010. (B) Top varieties in 2010, compared with 1990 and 2000. From Anderson, K., (assisted by Aayal, N.R.), 2013. Which Winegrape Varieties Are Grown Where? A Global Empirical Picture. University of Adelaide Press, Adelaide, Australia. www.adelaide.edu.au/wine-econ/databases/winegrapes-front-1213.pdf, reproduced by permission.

Grapevine cultivars

Garnacha (Grenache, Cannonau) is a widely planted cultivar in Spain, southern France, southern Italy, Sardinia, Sicily, California, and Australia. It exists in several phenotypic color variants (white and gray), in addition to the standard red clones. The vine has an upright growth habit, suitable for head training and spur pruning. It is well adapted to hot, dry conditions, but tends to be excessively productive with irrigation. Clusters are broad and compact, varying from pink to red, depending on the crop load. The cultivar is not well adapted to mechanical harvesting. By itself, it is often used to make rose´ or fortified wines. It may be blended with other varieties to speed maturation. Although sensitive to powdery mildew, bunch rot, and coulure, Garnacha is relatively resistant to downy mildew. Graciano is principally cultivated in Rioja and Navarra, but little grown outside northern Spain. Although relatively low in yield, it is comparatively drought tolerant and resistant to most fungal diseases. Regrettably, the peduncle (fruit stalk) is relatively woody, making the cultivar ill suited to mechanical harvesting. The fruit is one of the most aromatic of Spanish varieties and possesses good acidity. It is an integral component of many of the best northern Spanish wines. Merlot has the advantage of accepting cooler, moister soils than Cabernet Sauvignon, but is more susceptible to coulure (a physiological disruption causing excessive and early flower and/or fruit dehiscence). It is related to Cabernet Sauvignon, also having Cabernet franc as one of its parents. The other parent is a (now obscure) variety, Magdeleine Noire des Charentes (Boursiquot et al., 2009). The tendency of Merlot to mature more quickly has made it a popular substitute for Cabernet Sauvignon, with most of its flavor characteristics. Nebbiolo, grown principally in northwestern Italy, is generally acknowledged as producing one of the most highly regarded red wines. With traditional vinification, it produces a wine high in tannin and acid content, requiring years to mellow. Nonetheless, the color has a tendency to oxidize rapidly. Common varietal descriptors include tar, violets, and truffles. Nebbiolo yields well only when cane pruned (due to low basal fertility), but adapts well to a wide range of soil pHs and types. Surprisingly, based on its reputation, it has been cultivated little outside of northern Italy. This may be due to the variety’s weak skin and juicy fruit that make it ill suited to mechanical harvesting. It is susceptible to powdery mildew, but is relatively resistant to bunch rot. Pinot noir is the famous red grape of Burgundy. It is often considered to have arisen in southern Gaul about the time of the Romans. It possesses several distinctively feral characteristics. It is acknowledged to be one of the most environmentally sensitive varieties, and consists of a large number of distinctive clones. Fruit color mutants have given rise to Pinot gris and Pinot blanc. These changes arose from color mutants in one or both T layers

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of the fruit. Another variant is Pinot Meunier, commonly grown in Champagne. More prostrate, lower-yielding clones produce more flavorful wines, whereas upright, higher-yielding clones are more suited to producing rose´ and sparkling wines. Pinot noir produces an aromatically distinctive wine under optimal conditions; otherwise, it produces disappointingly nondistinctive wines. Various authors have proposed terms for its aroma, such as beets, peppermint, or cherries, but none seems sufficiently appropriate or adequate. The cultivar produces modest-sized clusters of small- to medium-sized fruit with large seeds. If the clusters are compact, it is particularly sensitive to bunch rot. Crossed with Cinsaut, it has produced one of the most distinctive of South African cultivars, Pinotage. The Californian cultivar Gamay Beaujolais is a clone of Pinot noir (Bowers et al., 1993). Sangiovese is probably an ancient cultivar, composed of an extensive number of distinctive clones. It is grown extensively throughout central Italy. It is best known for generating the light- to full-bodied wines from Chianti, and produces many of the finest red wines in Italy. Sangiovese is also grown under local synonyms, such as Brunello and Prugnolo. Sangiovese is relatively vigorous, but variable in yield. Its clusters are small- to medium-sized, possessing oval berries. The fruit is compatible with mechanical harvesting. Under optimal conditions, it yields a wine possessing an aroma reminiscent of cherries, violets, and licorice. Sangiovese has achieved little of the international recognition it deserves, the regrettable fate shared by most of its Italian brethren. Sangiovese is particularly sensitive to both bunch rot and powdery mildew. It appears to be the progeny of two ancient, southern Italian cultivars, Ciliegiolo and Calabrese di Montenuova (Vouillamoz et al., 2007). Syrah (syn. Shiraz) is the most renowned French cultivar from the Rhoˆne Valley. Lower-yielding strains produce a deep red, tannic wine, with long-aging potential. Until recently, Syrah was hardly grown outside France, except in Australia, where its fame has grown extensively. The variety is a vigorous grower with a spreading growth habit. The fruit clusters are elongated with small round to oval berries. It yields deep-colored, flavorful wines with aspects reminiscent of violets, raspberries, and currants, with a peppery finish. Syrah is particularly prone to drought, bunch rot, and infestation by grape berry moths. Most vines labeled Petit Sirah in California are misnamed clones of the Rhoˆne cultivar Durif (Meredith et al., 1999). The misidentification may not be too surprising as Petit Sirah has been used to refer to at least four different cultivars in France (Galet, 1990). Tempranillo (Ull de Llebre) is possibly the finest, at least the most renowned, Spanish red grape variety. Under favorable conditions, it yields a fine, subtle wine that ages well. In addition, it can produce delicate nouveaustyle wines. It is the most important red cultivar in Rioja

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2. Grape species and varieties

(occupying about 33,000 ha), and is grown extensively throughout much of Spain. Outside Spain, it is primarily grown in Argentina. In California, it usually goes under the synonym Valdepen˜as. Tempranillo generates an aroma distinguished by a complex, berry jam fragrance, with nuances of citrus and incense. Tempranillo produces midsized, thick-skinned fruit that is subject to both powdery and downy mildews. The vine is comparatively vigorous and produces upright shoots. It is probably the progeny of Albillo Mayor, a cultivar still well known in central Spain, and a (now obscure) cultivar, Benedicto (Iba´n˜ez et al., 2012). Touriga Nacional is one of the preeminent Portuguese grape varieties. It is grown predominantly in the Upper Douro, where it is used extensively in the production of port. It is also cultivated in other regions to produce red table wines. The wine is deep in color and richly flavored. The vine is fairly vigorous, with a trailing growth habit, but low in yield. Its clusters are of small to medium size, containing small berries. Zinfandel is extensively grown in California. It is synonymous with the Italian variety Primitivo (Bowers et al., 1993) and its Croatian equivalent, Crljenak kastelanski (Fanizza et al., 2005). Zinfandel is used to produce a wide range of wines, from ports to light blush wines. In rose´ versions, it shows a raspberry fragrance, whereas full-bodied red wines possess rich berry flavors. Some of the difficulties with Zinfandel are the uneven manner with which the fruit ripens and its tendency to produce a second crop later in the season. Both properties complicate harvesting fruit of uniform maturity. White cultivars Chardonnay is the most widely grown white French cultivar. This presumably derives from its appealing fruit fragrance and tolerance of diverse climatic conditions. Not only does it produce fine table wines, but it also yields one of the finest sparkling wines (champagne). The fruit is comparatively small, is round, and forms compact clusters. Under optimal conditions, the wine develops aspects reminiscent of various fruits, including apple, peach, and melon. The vine and fruit are predisposed to powdery mildew and bunch rot. Chenin blanc comes from the central Loire Valley of France. There it yields fine dry and sweet table wines, as well as sparkling wines. Chenin blanc is also grown extensively in Australia, California, and South Africa (in the last, occasionally under the synonym Steen). Fine examples of its wine often exhibit a delicate fragrance loosely said to resemble guava fruit or camellia blossoms. The fruit is tough skinned and of medium size. It is especially susceptible to both downy and powdery mildews, bunch rot, and grape berry moths. Ehrenfelser is one of the best, newer German varieties, being derived from a Riesling  Silvaner cross. It

has many of the characteristics of its Riesling parent, such as its flavor, acidity, disease resistance, and cold hardiness. In addition, it has the advantage of ripening somewhat earlier. Ehrenfelser is still largely cultivated in Germany, but is being grown with considerable success in Canada. Mu¨ller-Thurgau is possibly the most well known modern V. vinifera cultivar, once constituting nearly 30% of German hectarage. It was developed by H. Mu¨ller-Thurgau in 1882, supposedly from a crossing between Riesling and Silvaner. Nevertheless, microsatellite analysis has indicated that Mu¨ller-Thurgau is a crossing between Riesling and Madeleine Royale (Vouillamoz and Arnold, 2010). The latter variety itself appears to be derived from a cross between Pinot noir and Schiava Grossa. The first commercial plantings of Mu¨llerThurgau began in 1903. It is now cultivated in many cool regions of Europe and was formerly in New Zealand. Its mild acidity and subtle fruity fragrance are ideal for producing light wines. Mu¨ller-Thurgau is a high-yielding cultivar that often produces lateral (side) clusters of midsize fruit. It grows best in rich porous soils. The fruit is subject to both powdery and downy mildews and bunch rot. Muscat blanc is one of many related Muscat varieties grown throughout the world. Their aroma is so marked and distinctive that it is the only cultivar whose aroma is described in terms of itself, muscaty. Because of the intense flavor, slight bitterness (due to the high level of flavonoid extraction during maceration), and tendency to oxidize, Muscat grapes have most commonly been used in the production of sweet wines. Muscat grapes are also characterized by the presence of high levels of soluble proteins. Consequently, special precautions must be taken to avoid haze formation. The new Muscat cultivar Symphony is less bitter, and its lower susceptibility to oxidation gives it better aging ability. Muscat blanc (Moscato bianco in Italy) is the primary variety used in the flourishing sparkling wine industry in Asti. Other named Muscat varieties include the Orange Muscat (Muscato Fiori d’Arinico in Italy), Muscat of Alexandria, Muscat Ottonel, and the darkly pigmented Black Muscat (Muscat Hamburg). Parellada is a wonderful variety distinctive to the Catalonian region of Spain. It produces an aroma that is apple- to citrus-like in character, occasionally having been reported to show hints of licorice or cinnamon. Pinot gris and Pinot blanc (respectively Rula¨nder and Weissburgunder in Germany and Pinot grigio and Pinot bianco in Italy) are color mutants of Pinot noir. Despite being clones of Pinot noir (for details of their genetic differences see Carrier et al., 2012), their wines are unlike Pinot noir in flavor as well as color, justifying their own varietal names. Both are cultivated throughout cool climatic regions in Europe for the production of dry,

Grapevine cultivars

botrytized, and sparkling wines. In recent years, Pinot grigio has gained particularly in popularity, as has Sauvignon blanc. Pinot gris can vary in color from bluish to white, depending on the microclimatic conditions of the cluster. It typically yields subtly fragrant wines with aspects of passion fruit. Pinot blanc yields more fruity wines, supposedly with aspects resembling hard cheese. Riesling (White Riesling or Johannisberg Riesling) is without doubt Germany’s most highly esteemed grape variety. It is one of multiple progeny having Heunisch Weiss (syn. Gouais blanc) as one of its parents, appearing first in print in 1493 (Ambrosi et al., 1994). Outside Germany, its largest plantings are in California and Australia. It can produce fresh, aromatic, well-aged wines, which can vary from dry to sweet. Its floral aroma, commonly reminiscent of roses, has not made it unpopular throughout central Europe, and much of the world. This renown is reflected in the number of cultivars whose names have incorporated the word Riesling (i.e., Hunter Riesling, Goldriesling, Frankenriesling, and Wa¨lschriesling), although often bearing little if any genetic or aromatic similarity to Riesling. Riesling produces clusters of small- to medium-sized berries, which are particularly sensitive to powdery mildew and bunch rot. The fruit is cold hardy, and matures relatively late. Yield tends to be moderate. Sauvignon blanc is one of the primary white varieties in Bordeaux, and the main white cultivar grown in the upper Loire Valley. It has become popular in California and New Zealand in recent years. It also is grown in northern Italy and eastern Europe. Often, its aroma shows elements of green peppers, as well as an herbaceous aspect, especially in cooler climates. Optimally, the wine also expresses a subtly floral character, often considered to be passion fruit-like. One of its characteristic thiols has an aspect often described as reminiscent of cat urine. Its modest clusters produce small berries that are sensitive to powdery mildew and black rot, but possess partial resistance to bunch rot and downy mildew. Se´millon is another white Bordeaux variety, most well known for its use in producing Sauternes wines. In Bordeaux, it is commonly blended with Sauvignon blanc. Significant plantings also occur in Australia and Argentina. Se´millon produces small clusters of mediumsized fruit, susceptible to bunch rot. The vine is also especially sensitive to fanleaf degeneration. When fully mature, and without the intervention of noble rot, Se´millon yields a dry wine, said to contain nuances of fig or melon. These aspects develop primarily during aging. Traminer is often considered to be an ancient cultivar, some clones of which possess a distinctive aromatic character. The cultivar is grown primarily in cooler climatic regions. Its clones can be grouped into two color classes: white and pink to reddish. Pink strains appear to possess incomplete expression of the Gret1 retrotransposon

65

(which inhibits anthocyanin synthesis), permitting partial berry coloration (Pelsy et al., 2010). Regardless of the grape’s color, all clones are processed as white grapes, producing dry to sweet wines, depending on regional preferences. The name Traminer is usually applied to white clones with a mild fragrance, while relatively aromatically neutral clones are often called Savagnin, notably in southeastern France. Intensely fragrant, pinkish Gewu¨rztraminer clones may produce wines possessing an intense aroma, resembling that of litchi fruit. Nonetheless, depth of coloration and aromatic character may depend more on cultural and climatic conditions than on the clone’s color (Bourke, 2009; Ducheˆne et al., 2009). All forms produce modest clusters of small fruit with tough skins. The variety is prone to powdery mildew and bunch rot, and often expresses coulure. Albarin˜o (Spain) and Alvarinho (Portugal) were once thought to be additional synonyms, but have been shown to be unrelated (Santiago et al., 2007). Viognier has been a variety largely restricted to the Condrieu appellation in the Rhoˆne Valley. Comparatively recently, it has caught the fancy of several North American and Australian producers. The variety has a tendency toward poor fertility and, correspondingly, low yield. Thus, it needs to be cane pruned. The vine generally requires excellent drainage. Viognier produces small round berries possessing a muscaty fragrance. Correspondingly, the wine matures quickly. The variety is also reported to show peach or apricot resemblances. Viura is the main white variety in Rioja. It produces few clusters, but they are of considerable size. In cool regions, it produces a fresh wine possessing a subtle floral aroma with aspects of lemon. After prolonged aging in wood, it develops a golden color and rich butterscotch or banana fragrance. These characterize the traditionally aged white wines of Rioja, now regrettably rare.

Interspecies hybrids American cultivars and their hybrids Although decreasing in significance throughout North America, early selections from native American grapevines or accidental interspecies hybrids are still extensively grown. American hybrids constitute major plantings in eastern North America, particularly Washington State, and they are grown commercially in South America and Asia. Most are accessions from feral vines or crosses involving V. labrusca or V. rotundifolia. Both have fruit that are within the size range typical of vinifera cultivars, in contrast to most other indigenous North American Vitis spp. Nonetheless, V. rupestris, V. riparia, and V. berlandieri have been important sources of resistance to many grapevine diseases and pests, notably downy and powdery mildews, and phylloxera.

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2. Grape species and varieties

Of American cultivars, the most important are Vitis labrusca derivatives. They possess a wide range of flavors. Some, such as Niagara, are characterized by the presence of a foxy aspect. The various proposed derivations of this cultivar are detailed in Bailey (1898). Other flavors expressed in labrusca-related cultivars are strawberry (Ives), the grapy aspect of methyl anthranilate (Concord), or a strong floral aroma (Catawba). Although these cultivars became associated with decidedly sweet wines, and denigrated by those habituated to dry European wines, there is no reason for this to persist. Their flavors are no more intense than some V. vinifera cultivars, and as amenable to producing dry wines. Wines produced from V. riparia and V. cinerea do not possess detectable concentrations of methyl anthranilate, or 2-aminoacetophenone. In contrast, they are characterized by high concentrations of methoxypyrazines, the same compounds that characterize Cabernet Sauvignon and related cultivars. They are also characterized by herbaceous flavors, associated with eugenol, cis-3henenol, and 1,8-cineole (Sun et al., 2011). The high acidity and low sugar content of American cultivars, and their hybrids, have made chaptalization (the addition of sugar to the juice) necessary for standard table wine production. Various methods have been used to diminish the supposed excessive flavor of American cultivars. Long aging results in the dissipation of these flavors. This is generally impractical, as most V. labrusca wines have traditionally been consumed young. The presence of high levels of carbon dioxide, as in sparkling wines, tends to mask most labrusca fragrances. Processing the grapes via carbonic maceration was another technique investigated as a means of reducing (or masking) the intensity of labrusca flavors (Fuleki, 1974). However, the most generally accepted mechanism of aroma diminution has been early picking and cold fermentation. Hedrick (1908) noted that, when of moderate intensity, the flavors of American hybrids were often agreeable, possessing exotic fruit flavors. He viewed the “universal condemnation of this taste by the French and some other Europeans” as pure prejudice. Other factors leading to the opinion that American hybrid grapes could not produce acceptable wines may have come from their former exclusive use in very sweet winesdthe preference by many consumers in North America in the past. This is often considered an anathema to many aficionados (except when the wines are botrytized and fortified). Only now, and on a very limited scale, some vintners are producing small quantities of dry versions of labrusca hybrids or other accessions. The varieties Norton and Cynthiana (possibly clonal variants; Reisch et al., 1993) are predominantly of Vitis aestivalis origin, one of the few other native species

with medium-sized fruit. These cultivars are primarily cultivated in the lower Midwest (Arkansas, Kentucky, Illinois, and Missouri) and mid-Atlantic states (Maryland and Virginia). Locally derived from wild vines, and possibly introgressed to V. vinifera, they possess resistance to the local indigenous diseases and pests that complicate cultivation of vinifera varieties in these states. The other major group of American cultivars are those derived from V. rotundifolia. Although Scuppernong is the most well recognized varietal name, it is rarely grown today. New self-fertile varieties, possessing different aromatic properties, are the principal muscadine cultivars grown in commercial vineyards (Morris and Brady, 2007). The excellent resistance of these cultivars to indigenous diseases, especially Pierce’s disease, has allowed them to flourish in the southeastern coastal United States. Similar to the V. labrusca cultivars, the low sugar content of the fruit usually requires chaptalization before vinification. The pulpy texture, tough skin, differential fruit maturation, and separation of fruit from the pedicel on maturation complicate their use in winemaking. Most muscadine cultivars have a distinctive and marked fragrance, containing aspects of orange blossoms and roses. Some fertile crossings with V. vinifera show vinifera-like flavors, combined with the fruiting characteristics and disease resistance of their muscadine parentage. French-American hybrids (direct producers) French-American hybrids were initially developed to avoid the necessity, complexity, and expense of grafting V. vinifera cultivars to phylloxera-resistant rootstocks. This, along with greater resistance to several leaf pathogens (enhanced production of pathogenesis-related [PR] proteins) and higher yield, made them popular with many grape growers in France. The tendency of base buds to grow and bear fruit led to frequent overcropping. This exacerbated the increasingly serious problem of grape overproduction in France, a problem that still plagues much of Europe. This factor, combined with the nontraditional fragrance of these hybrids, led to a general ban on new plantings. Their use in appellation control wines was also prohibited. Restrictions against FrenchAmerican hybrid use subsequently spread throughout the European Union. The remaining, temporary, exception is the cultivation of Baco blanc for Armagnac production, which is supposed to have expired. In North America, with the exception of most of the southern, gulf, and western coastal states, FrenchAmerican hybrids formed the basis of the expanding wine industry in the early 1960s. They are still grown extensively in some South American and Asian countries. In Europe, as well as other areas, French-American hybrids are often used in breeding programs as a source of resistance to several foliar, stem, and fruit pathogens. Many French-American hybrids suffer from sensitivity

Grapevine cultivars

to soil-borne virus diseases, such as those of the tomato ringspot group (Alleweldt, 1993). Where this is a problem, grafting to resistant rootstocks is required. Unlike American hybrids, few French-American hybrids possess Vitis labrusca parentage and therefore any hint of the “foxy” character that seems so repellant to most critics. Oddly, critics do not complain about the supposed “cat piss” attribute often attributed to Sauvignon blanc (due to 3-sulfanylhexan-1-ol), the intense flavors of Gewu¨rztraminer, or the ethyl acetate or Brett. character of some prestigious white and red wines, respectively. French-American varieties typically contain some Vitis rupestris, V. riparia, and V. aestivalis var. lincecumii parentage, sources of disease resistance and/or cold tolerance. Their parentage is usually confirmed by the basis of their DNA (Pollefeys and Bousquet, 2003). Despite aspersions cast on wines made from FrenchAmerican and other interspecies hybrids, blind comparative tastings between these and vinifera cultivars often indicate that hybrid-derived wines are as appreciated or considered superior to vinifera-derived wines (Van Der Meer et al., 2010; Pedneault et al., 2012; Rousseau et al., 2013). For those desiring to use organic viticulture, the comparative disease resistance of these cultivars reduces the need and expense of pesticides (Pedneault and Provost, 2016). Although a cost savings, reduced market demand for wines carrying unfamiliar names can limit grower and winemaker profits. Other potential problems in wine production can arise from issues due to nonuniform fruit ripening (due to yield from lateral buds that flower later in the season), a tendency to possess higher pectin contents (necessitating the expense of using enzymes to facilitate juice extraction), and enhanced tannin precipitation by PR proteins. Nonetheless, many hybrid cultivars possess flavor compounds similar to those considered desirable in pure vinifera cultivars. For techniques designed to accentuate these positive attributes of interspecific hybrids, see Pedneault and Provost (2016). Baco noir is a Folle blanche  V. riparia hybrid. Its acidity, flavor, and pigmentation yield a wine with considerable aging potential. It develops a fruity aroma associated with aspects of herbs. It is sensitive to bunch rot and several soil-borne viruses. Poor cane maturity is often a problem in cold climatic regions due to vine vigor. De Chaunac is a Seibel crossing of unknown parentage. Once widely planted in eastern North America, due to its cold hardiness and high yield, its tendency to produce wine of neutral character and its susceptibility to several soil-borne viruses have resulted in a loss of favor. Chambourcin is a Joannes Seyve hybrid of unknown parentage. Its popularity increased markedly in the Loire Valley during the 1960s and 1970s. It is considered one of the best of French-American hybrids. Chambourcin has also done remarkably well in Australia, possibly due to the long growing season. This permits full grape

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maturity and the development of a rich, wonderfully complex flavor. Chambourcin has also found a following in eastern North America. The variety possesses good resistance to both downy and powdery mildews. Mare´chal Foch is a Kuhlmann hybrid, derived from crossing a V. riparia  V. rupestris selection with Goldriesling (Riesling  Courtiller musque´). It yields deeply colored, berry-scented, early-maturing wines. The variety’s characteristics of winter hardiness, productiveness, early maturity, and resistance to downy mildew have also given it appeal in eastern North America. Delaware is one of the finest, early ripening, light-red American hybrids. It is generally thought to be a V. labrusca  V. aestivalis  V. vinifera hybrid. Nevertheless, its susceptibility to phylloxera and various fungal pathogens, tendency to crack, and need for welldrained soil has limited its widespread cultivation. It was once extensively used in sparkling wine production. Dutchess is another highly rated, older, late-ripening, white American hybrid. This Vitis labrusca  V. vinifera hybrid has a mild, fruity aroma with little labrusca character. As with Delaware, difficulty in growing the cultivar negates most of its enologic qualities. Magnolia is one of the more popular, new, muscadine cultivars. It produces bisexual flowers and is self-fertile. It yields sweet, bronze-colored fruit. Noble is a dark-red muscadine cultivar. Its deep-red color and bisexual habit have made it popular in the southeastern United States. Seyval blanc is a Seyve-Villard hybrid of complex V. vinifera, V. rupestris, and V. aestivalis var. lincecumii parentage. The variety yields a mildly fruity white wine, with a pomade fragrance and bitterish finish. Although susceptible to bunch rot, it is relatively winter hardy, tolerant of many soil types, and a consistent producer. Vidal blanc is possibly the best of the white FrenchAmerican hybrids. This Vidal hybrid of complex ancestry has both excellent winemaking and excellent viticultural properties. Under optimal conditions Vidal blanc yields a wine of Riesling-like character. Its tough skin and late maturity assist in its being used in the production of ice wines of excellent quality. The variety is relatively cold hardy, but less so than Seyval blanc. Although most interspecies hybridization ceased in France by the 1920s, it has continued unabated in Germany. Varieties such as Orion, Phoenix, and Regent show winemaking qualities equal or superior to several currently used V. vinifera cultivars. Breeding has also continued in eastern North America. Veeblanc is one of the newer white cultivars of complex parentage developed in Ontario. It generates a mildly fruity wine of good quality. New York also has an active breeding program. One of their most commercially successful introductions to date has been Cayuga White. It is well

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adapted to a range of wine styles; possesses a fruity fragrance resembling apples, citrus, and tropical fruit; and produces a wine without bitterness and a rich mouthfeel. When harvested early, it produces an excellent base for sparkling wines. Although very productive, Cayuga White seldom requires cluster thinning. It is also resistant to most common fungal diseases and, thus, needs little fungicidal protection. Another popular new introduction from Cornell is Traminette. It possesses much of the aromatic character of its Gewu¨rztraminer parent, but is more cold hardy. Other introductions with desirable flavors, combined with excellent disease resistance characteristics, are Aromella and Arandell. With the increasing interest in organic viticulture, such additions are of particular value. In cold climatic regions, such as the Midwestern and Northeastern United States and Canada, other interspecific hybrids of considerable interest are Frontenac and Marquette. They are progeny of the breeding program at the University of Minnesota.

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Suggested reading

Suggested reading Taxonomy, evolution and cultivar origins Go¨kbayrak, Z., So¨ylemezoglu, G., 2010. Grapevine throughout the history of Anatolia. Int. J. Bot. 6, 465e472. Gross, D.L., Olsen, K.M., 2010. Genetic perspectives on crop domestication. Trends Plant Sci. 15, 529e537. Heinitz, C.C., Uretsky, J., Peterson, J.C.D., Huerta-Acosta, K.G., Walker, M.A., 2019. Crop wild relatives of grape (Vitis vinifera L.) throughout North America. In: Greene, S.L., Williams, K.A., Khoury, C.K., Kantar, M.B., Marek, L.f. (Eds.), North American Crop Wild Relatives, vol. 2. Springer Nature, Cham, Switzerland, pp. 329e351. Levadoux, L., Boubals, D., Rives, M., 1962. Le genre Vitis et ses espe`ces. Ann. Ame´lior. Plantes 12, 19e44. Olmo, H.P., 1995. The domestication of the grapevine Vitis vinifera L. in the Near East. In: McGovern, P.E. (Ed.), The Origins and Ancient History of Wine. Gordon and Breach, Luxembourg, pp. 31e44. Wen, J., 2007. Vitaceae. In: Kubitzki, K. (Ed.), The Families of Vascular Plants, vol. IX. Springer, Berlin, Germany, pp. 467e479. Zecca, G., Abbott, J.R., Sun, W.-B., Spada, A., Sala, F., Grassi, F., 2012. The timing and the mode of evolution of wild grapes (Vitis). Mol. Phylogenetics Evol. 62, 736e747.

Rootstock Catlin, T., 1991. Alternative Rootstock Update. ASEV Technical Projects Committee and UC Cooperative Extension. Univerisity of California Agriculture Publications, Oakland, CA. May, P., 1994. Using Grapevine Rootstocks e the Australian Perspective. Winetitles, Adelaide, Australia. Pongra´cz, D.P., 1983. Rootstocks for Grapevines. David Philip, Cape Town, South Africa. Wolpert, J.A., Walker, M.A., Weber, E. (Eds.), 1992. Proceedings of the Rootstock Seminar: A Worldwide Perspective. American Society of Enology and Viticulture Davis, CA.

Grape breeding, selection, propagation Costantini, F.M., Moreira, E., Zyprian, E., Martı´nez-Zapater, J.M., Grando, M.S., 2009. Molecular maps, QTL mapping and association mapping in grapevine. In: Roubelakis-Angelakis, K. (Ed.), Molecular Biology and Biotechnology of the Grapevine, second ed. Kluwer Adacemic Publ., Dordrecht, Netherlands, pp. 535e564. Di Gaspero, G., Foria, S., 2015. Molecular grapevine breeding techniques. In: Reynolds, A.G. (Ed.), Grapevine Breeding Programs for the Wine Industry. Woodhead Publishing, Cambridge, UK, pp. 23e37. Eibach, R., To¨pfer, R., 2015. Traditional grapevine breeding techniques. In: Reynolds, A.G. (Ed.), Grapevine Breeding Programs for the Wine Industry. Woodhead Publishing, Cambridge, UK, pp. 3e22.

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Knott, G.J., Doudna, J.A., 2018. CRISPR-Cas guides the future of genetic engineering. Science 361, 866e869. Liu, J., Osbourn, A., Ma, P., 2015. MYB transcription factors as regulators of phenylpropanoid metabolism in plants. Mol. Plant 8, 689e708. Martinelli, L., Gribaudo, I., 2009. Strategies for effective somatic embryogenesis in grapevine: an appraisal. In: Roubelakis Angelakis, K. (Ed.), Molecular Biology and Biotechnology of the Grapevine, second ed. Kluwer Adacemic Publ., Dordrecht, Netherlands, pp. 461e494. Miguel, C., Marum, L., 2011. An epigenetic view of plant cells cultured in vitro: somaclonal variation and beyond. J. Exp. Bot. 62, 3713e3725. Sefc, K.M., Pejic, I., Maletic, E., Thomas, M.R., Lefort, T., 2009. Microsatellite markers for grapevine: tools for cultivar identification and pedigree reconstruction. In: Roubelakis-Angelakis, K.A. (Ed.), Molecular Biology & Biotechnology of Grapevine, second ed. Springer, Dordrecht, The Netherlands, pp. 565e596.

Grape cultivars Alleweldt, G., 1989. The Genetic Resources of Vitis: Genetic and Geographic Origin of Grape Cultivars, Their Prime Names and Synonyms. Federal Research Centre for Grape Breeding, Geilweilerhof, Germany. Bettiga, L., 2003. Wine Grape Varieties in California. Division of Agriculture and Natural Resources, University of California, Oakland, CA. Pub. # 3419. Chitwood, D.H., Ranjan, A., Martinez, C.C., Headland, L.R., Thiem, T., Kumar, R., et al., 2014. A modern ampelography: a genetic basis for leaf shape and venation patterning in grape. Plant Physiol. 164, 259e272. Galet, P., 1979. In: Morton, L.T. (Ed.), A Practical Ampelography e Grapevine Identification. Cornell University Press, Ithaca, NY. Galet, P., 1988. 1990. Ce´pages et Vignobles de France: Vol. 1 (Les Vignes Ame´ricaines), Vol. 2 (L’Ampe´lographie Franc¸aise), Vol. 3 (Les Ce´pages de Cuves), second ed. Galet, Montpellier, France. Hillebrand, W., Lott, H., Pfaff, F., 2002. Taschenbuch der Rebsorten. Fachverlag Fraund, Wiesbaden, Germany. Kerridge, G., Antcliff, A., 1996. Wine Grape e Varieties of Australia. CSIRO, Collingwood, Australia. Mackay, J.F., Wright, C.D., Bonfiglioti, R., 2008. A new approach to varietal identification in plants by microsatellite high resolution melting analysis: application to the verification of grapevine and olive cultivars. Plant Methods 4, 8. Maghradze, D., Rustioni, L., Turok, J., Scienza, A., Failla, O. (Eds.), 2012. Caucasus and Northern Black Sea Region Ampelography. Vitis Special Issue, p. 488. http://anl.az/el_en/book/2014/I39338.pdf. Olien, W.C., 1990. The muscadine grape: Botany, viticulture, history, and current industry. Hortscience 25, 732e739.

C H A P T E R

3 Grapevine structure and function O U T L I N E Structure and function

77

The root system The young root Mycorrhizal and endophytic associations Secondary tissue development Root-system development

78 78 81 82 83

The shoot system Buds Shoots and shoot growth Tissue development

85 85 88 89

Tendrils

92

Leaves Photosynthesis and other light-activated processes Transpiration and stomatal function

92

Reproductive structure and development Inflorescence (flower cluster) Inflorescence morphology and development Flower development Timing and duration of flowering Pollination and fertilization Flower type and genetic control

94 98 99 99 100 102 103 104 106

Structure and function

107 107 112 112 114 115 116 117 120 120 127 127 128 129

Cultural and climatic influences on berry maturation Yield Sunlight Temperature Inorganic nutrients Water

130 130 134 137 138 138

References

139

Suggested reading

149

nucleus. As the cells differentiate, plasmodesmal and cytoplasmic organelles often deteriorate, and the cells may specialize into functional but nonliving cells. The result is that the plant body begins to resemble layers of overlapping, longitudinal, semiindependent cones of specialized tissues. Most conductive (vascular) cells elongate longitudinally, permitting the rapid movement of water and nutrients between superimposed sections of root and shoot tissue. In contrast, conduction between horizontally adjacent cells is limited, occurring largely apoplasmically (by diffusion through intercellular spaces). Direct

The uniqueness of plant structure is obvious to even the most casual observer. However, some of the most distinctive and unique features become apparent only when viewed microscopically. Unlike animal cells, plant cells are enclosed in a rigid cell wall. Nevertheless, each cell initially possesses direct cytoplasmic connections with adjacent cells through minuscule channels called plasmodesmata. Thus, embryonic plant tissue resembles a huge cell divided into multiple interconnected compartments, each possessing its own cytoplasm, organelles, and

Wine Science, Fifth Edition https://doi.org/10.1016/B978-0-12-816118-0.00003-9

Berry growth and development Berry structure Seed morphology Chemical changes during berry maturation Growth regulators Water uptake Sugars Acids Potassium and other minerals Phenolics Pectins Lipids Nitrogen-containing compounds Aromatic compounds

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© 2020 Elsevier Inc. All rights reserved.

78

3. Grapevine structure and function

translocation between tissues on opposite sides of shoots and roots is often nonexistent (except where vascular tissues do not elongate perfectly vertically). Spiraling of the vascular tissues can permit some indirect lateral translocation. Not only do the vascular tissues permit essential translocation between the shoot and the root, but the xylem also provides the water essential for plant cooling. Thus, the physical characteristics of the vascular system place absolute limits on plant productivity and size that biochemical pathways cannot obviate or circumvent. The vascular system in most plants consists of two structurally and functionally distinct components. The main water and mineral conducting elements, the xylem tracheae and tracheids, become functional on the disintegration of their cytoplasmic contents. Their empty lumens act as passive conduits. Loading and unloading functions appear to be under the control of adjacent, living contact (parenchyma) cells. The primary cells translocating organic nutrients are the sieve tube elements of the phloem. They become functional when the nucleus disintegrates and central vacuole disappears. Cytoplasmic organelles and clusters of proteins are appressed against the cell membrane. Metabolic control is thought to be associated with adjacent companion (parenchyma) cells. These retain their nucleus, central vacuole, and cytoplasm, which is packed with mitochondria, plastids, and other organelles (van Bel, 2003). Vascular plants (such as the grapevine) also show a distinctive growth habit. Growth in length is primarily restricted to specialized embryonic (meristematic) cells located in shoot and root tips. Growth in breadth is initially limited to that resulting from the enlargement of the cells produced in the shoot or root apices (primary tissues). Further growth in diameter occurs when a band of cells, the vascular cambium, differentiates between the phloem and xylem. As this lateral section of meristematic tissue becomes active, it produces cells both laterally (to the sides) and radially (to the inside and outside). Thus, as the vine grows, the shoots and roots increasingly resemble annually produced, superimposed, elongated, thin cones of secondary phloem and thicker zones of secondary xylem extending from root tip to shoot tip. The oldest (primary) phloem is located outermost, and the oldest (primary) xylem is located innermost. Phloem cells may remain functional for up to 4 years, whereas xylem vessels may continue to translocate for up to 7 years. In addition, plants show distinctive growth patterns that generate leaves and their evolutionary derivativesd flowers, tendrils, etc. In leaves and flowers, sites of growth (plate meristems) occur dispersed throughout the young leaf or flower part. Their nonuniform rates and patterns of growth generate the characteristic shape of the respective plant parts (see Fig. 3.13) and

generate the patterns often used in cultivar identification (ampelography).

The root system The root system possesses several cell types, tissues, and regions, each with a particular structure and function. It also consists of a series of root types: leader, fine lateral, and large permanent. Each possesses a particular function and relative life span. Thus, the root system works as a community of distinct but interconnected components, each contributing to the success of the whole. The root tip performs many of the most significant root functions, such as water and inorganic nutrient uptake, synthesis of several growth regulators, and the elongation that pushes the root tip into regions untapped of water and nutrient supplies. Not surprisingly, this region is the most physiologically active and has the highest risk of early mortality (Anderson et al., 2003). In addition, the root tip is the site for the initiation of mycorrhizal associations, while secretions from the root cap soften the soil in advance of penetration. Root cap secretions also promote the development of a unique rhizosphere microbial flora on and around the root tip. Older, mature portions of the root transport water and inorganic nutrients upward to the shoot system and organic nutrients downward for storage or root growth. The outer, secondary tissues of the mature root restrict water and nutrient loss to the soil and help protect the root from parasitic and mechanical injury. Permanent parts of the root system anchor the vine and act as a major storage organ during the winter (Yang et al., 1980; Zapata et al., 2001). Nutrient reserves decline during the winter and spring months associated with root maintenance, and shoot and leaf production, respectively. Export to the shoot in the spring involves translocation via both the xylem and the phloem (Glad et al., 1992a,b). Nutrient accumulation recommences after shoot growth slows in midsummer to late summer, reaching a peak in autumn. This may explain why roots produced before flowering have the shortest life span (due to the lowest carbohydrate reserve) (Anderson et al., 2003). Because of significant differences in the structure and function of young and mature roots, they are discussed separately.

The young root Structurally and functionally, the young root can be divided into several zones. The most apical is the root tip, containing the root cap and apical meristem (the metabolic “hot spot” of the root system). The latter consists of cells that remain embryonic as well as those that differentiate into the primary tissues of the root. Primary tissues are defined as those that develop from apically

The root system

located meristematic cells. Secondary tissues develop from laterally positioned meristematic tissues (cambia). The embryonic cells of the root tip are concentrated in the center of the meristematic zone, which was erroneously termed the quiescent (transition) zone. In addition, the region is a major site for the synthesis of certain growth regulators, notably cytokinins, abscisic acid, ethylene, and gibberellins. Their production is promoted by auxins translocated down from the shoot. In addition, auxins direct cell division, elongation, and differentiation in the root tip as well as stimulate root hair initiation and development and lateral root induction. Together, root- and shoot-derived regulators balance shoot vs. root growth. They also influence stomatal function (notably abscisic acid), inflorescence initiation, and fruit development. Hormonal interaction in plants is notoriously complex and site-specific, often depending on gradients established in different tissues of the shoot or root. An example is the cyclical flow of auxins in the inner and outer columns of the root tip generated by different plant-specific phytohormone transport proteins in the root tip (Baluska et al., 2010). Hormonal specificity also depends on the selective production of receptor proteins (e.g., Westfall et al., 2012). Surrounding the permanently embryonic center of the transition zone are rapidly dividing cells and their derivatives in early stages of differentiation. Cells positioned toward the apex develop into root cap cells. These are short-lived and produce mucilaginous polysaccharides that ease root penetration into the soil. Internal (columella) cells respond to environmental signals and direct root growth. The different independent mechanisms by which gravitational, nutritional, thigmotropic, and moisture differentials are detected (Jaffe et al., 1985) may explain why primary (tap) roots grow downward while secondary (lateral) roots initially grow outward. Their differential response to environmental signals leads to the three-dimensional but primarily lateral spread of the root system through the soil. In contrast, plant tissue polarity (shootward vs. rootward) is fixed early in embryogenesis. It is based on the orientation of a set of PIN proteins that remains stable. They take up a basal position perpetuated in all subsequent cells produced (Friml et al., 2006). PIN proteins direct the movement of auxins that establish apical dominance. The cap also appears to cushion embryonic cells from physical damage during penetration into the soil. Laterally, cells differentiate into the epidermis, the hypodermis, and cortex, the cortex consisting of several layers of undifferentiated parenchyma cells. Behind the apical meristem, vascular tissues begin to differentiate and elongate. Cell enlargement occurs primarily along the axis of root growth and forces the extension of the root tip into the soil. This is primarily limited to a short (w2 mm) region called the elongation zone. Except for

79

some enlargement in cell diameter, most lateral root expansion results from the subsequent production of new (secondary) tissue behind the root apex. Most newly generated cells complete their differentiation just behind the elongation zone. This is also the zone in which water and mineral absorption is most pronounced. Thus, the region is variously termed the differentiation or absorption zone. It is often about 10 cm in length. The next region is called the root-hair zone due to the fine cellular extensions (root hairs) that develop outward from the epidermal covering. The formation, length, and life span of root hairs depend on many factors. For example, alkaline conditions suppress root-hair development as does the formation of a symbiotic association with mycorrhizal fungi. Root hairs increase rootesoil contact and thereby potential water and mineral uptake. However, due to their absence or limited production under field conditions (McKenry, 1984), especially in mycorrhizal roots, their relative significance to water and nutrient uptake may have been exaggerated. Root hairs also release, and liberate upon their decay, organic constituents into the soil solution, thus promoting further development of a unique microbial flora initiated by root cap secretions on and around the root. This may be particularly significant in deeper, organic, and nutrient-poor layers of the soil. The rhizosphere flora can also assist in protecting the root from soil-borne pathogens, favors solubilization of inorganic soil nutrients, and may even assist in combating leaf pests and pathogens (Pineda et al., 2010). The first of the vascular tissues to differentiate is the phloem. Its early development facilitates the translocation of organic nutrients to the dividing and differentiating cells at the root tip. The xylem, involved in long-distance transport of water and inorganic nutrients, develops further back in the differentiation zone. This sequence probably evolved to avoid the removal of water and inorganic nutrients from the growing root tip. These compounds, locally absorbed, are required for the cell growth that typifies the region. The primary xylem and phloem cells mature in distinct packets, termed vascular bundles, separated by columns of undifferentiated parenchyma cells. Adjacent to the primary vascular bundles and forming the innermost layer of the cortex (outer tissues of the young root) is the endodermis. The endodermis deposits a band of wax in, and lignin around, its radial walls, called the Casparian strip. This restricts the apoplasmic diffusion of water and nutrients between the cortex and vascular cylinder. Thus, movement of water and nutrients between the cortex and vascular cylinder must pass through the cytoplasm of the endodermal cells (protoplasmically). This gives the root metabolic control over transport into and out of the vascular tissues. Further back in the differentiation zone, the pericycle develops

80

3. Grapevine structure and function

just inside the endodermis. It generates a protective cork layer further isolating the inner vascular tissues from the cortex, and eventually the soil if the root continues to grow and differentiate. The pericycle is also the site for the initiation of lateral root production. Uptake of water and inorganic nutrients is primarily under metabolic control and involves specific transport proteinsdfor example, aquaporins, membrane-bound proteins, and selective transport of water into the root (Zhao et al., 2008). Activity of its genes decreases markedly along the root length, being highest next to the vascular tissues (Gambetta et al., 2013). Production can also vary diurnally in response to drought stress and is cultivar specific (Vandeleur et al., 2009). Thus, although the root-hair zone is the major site for water and mineral uptake, this is not exclusively so. Behind the root-hair zone, the epidermis may soon die, turning brown. The underlying hypodermis becomes encased in waxy suberin. Suberization occurs rapidly during the summer and may advance to include the root tip under dry conditions (Pratt, 1974). The endodermis also thickens and becomes suberized behind the root tip. Although these modifications limit water uptake, they do not prevent apoplasmic movement (via or between cell walls). The extensive woody component of the root system can constitute a significant site for water uptake (Gambetta et al., 2013). The influence of suberization on mineral uptake depends on the ion involved and the amount of wax in the suberin. Water and potassium uptake by heavily suberized woody roots approximate 30% and 1%e4%, respectively, of that absorbed by young unsuberized roots (Queen, 1968). In maturing sections of feeder roots (short-lived lateral roots), the cortical tissues collapse and the root becomes encased in cork, especially in dry soil. However, as only the inner wall surfaces of cork cells are suberized, and may be penetrated by plasmodesmata, diffusion routes for water and solutes through pores in the walls between adjacent cells are possible (Atkinson, 1980). This could be of considerable importance. As few feeder roots survive from one year to the next, most water and nutrient uptake in the early spring occurs via older structural components of the root system. Feeder-root production may not reach its maximum until shoot growth has ceased. Developmental and anatomical changes in root structure behind the root tip are reflected in a rapid decline in nitrate uptake and respiration along the root length. Nevertheless, reduction in nitrogen uptake is more precipitous than would be expected based solely on morphological changes. Volder et al. (2005) found that nitrate uptake declined by about 50% within 2 days of emergence and fell to 10%e20% within a few days. Uptake subsequently remained relatively constant for a month or more. Respiration followed a similar trend.

These changes may owe their origin to rapid nitrate depletion in the immediate vicinity of the growing tip. In addition, development of the xylem and initiation of nitrogen transport to other parts of the vine may also help explain the decline in net metabolic rate. Nutrient and water uptake are partially regulated by the activation, timing, and duration of new root production. Root life span depends on multiple factors. These include soil depth, timing of emergence, and root diameter. For example, fine lateral roots often remain viable for little more than 50 days, whereas deeper and coarser roots usually survive much longer (Anderson et al., 2003). McLean et al. (1992) obtained similar results, finding that no more than 60%e80% of fine roots survive the first season. Pruning treatment and grape yield also influence root longevity. For example, higher grape yields were correlated with reduced root viability, whereas roots of minimally pruned vines tended to remain metabolically active longer than those of balance-pruned vines (Comas et al., 2000). Regardless of the site, water uptake is induced by passive forces. In the spring, absorption and movement may be driven osmotically by the conversion of stored carbohydrates into soluble sugars. The negative osmotic potential produced generates what is termed root pressure. It produces the “bleeding” that occurs at the cut ends of spurs and canes in the spring. Once the leaves have expanded sufficiently, transpiration creates the negative pressure (suction) that maintains the upward movement of water and inorganic nutrients in the xylem throughout the remainder of the growing season. Unlike water uptake, mineral uptake and unloading into the xylem is an active process under the control of tightly appressed contact cells (parenchyma). Metabolic energy is also required for loading growth regulators synthesized in the root tip. Subsequent to loading, movement appears to be passive, being carried along with the water flowing in the xylem. In contrast, organic compounds are seldom absorbed from the soil and translocated in the conducting elements of the root system. Exceptions include systemic fungicides and herbicides as well as some microbial toxins and growth regulators. Xylem-inhabiting microbes may also be passively translocated in the transpiration stream. Although essential to plant growth, regulation of mineral uptake and nutrient loading into the xylem sap remains poorly understood. For example, Mapfumo et al. (1994) discovered large lignified xylem vessels possessing protoplasm up to 22.5 cm back from the root tip. In several crops such cells have been found to be involved in potassium accumulation before transport (McCully, 1994). Whether this is true for grapevines is unknown. In addition, marked remobilization of stored Ca, K, Mg, Zn, and Fe can occur in the spring. Their movement out of the root seems dependent on the

81

The root system

xylem flow rate. In contrast, NH4, NO3, P, Cu, Na, and Cl movement appear to be independent of xylem flow rate (Campbell and Strother, 1996a, 1996b).

Mycorrhizal and endophytic associations An important factor influencing mineral uptake is the symbiotic association formed between the root and a mycorrhizal fungus. Grapevines are among the more than 80% of plant species that develop mycorrhizal associations. Plants with weedy tendencies are an exception. Weeds tend to depend on their ability to germinate rapidly and produce a fine root-hair system to establish themselves in disturbed sites. In contrast, most plants germinate and grow more slowly depending on mycorrhizal associations to exploit water and nutrient reserves. Of the three major groups of mycorrhizal fungi, only vesicularearbuscular (V-A) fungi invade grapevine roots (Schubert et al., 1990). The latter appear to be one of the most ancient fungal groups, exhibiting no known sexual stage, being multinuclear and obligately mycorrhizal, and typically possessing one or more endosymbiotic bacteria. Species of Glomus have often been considered the primary V-A genus associated with grapevines. Nonetheless, several other genera may be involved, for example Acaulospora, Gigaspora, Scutellospora, and Sclerocystis. The new understanding follows application of genomic analysis independent of the vagaries of morphology (Holland et al., 2014). These fungi produce chlamydospores or sporocarps (containing many

(A)

Non-mycorrhizal root

(B)

chlamydospores) from which infective hyphae invade the root epidermis. From this site, the fungus penetrates and colonizes the cortex. Here, the fungus produces large swollen vesicles and highly branched arbuscules, the two features that give these fungi their name. The fungi do not markedly alter root morphology, as do other mycorrhizal fungi (Fig. 3.1). Nonetheless, V-A mycorrhizae do reduce root-hair formation, increase lateral root production, and promote dichotomous root branching. Invasion often results in the normally white root tip possessing a yellowish-brown cast and becoming misshapen. Although mycorrhizal invasion reduces root elongation and soil exploration, it produces a more economical root system for nutrient acquisition (Schellenbaum et al., 1991). New mycorrhizal associations begin near the root tip in response to strigolactones liberated by young roots. In turn, the V-A fungus produces chitin oligomers and lipochitooligosaccharides. These induce changes in root physiology that permit the fungus to penetrate and generate an infection thread into the cortical tissues (Gobatto, 2015). This phenomenon continues as the root tip advances while mycorrhizal activity declines and ceases rearward (as the root matures and the cortex is sloughed off). Mycorrhizal roots are strong sinks for plant carbohydrates, which may help explain why these roots tend to live longer than nonmycorrhizal roots. As the fungus establishes itself in the cortex, wefts of hyphae ramify into the surrounding soil, extending outward up to 2 cm. The hyphal extensions both absorb and translocate water and minerals back to the root.

Endomycorrhiza Arbuscules

Lysed epidermal and cortical cells

Root hair

Microbes and plant mucilages Rhizoplane Rhizosphere soil

Soil aggregates External hyphae Thick-walled spores

Sloughed root cap cells

FIGURE 3.1 Morphological and ecological features of mycorrhizal and nonmycorrhizal roots. Mycorrhizal roots develop an equivalent of the rhizosphere called the mycorrhizosphere. From Linderman, R.G., 1988. Mycorrhizal interactions with the rhizosphere microflora: the mycorrhizosphere effect. Phytopathology 78, 366e371; reproduced by permission.

82

3. Grapevine structure and function

Mycorrhizal fungi are more effective at mineral absorption than are root hairs, at least partially due to their high surface area/volume ratio. The small diameter of their hyphae also permits their penetration into more minute soil pores than root hairs. Mineral mobilization and uptake are further aided by the release of hydroxamates (peptides), oxalate, citrate, and malate by the hyphae. This feature is particularly valuable with poorly soluble inorganic nutrients, such as phosphorus, zinc, and copper. Phosphorus is one of most immobile of essential soil nutrients, and its diffusion in dry soils may decline to 1%e10% of that in moist soils. Carbohydrates supplied by the vine sustain fungal growth. These are “exchanged” for inorganic nutrients, notably phosphorus and ammonia, but also nickel, sulfur, manganese, boron, iron, zinc, copper, calcium, and potassium. Mycorrhizal associations also provide the vine with some protection from the toxic effects of potential soil contaminants, notably lead and cadmium (Karagiannidis and Nikolaou, 2000). The benefits of mycorrhizal associations are particularly noticeable in soils low in readily available nutrients and under dryland-farming conditions. Under limiting growth conditions, the shoot conducts additional carbohydrates to the roots, which encourages mycorrhizal development. In contrast, where access to inorganic nutrients is adequate, less carbohydrate is allocated to the roots, and mycorrhizal expansion is limited. Under drought conditions, mycorrhizal fungi augment water uptake and transport to the host, enhance stomatal conductance and transpiration, and accelerate recovery from stress (see Allen et al., 2003). Both root and mycorrhizal exudates influence soil texture and the soil flora. These effects tend to be beneficial, partially due to the selective favoring of nitrogenfixing and ethylene-synthesizing bacteria around the roots (Meyer and Linderman, 1986). These changes increase root resistance, or at least tolerance to fungal and nematode infections. For example, root inoculation with Glomus mosseae reduced the incidence of grapevine “replant disease” in affected nursery soils (Waschkies et al., 1994). Mycorrhizal associations also tend to reduce vine sensitivity to salinity and mineral toxicities. By affecting the level of growth regulators, such as cytokinins, gibberellins, and ethylene, mycorrhizal fungi further influence vine growth. An additional indirect effect may be the promotion of chelator production, such as catechols by soil bacteria. These help keep minerals, such as iron, in a biologically available form. Finally, mycorrhizae contribute to the formation of a stable soil-crumb structure. In most vineyard soils, mycorrhizal associations arise spontaneously from naturally occurring soil inocula. Inoculating cuttings in the nursery may be ineffectual

due to extensive rootlet mortality following transplantation (Conner and Thomas, 1981) and the ample presence of indigenous inocula. Specific inoculation tends to be of more value when vines are planted in fumigated soils devoid of an indigenous population of VeA fungi. Spontaneous infection is poorly understood, with spore populations tending to depend on the soil type and cover (being higher in sandy soils with groundcover). Establishment is also facilitated by a favorable rhizosphere flora but impeded by fungicidal applications. In addition, soils low in available phosphorus encourage mycorrhizal establishment (Karagiannidis et al., 1997), whereas soils rich in this nutrient limit its development. Differences in the incidence of colonization also occur among different rootstocks, usually being higher in cultivars that impart greater vigor to the scion. Nonetheless, these differences are small in comparison to factors such as crop load. Higher yield is associated with reduced numbers of roots with arbuscules (Schreiner, 2003), possibly due to a diminished translocation of carbohydrates to the roots. Although mycorrhizal associations are usually beneficial, there are situations where its formation may be ineffective or detrimental. If phosphorus is seriously deficient, colonization by mycorrhizal fungi may be unable to overcome the deficiency (Ryan et al., 2005). Under such conditions, metabolism of plant carbohydrates by the mycorrhizal fungus may reduce crop yield due to its “parasitic” action. In addition, mycorrhizae may increase the availability of, and thereby toxicity of, trace elements such as aluminum. Mycorrhizal fungi may also occasionally be associated with an increased incidence of lime-induced chlorosis (Biricolti et al., 1992).

Secondary tissue development As noted, the epidermis and root cortex often become infected by mycorrhizal fungi. These tissues may also succumb to microbial and nematode attack. This may result in the brown and collapsed regions commonly observed along otherwise healthy young roots. Regardless of health, the cortex and outer tissues soon die, especially if the central region of the root commences secondary (lateral) development (Fig. 3.2). Secondary growth includes the production of a band of cork (periderm) around the root just inside the endodermis. Its formation restricts and eventually cuts off nutrient flow to the cortex and epidermis. Another lateral meristem (vascular cambium) develops as a circular band around the root in the zone between the initial (primary) xylem and phloem. The cambium produces new (secondary) vascular tissuesdxylem and phloem to the inside and outside. This results in the formation of

83

The root system 1°xy 2°xy m 2°ph cc ck c

(E)

E

E

1°xy 2°xy 2°ph cc 1°ph en h ep

(D)

D

(C)

xy ph pe en c h ep

C

(B)

(A)

D

C

pI xy ph pe en c h ep

B

B

A

A

pI xy ph pe en c ep

FIGURE 3.2 Diagrammatic representation of a grapevine root showing anatomical and structural changes along its length. (A) White primary root, epidermis intact with root hairs. (B) Epidermis collapsed gives a brown appearance, hypodermis and endodermis suberized. (C) Cortical collapse (browning) and degeneration of all tissues. (D) Cortical collapse with expansion of the stele (vascular zone) due to development of secondary growth. (E) Woody, secondary growth with remnants of the cortex still attached. Abbreviations: ep, epidermis; h, hypodermis; c, cortex; en, endodermis; pe, pericycle; ph and 1 ph, primary phloem; xy and 1 xy, primary xylem; pi, pith; cc, cork cambium; ck, cork; m, medullary ray; 2 ph, secondary phloem; 2 xy, secondary xylem. From Richards, D., 1983. The grape root system. Hortic. Rev. 5, 127e168; reproduced with permission.

complete bands of xylem and phloem that encircle the root (Fig. 3.3). Periodically, cells produced in the vascular cambium differentiate into ray parenchyma instead of xylem or phloem tissues. Ray cells elongate along the radial axis of the root, forming what are termed medullary rays. They facilitate water and nutrient exchange between contiguous regions of the phloem and xylem. As the root enlarges in diameter, new cork layers may develop in the secondary phloem, cutting off nonfunctional phloem and older cork layers.

Root-system development In exploiting the soil for water and nutrients, the root system employs both extension and branching. Extension entails the rapid growth of thick leader roots into unoccupied soil, with the direction and extent of growth being dependent as much on water availability, soil penetrability, vine density, and rootstock/scion combination as positive geotropism (Seguin, 1972; McKenry, 1984). In favorable sites, many thin, highly branched,

84

3. Grapevine structure and function

FIGURE 3.3 Diagrammatic representation of the secondary growth in grapevine roots: 1, epidermis; 2, cortex; 3, endodermis; 4, pericycle; 5, primary phloem; 6, primary xylem; 7, vascular cambium; 8, secondary phloem; 9, secondary xylem; 10, medullary rays; 11, periderm; 12, pith. From Swanepoel, J.J., de Villiers, C.E., 1988. The anatomy of Vitis roots and certain abnormalities. In: Van Zyl, J.L. (ed.), The Grapevine Root and its Environment, pp. 138e146. Department of Agricultural Water Supply Techical Communication No. 215. Pretoria, South Africa, reproduced by permission

lateral roots develop from leader roots. Most of the lateral feeder roots are short-lived and replaced by new laterals. Thus, few laterals survive and become a part of the permanent structural framework of the root system. Large roots are retained much longer than most young roots but are themselves subject to periodic abortion (McKenry, 1984). The largest roots generally develop within a zone 0.3e0.35 m below ground level. Their number and distribution tend to stabilize a few years after planting (Fig. 3.4). From this basic framework, smaller permanent roots spread outward, with a restricted number angled downward. These in turn produce most of the shortlived feeder roots of the vine. Tentative evidence suggests

Depth of soil (cm)

125 105

58-year-old vine 12-year-old vine

85 65 45 25

10

30

50

Percentage of roots by weight

FIGURE 3.4 Influence of vine age on root-system distribution. From Branas, J., Vergnes, A., 1957. Morphologie du syste`me radiculaire de la vigne. Prog. Agric. Vitic. 147, 1e47.

that a ratio of fine-to-larger roots (2 mm) greater than 3.5 indicates an effective root system (Archer and Hunter, 2005). This permits the vine to quickly take advantage of favorable conditions to fully mature its fruit. Root-system expansion is dependent on both environmental and genetic factors. Distribution may be limited by layers or regions of compacted soil, high water tables, or saline, minera - l, and acidic zones. Thus, it is judicious that the soil be properly assessed and corrective measures taken where necessary before planting. Tillage, mulching, and irrigation favor the optimal positioning of major root development but cannot offset unfavorable soil conditions. Increasing planting density tends to decrease root mass but increase root density (roots per soil volume). Differences in the angle and depth of penetration vary with the genetic potentialities of the rootstock and its interaction with the scion. For example, Vitis rupestris rootstocks are generally thought to sink their roots at a steeper angle and penetrate more deeply than Vitis riparia rootstocks, while the latter produce a shallower, more spreading root system (Bouard, 1980). However, an extensive review of the literature suggests that local soil conditions are more significant than genetic factors in affecting root distribution (Smart et al., 2006). Grapevine roots are characterized by their extensive colonization of the soil, principally horizontally, but at low rooting densities (Zapriagaeva, 1964). Despite extensive ramification, grapevine roots commonly occupy only about 0.05% of the available soil volume (McKenry, 1984). Most root growth develops within a zone 4e8 m around the trunk. Depth of penetration varies widely but is largely confined to the top 60e80 cm. Nevertheless, roots up to 1 cm in diameter

85

The shoot system

can penetrate to a depth of more than 6 m. The tendency for deep penetration may reflect evolutionary pressures related to competition with the roots of trees on which they tended to climb. The finest roots, which do most of the absorption, generally occur within the uppermost 0.1e0.6 m. This portion of the root system may be located lower depending on the depth of tilling or the presence of a permanent sward. Conversely, mulches or clean, no-till conditions allow root growth nearer the soil surface. Unlike many woody perennials, the initiation of spring root growth lags significantly behind shoot growth. This may relate to the requirement for auxin translocation from germinating shoot buds. The latter occurs later than in most other perennial plants. Auxins are required to activate cytokinin synthesis in the root tip, which in turn stimulates root growth. Root production in Russia commences slowly after bud break, when soil temperatures rise above 6
C (Khmelevskii, 1971). Mohr (1996) observed that root growth commenced slowly in the Mosel Valley, occasionally reaching a peak only in late summer, while Comas et al. (2005) in Pennsylvania found root production reached a peak near midseason (between bloom and ve´raison) (Fig. 3.5). Root production may closely follow canopy bud burst flowering

maturity

production but is also significantly influenced by yearly climatic fluctuations (Comas et al., 2010). For example, restricted water availability increased the root-to-shoot ratio (Hofa¨cker, 1976). Heavy pruning and delayed canopy development also affect root formation. A second, autumnal root-growth period may occur in warm climatic regions where the postharvest growth period was long (in South Africa) (van Zyl and van Huyssteen, 1987). In contrast, root growth may be unimodal in temperate climates. Root production may be pronounced in years following high yields, when starch levels in the canes are low. Signs of root mortality appear to correspond with fruit development and ripening. The effect of soil moisture on root production may be partially indirect by affecting soil aeration and mechanical resistance. Some cultivars appear to be particularly sensitive to anaerobiosis, with oxygen tensions as low as 2% becoming toxic (Iwasaki, 1972). Waterlogging also enhances salt toxicity (West and Taylor, 1984). Furthermore, soil moisture content affects the rate at which soil warms in the spring, slowing the resumption of root growth and the availability and movement of nutrients in the soil. Even relatively short periods of waterlogging (2 days) can significantly retard root growth (McLachlan et al., 1993). Root growth and distribution are also markedly influenced by soil cultivation, compaction, and irrigation.

leaf fall

The shoot system

160

Grapevine shoots possess an unusually complex developmental pattern. This complexity provides the vine with a remarkable ability to adjust to changing conditions throughout the growing season. It has also generated an equally complex terminology. Three or more successive sets of shoots may develop in a single year. In addition, dormant buds from previous seasons may become active. In recognition of this complexity, shoots may be designated according to their origin, age, position, or length. The buds, from which shoots arise, may also be identified relative to their position, germination sequence, and fertility.

Shoot length (cm)

120

80

Buds S

O

N

D

J

F

M

A

M

FIGURE 3.5 Relationship between the state of shoot growth (-) and root growth (:) for the variety Shiraz grown in the Southern Hemisphere. After Richards, D., 1983. The grape root system. Hortic. Rev. 5, 127e168; Richards, D., 1983. The grape root system. Hortic. Rev. 5, 127e168; from Freeman, B.M., Smart, R.E., 1976. A root observation laboratory for studies with grapevines. Am. J. Enol. Vitic. 27, 36e39, reproduced by permission.

All buds can be classed as axillary budsdbeing formed in the axils of foliar leaves or their modifications (bracts). Axils are defined as the positions along shoots where leaves develop. As such, axils are a part of a circular region of the stem called a node (see Fig. 2.1). Most structures that develop from shoots (leaves, buds, tendrils, and inflorescences) develop at nodes. The first two nodes of a shoot are very close and subtend bracts. Only the subsequent nodes, which generate

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3. Grapevine structure and function

LAT

3

1

2

LS

FIGURE 3.6 Transverse section through an axillary bud complex. LS, leaf scar; LAT, lateral bud; primary (1), secondary (2), and tertiary (3) buds of the compound bud. Bracts of the lateral (solid) and primary (hatched) structures are also shown. From Pratt, C., 1959. Radiation damage in shoot apices of Concord grape. Am. J. Bot. 46, 103e109, reproduced by permission.

photosynthetic leaves, are usually considered when counting shoot nodes. These are frequently referred to as count nodes. Most stem elongation occurs in the region separating adjacent nodes, the internodes. Buds occurring in the axils of bracts (leaves modified for bud protection) are often termed base buds. They typically remain dormant and thus are termed latent buds. Depending on their location, they may give rise to water sprouts or suckers (see Fig. 4.11) based on whether they originate above or below ground. Each node can potentially produce an axillary-bud complex consisting of four buds on a foreshortened shoot. These include the lateral (prompt or true axillary) bud positioned to the dorsal side of the shoot and a compound bud that is positioned more ventrally (Fig. 3.6; Plate 3.1). It typically remains dormant until the

PLATE 3.1 Longitudinal section of a grape bud showing the three growing points or buds. Photo courtesy of the late R. Pool, New York State Agricultural Experimental Station, Cornell University, Geneva, NY.

subsequent season. The compound bud typically possesses three buds in differing states of development referred to as the primary, secondary, and tertiary buds. In a notation system proposed by Bugnon and Bessis (1968), the shoot is termed N, the first (lateral) bud is denoted Nþ1, the second (primary) bud is designated Nþ2, and the third (secondary) and fourth (tertiary) buds are considered Nþ31 and Nþ32, respectively. Depending on genetic and environmental factors, lateral buds may differentiate into shoots during the season in which they are produced. More commonly they remain dormant, becoming latent buds. Shoots developing from lateral buds (Nþ1) in the year of their formation are termed lateral (summer) shoots. There is considerable dissension among grape growers as to whether lateral shoots should be retained, trimmed, or removed. Initially, lateral shoots act as a drain on vine photosynthate. However, they later can act as net carbohydrate exportersdwhen possessing two or more fully expanded leaves (Hale and Weaver, 1962). Lateral shoots that terminate their growth in midseason can benefit fruit ripening (Candolfi-Vasconcelos and Koblet, 1990) but may reduce fruit set earlier in the season (Vasconcelos and Castagnoli, 2000). These divergent effects may be explained by the relative maturity of the leaves at different stages during fruit production. In vigorous vines, lateral shoots can lead to dense canopies, generating excessive shading, enhancing disease susceptibility, and reducing fruit quality. Nonetheless, moderate shading can be beneficial in very sunny climates as well as in modifying fruit flavors in ways that may suit particular wine styles. In addition, fruitbearing lateral shoots may produce what is called a “second” crop. In most vinifera cultivars, lateral buds typically remain dormant unless damage or early shoot pruning removes the source of auxins that induce their dormancy. Lateral buds in other Vitis spp. and Frenche American hybrids are more likely to germinate during the growing season. Secondary lateral buds, formed in the leaf axils of lateral shoots, also possess the potential to develop during the current year. If they do, they may generate a second series of lateral shoots. The primary (Nþ2) bud develops in the axil of the bract (prophyll) produced by the lateral bud (Nþ1) of a compound bud. Secondary and tertiary buds develop in the axils of the bracts produced by the primary and secondary buds, respectively. All three buds typically remain inactive during the growing season in which they develop unless severe summer pruning or other trauma removes apical (auxin-induced) exogenous dormancy. By autumn, compound buds develop endogenous (self-imposed) dormancy. By this time the compound bud is vernacularly referred to as an “eye.” Concurrent with the development of endogenous dormancy, bud moisture content declines from about

The shoot system

80% to 50%, starch grains become prominent, signs of mitosis disappear, catalase activity declines, and respiration falls to its lowest level (Lavee and May 1997). The sequence of steps by which dormancy establishes itself is still unclear, but it is associated with an increase in the content of cis-abscisic acid (Koussa et al., 1994) and the production of a particular set of glycoproteins (Salzman et al., 1996). Release from dormancy typically requires exposure to low temperatures (Fig. 3.7). In temperate zones, this may commence in late fall and finishes during the winter months. Although the biochemical processes involved are unknown, they appear to involve an increase in hydrogen peroxide production and the 100

Control

80 60

Chill hours (3°C) 0 50 100 200 400 800

40 20 0 Cumulative budbreak (%)

100

1.25% H2CN2

80 60 40 20 0 100 2.50% H2CN2

80 60 40 20 0

0

10

20

30 40 Days at 22°C

50

60

FIGURE 3.7 Influence of chilling exposure and hydrogen cyanamide concentration on the cumulative budbreak of Perlette cuttings. Cuttings were kept under continuous white light (photon flux density ¼ 100 mmol m2 s1) at 22
C. From Dokoozlian, N.K., Williams, L.E., Neja, R.A., 1995. Chilling exposure and hydrogen cyanamide interact in breaking dormancy in grape buds. Hortscience 30, 1244e1247, reproduced by permission.

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development of oxidative stress associated with low levels of catalase. This is consistent with the action of hydrogen cyanamide (Sudawan et al., 2016). It may be applied in situations where early bud break is desired (Fig. 3.7). Hydrogen cyanamide depresses transcription of the catalase gene (Or et al., 2002). Hydrogen cyanamide application is also associated with the activation of genes that produce a dormancy-breaking protein kinase and genes producing pyruvate decarboxylase and alcohol dehydrogenase in buds (Or et al., 2000). Genes encoding enzymes such a glutathione reductase, glucose-6-phosphate dehydrogenase, and 1,3,-b-glucanase also appear to be involved (Vergara and Pe´rez, 2010), possibly by increasing the availability of NADH (Pe´rez et al., 2008). In tropical and subtropical regions, severe pruning usually can induce premature bud break. However, it is more effective and uniform if combined with hydrogen cyanamide application. Garlic extract may be substituted but is less effective (Botelho et al., 2007). Sublethal heat shock can also facilitate bud break under greenhouse conditions and is being proposed for vineyard application (Zion et al., 2012). Assuming that the primary buds are not destroyed by freezing temperatures, insect damage, or pathogenic or physiological disturbances, they generate the primary shoots (major shoot system) of each year’s growth. The secondary and tertiary buds become active only if the primary and secondary buds, respectively, die. The primary bud is the most developed in the compound bud. It usually possesses several primordial (embryonic) leaves, inflorescences, and lateral buds before becoming dormant by the end of the growing season (Morrison, 1991). The secondary bud occasionally is fertile, but the tertiary bud is typically infertile (bears no inflorescences). The degree of inflorescence differentiation prior to the onset of autumnal dormancy is a function of the genetic characteristics of the cultivar, vigor of the rootstock, bud location, and the immediate surrounding environment during bud formation. Axillary buds that bear nascent (primordial) inflorescences are designated as fruit (fertile) buds to differentiate them from those that do not. The latter are referred to as leaf (sterile) buds. In most Vitis vinifera cultivars, fruit buds form with increasing frequency distal to the basal leaf, often reaching a peak between the fourth and tenth count nodes. However, this property varies considerably among cultivars (Fig. 3.8). Some cultivars, such as Nebbiolo, do not form fruit buds at the base of the shoot. Such cultivars are not spur-pruned, as this would drastically limit fruit production. Bud fruitfulness also influences pruning procedures. For example, the production of small fruit clusters by Pinot noir usually requires the retention of long canes (and thereby more fruit buds).

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3. Grapevine structure and function

Fertility (number of inflorescences)

2.2 2.0 1.8 1.6 1.4 1.2 Silvaner Riesling

1.0

Gewurztraminer

0.8

Pinot Blanc

0.6 1

2

3

4

5

6

7

8

9

10

11

12

13

14

Bud position along the cane

FIGURE 3.8 Influence of bud position on shoot fertility. Data from Huglin, P., 1986. Biologie et e´cologie de la Vigne. Payot Lausanne, Paris, France.

Shoots derived from fruit buds generally produce two (one to four) flower clusters. These typically differentiate opposite the third and fourth, fourth and fifth, or fifth and sixth leaves on the shoot. Although inflorescence number and position are predominantly scion characteristics, they may be modified by hormonal signals coming from the rootstock (see Richards, 1983).

Shoots and shoot growth Components of the shoot system obtain their names from the type and position of the buds from which they are derived as well as their age, position, and relative length (see Fig. 4.11). By the end of the season, the outer photosynthetic subepidermal tissues degenerate, turning brown. At this point the shoot is termed a cane. It retains this name if left long, or a spur if pruned short, retaining but a few buds. Canes retained for 2 years or more and supporting fruiting wood (spurs or canes) are called arms. When these form the major upright structure of the vine, they are referred to as a trunk, whereas if positioned horizontally, they are called cordons. When old and thick, they may no longer need support. Nevertheless, most trunks, cordons, canes, and growing shoots are supported by a trellis. All stem tissue two or more years old is termed “old wood.” Shoot growth is controlled primarily by environmental factors. Because grapevines do not produce terminal buds, growth could theoretically continue as long as climatic conditions permitted. This growth habit, termed indeterminate, is much more characteristic of tropical than temperate woody plants. Shoot growth is favored by warm conditions, especially warm nights. Low-light conditions promote shoot elongation but are detrimental

to inflorescence induction. Genetic factors, probably acting through hormone production, generate the distinctive growth patterns typical of various cultivars. These tendencies can be modified by pruning and other cultural practices. For example, minimal pruning induces more but shorter thinner shoots. Bud break and shoot growth have generally been thought to begin when the mean daily temperature reaches >10
C following the loss of endogenous dormancy. However, bud break in some varieties may commence at temperatures as low as 0.4
C (average 3.5
C), and leaf production may begin at 5
C (average 7
C) (Moncur et al., 1989). The rate of bud break and shoot growth increases rapidly above the minimum temperature (Fig. 3.9). Once initiated, growth quickly reaches a maximum, after which growth tends to slow and may cease. Further shoot growth during the summer generally originates from the activation of lateral buds. The slow initiation of growth in the spring and the potential for lateral shoot growth throughout the season probably reflect the ancestral growth habit of the vine. Slow bud activation would have delayed leaf production until its supporting tree had completed most of its foliage production. Thus, the vine could position its leaves, relative to the foliage of the host, in locations optimally suited for its own photosynthesis. In eastern North America, growth can be sufficiently rampant to eventually result in the death of the host tree. In addition, the potential to generate several series of lateral shoots allows the vine to position new leaves in favorable sites when and where necessary. Older portions of the vine provide the support and translocation needs for growing shoots, leaves, and fruit. The woody parts of the vine constitute the majority of its structure. Mature wood also acts as a significant storage

The shoot system

but is most marked after fruit ripening (Conradie, 1990, 1991a). It appears that some of the first nutrients mobilized in the spring are those last stored in the fall. Although shoot growth can continue into the fall, this is usually undesirable. Not only is shoot maturation delayed and bud survival reduced, but it also draws nutrients away from ripening fruit. Ideally, shoot growth termination should correlate with the onset of ve´raison. Various procedures may be used to promote this termination. These may include inducing limited water deficit, trimming of shoot tips, or the use of devigorating rootstocks. In hot Mediterranean climates, shoot growth may terminate shortly after flowering when little more than a meter long. In contrast, shoots in more temperate, moister climates may grow 4 m or more.

0.06 Rate of bud break (buds/day)

89

0.05

0.04

0.03

0.02

0.01

5

10 15 20 25 Weighted-mean temperature °C

30

FIGURE 3.9 Determination of base temperature (Y) for bud break of Pinot noir from the effect of temperature on the rate of bud break. From Moncur, M.W., Rattigan, K., Mackenzie, D.H., McIntyre, G.N., 1989. Base temperatures for budbreak and leaf appearance of grapevines. Am. J. Enol. Vitic. 40, 21e26, reproduced by permission.

organ, thereby helping to cushion the effects of unfavorable growth conditions on fruit production during any one season. These reserves also tend to increase average vine productivity. Berry sugar content and pH increase slightly relative to the portion of old wood (Koblet et al., 1994). In addition, increased berry aroma and fruit flavor have been correlated with vine age (Heymann and Noble, 1987; Du et al., 2012) or with the proportion of old wood (Reynolds et al., 1994). Whether these associations are direct effects of vine age (e.g., increased old tissue relative to new shoot and foliage tissue, more profound root penetration), indirect effects of age-related reduced vigor (e.g., accumulated effects of chronic fungal or viral infections), other factors (e.g., increased flavor diversity of fruit for differently aged vine), or some combination is unestablished. Early shoot growth depends primarily on nutrients previously stored in the vine’s woody parts (May 1987). Nitrogen mobilization and translocation to the buds initiates in the canes and progresses downward, successively coming from older parts of the vine and finally reaching the roots (Conradie, 1991b). Significant nutrient mobilization and translocation from the older parts of the vine to the developing fruit may also occur following ve´raison (the onset of color change in the fruit). The vine stores organic nutrients, primarily as starch and arginine as well as inorganic nutrients, such as potassium, phosphorus, zinc, and iron. The movement of nutrients, such as nitrogen, downward and into woody parts of the vine probably occurs throughout the growing season

Tissue development Shoot growth develops from an apical meristem that consists of two layers of ill-defined tunica cells covering an inner collection of corpus cells (Morrison, 1991, Fig. 3.10). The outer layers produce the epidermal tissues of the stem, leaves, tendrils, flowers, and fruit and initiate the development of leaf and flower primordia. The corpus generates the inner tissues of the shoot and associated organs. The shoot apex, unlike its root equivalent, has a highly compressed, complex morphology. Buds, leaves, tendrils, and flower clusters all begin their development within a few millimeters of the apex (Fig. 3.11). Subsequent cell division, differentiation, enlargement, and elongation produce the mature structures of each organ.

FIGURE 3.10 Median longitudinal section through a dormant shoot apex of Vitis labrusca. AM, Apical meristem; Corp, corpus; D, diaphragm; L, leaf primordium; RM, rib meristem; T, tendril initiation; Tu, two-layered tunica. From Pratt, C., 1959 Radiation damage in shoot apices of Concord grape. Am. J. Bot. 46, 102e109, reproduced by permission.

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3. Grapevine structure and function

FIGURE 3.11 Longitudinal section of the shoot tip of Concord grape. From Pratt, C., 1974. Vegetative anatomy of cultivated grapes, a review. Am. J. Enol. Vitic. 25, 131e148; Pratt, C., 1974. Vegetative anatomy of cultivated grapes, a review. Am. J. Enol. Vitic. 25, 131e148, reproduced by permission.

As in the root, shoot phloem is the first vascular tissue to differentiate. The need for organic nutrients by the rapidly dividing apical cells undoubtedly explains the rationale for this morphogenic sequence. Cell elongation, which primarily entails water uptake, occurs later. Thus, xylem differentiation can be delayed and occurs further back. Early xylem development could also complicate shoot elongation. Even when they begin to differentiate, the first vessels possess only partial (annular or spiral) secondary wall thickening. This allows for some vessel elongation by stretching areas consisting primarily of the more flexible primary cell wall. Full, secondary, pitted and lignified thickening occurs only where elongation has ceased. In contrast, the earliest thin-walled phloem cells are destroyed as the shoot tip elongates. New phloem sieve tubes differentiate to replace those destroyed by cell elongation. The outer tissues of the young stem consist of a layer of epidermis and several layers of cortical cells (Fig. 3.12). The epidermis is initially photosynthetic and bears stomata, hair cells, and pearl glands, especially adjacent to where vascular bundles differentiate. Most young cortical cells also contain chloroplasts and are photosynthetic. Nonetheless, the cortex tends to function more as a temporary storage tissue, notably for carbohydrates (primarily as starch) but also proteins. The innermost cortical layer is distinguished by possessing large starch granules termed the starch sheath. Collections of cortical cells with especially thickened side walls, termed collenchyma, differentiate opposite the vascular bundles. These regions generate the ridges (ribs) of the young shoot. The region, internal to the zone where vascular bundles develop, is termed the pith. It consists of relatively undifferentiated

FIGURE 3.12 Transverse section of a seedling stem of Vitis vinifera. E, epidermis; C, cortex; F, collenchyma fibers; Ph, phloem; X, xylem; Pi, pith. Modified from Esau, K., 1948. Phloem structure in the grapevine, and its seasonal changes. Hilgardia 18, 217e296.

parenchyma cells. These become increasing large toward the center. When cell elongation ceases, a band of cells between the phloem and xylem begins to differentiate into a lateral meristem, the vascular cambium. It generates the secondary vascular and ray tissues of the maturing stem (Plate 3.2). The secondary phloem, similar to the primary, consists of translocating cells (sieve tubes), companion cells, fibers, and storage parenchyma. The secondary xylem consists of tracheids and larger diameter thickened xylem vessels as well as structural fibers and parenchyma cells. Ray cells elongate vertically along stem axis, providing for cross-flow transport between the xylem and phloem. Special vessel relays also

PLATE 3.2 Cross-section of a young shoot: basal internode that is no longer elongating but is growing thicker. Photo courtesy M. Goffinet, Department of Horticultural Sciences, New York State Agricultural Experimental Station, Cornell University, Geneva, NY.

The shoot system

connect adjacent xylem vessels both tangentially and radially via scalariform pitting (Brodersen et al., 2013). These vessel interconnections limit the effects of air embolism but facilitate xylem-borne pathogen dispersion. In the phloem, ray tissue expands laterally to form Vshaped segments (Plate 3.3). These cells store starch along with xylem parenchyma. As secondary phloem and xylem accumulate, the vascular bundles take on the appearance of elongated lenses in cross-section. The innermost stem tissue, the pith, consists predominantly of thin-walled parenchyma cells. In species belonging to the subgenus Vitis, the pith soon disintegrates in all but the nodal region, where it develops into the woody diaphragm (see Fig. 2.3A). Environmental conditions during growth can significantly affect xylem development. For example, leaf shading (Schultz and Matthews, 1993) and water deficit (Lovisolo and Schubert, 1998) slow growth, possibly as a consequence of reduced water conductance and disrupted hormonal movement. Positioning shoots downward also reduces vessel diameter while increasing their number (Schubert et al., 1999). This influence has occasionally been used to reduce vine vigor. The effects of shoot orientation on water conductance appear to be related to increased auxin levels (Lovisolo et al., 2002), promoting vessel division but limiting expansion. Reduced vessel diameter increases resistance to water flow but limits the likelihood of embolism (the formation of gas pockets in vessels that disrupts water flow). Cultivars differ markedly in their susceptibility to embolism (Alsina et al., 2007). Nonetheless, the significance given to embolism in the past may have been exaggerated. In some plants, it is a normal and daily occurrence,

PLATE 3.3 Internal anatomy of a cane: taken from a brown internode. Photo courtesy M. Goffinet, Department of Horticultural Sciences, New York State Agricultural Experimental Station, Cornell University, Geneva, NY.

91

with gas pockets reabsorbing shortly after forming despite the water column still being under negative water potentials (McCully, 1999). During shoot maturation, a layer of parenchyma cells in the phloem differentiates into a cork cambium (phellogen) (Plate 3.3). The cork (periderm) produced restricts nutrient and water supplies from reaching the outer tissues (mostly cortex and epidermis). These subsequently die, slough off, and create the brown appearance of maturing shoots. In most regions, with the probable exception of the tropics, the end walls (sieve plates) of the sieve tubes become plugged with callose in the autumn, terminating translocation of organic nutrients. This occurs as the leaves die in the autumn. When buds become active in the spring, phloem cells in the cane and older wood progressively regain their ability to translocate. Activation progresses longitudinally up and down the cane from each bud and outward from the vascular cambium (Aloni and Peterson, 1991). The rootward movement of auxins, produced by newly emerging leaves, appears to stimulate callose breakdown (Aloni et al., 1991). Although cambial reactivation is associated with bud activation, it shows marked apical dominance. Thus, activation starts in the uppermost buds and progresses downward and laterally around the canes and trunk until the enlarging discontinuous patches meet (Esau, 1948). The activated cambium recommences producing new xylem and phloem tissue (Plate 3.4). Unlike either the phloem or the cambium, mature xylem vessels contain no living material and are potentially functional whenever the temperature permits water to exist in a liquid state. Phloem cells in the trunk may remain viable for upwards of 4 years, but most sieve tubes are functional only during the year they form (Aloni and Peterson, 1991). In the xylem, vessel inactivation begins about 2e3 years after formation but is complete only after 6e7 years. Inactivation involves tyloses that grow into the vessel lumen from surrounding parenchyma cells. These paratracheal parenchyma cells also produce tyloses and gels that occlude vessels in response to wounding. Tyloses form primarily during the growing season, whereas gels are deposited principally during the winter (Sun et al., 2008). In woody stem sections, except for functional xylem and phloem cells, most cells function in storing nutrients, notably starch. This may even include old xylem vessels plugged with tyloses. In the spring, most starch is remobilized and translocated to growing shoots. In members of the subgenus Vitis, new cork cambia develop at infrequent intervals in nontranslocatory regions of the secondary phloem. Tissues external to the developing cork cambium, largely old phloem (and initially remnants of the cortex and epidermis), die

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3. Grapevine structure and function

opposite most leaves. On bearing shoots, flower clusters replace the tendrils in the lower two or more locations in most V. vinifera cultivars, and in the basal three to four tendril locations in V. labrusca cultivars.

Leaves

PLATE 3.4 Anatomy of perennial woody stem in spring. Photo courtesy M. Goffinet, Department of Horticultural Sciences, New York State Agricultural Experimental Station, Cornell University, Geneva, NY.

and turn brown. The tissues subsequently split and are eventually shed. In contrast, in the subgenus Muscadinia, the cork cambium forms under the epidermis and persists for several years. Thus, canes of the Muscadinia do not form shedding bark like Vitis species (see Fig. 2.3D).

Tendrils Tendrils are modified flower clusters inhibited from completing floral development by gibberellins (Srinivasan and Mullins, 1981; Boss and Thomas, 2002). Tendril morphogenesis also depends on the balance between gibberellins and cytokinins (Srinivasan and Mullins, 1979). Flower clusters are themselves viewed as modified shoots. Not surprisingly, tendrils bear shoot-like features. However, unlike vegetative shoots, tendril growth is determinant; that is, growth is strictly limited. Tendril development also passes through three developmental and functional phases. Initially, tendrils develop water-secreting openings called hydathodes at their tips. Subsequently, the hydathodes degenerate and pressuresensitive cells develop along the tendril. On contact with solid objects, these specialized cells activate elongation and cellular growth on the opposite side of the tendril. This induces twining of the tendril around the object touched. With differentiation of collenchyma in the cortex and xylem and lignification of ray cells, the tendril becomes woody and rigid at maturity. With the exception of Vitis labrusca and its cultivars, tendril production develops in a discontinuous manner in Vitis; that is, tendrils are produced opposite the first two of every three leaves distal to the first two to three basal leaves. In V. labrusca, tendrils are produced

Leaves develop as localized outgrowths just underneath the epidermis of the apical meristem. Cortical cells differentiate to connect the developing shoot vascular system with the vascular bundles in the developing leaf. These branches of the vascular system are termed leaf traces. They originate slightly below the leaf itself in the tightly compacted shoot tip (see Fig. 3.11). The first two leaf-like structures that develop in a bud develop into protective bracts. The internodes between the bracts remain truncated. Subsequent internodes elongate normally, and the leaf primordia expand into mature leaves. Maturing leaves consist of a broad, photosynthetically active blade, a supportive and conductive petiole, and two basal short-lived semicircular stipules. Unlike the growth of most plant structures, leaf expansion entails the action of many variously positioned plate meristems (Fig. 3.13) rather than apical or lateral meristems. This unique growth pattern generates the flat, distinctly lobed appearance that characterizes each cultivar. Like most vascular plants, grape leaves consist of an upper and lower epidermis, a palisade layer, several layers of spongy mesophyll (Fig. 3.14), and a few large (and multiple small) veins. The epidermis consists of a single layer of flattened cells with irregularly undulating edges resembling the pieces of a jigsaw puzzle. Within these basic aspects, there can exist considerable variation in cell shape and size among cultivars (Boso et al., 2011). The upper epidermal wall is covered by a cuticle consisting of an inner layer of cutin and an outer waxy component. The latter consists of upward-projecting wax plates. Cutin is composed of hydroxyl and hydroxy-epoxy fatty acids, whereas the waxy plates consist of a complex mix of long-chain aliphatic and cyclic compounds. The cuticle (notably its lipophilic, waxy outer layer) retards water and solute loss (Scho¨nherr, 2006), helps limit the adherence of pathogens, minimizes mechanical abrasion of the leaf, and slows the diffusion of chemicals into the leaf. The lower epidermis shows a less well-developed cuticle and possesses stoma. Although stomatal shape, density, and distribution are varietal characteristics (Fig. 3.15), these features are also influenced by environmental conditions. For example, stomatal density is reduced under warm soil conditions, increased CO2 concentration, and higher starch levels in the root and trunk (Rogiers et al., 2001). The undersides of leaves also typically possess

Leaves

FIGURE 3.13 Illustration of a young grape leaf (Pinot noir C.3309) showing the directions of blade expansion and xylem differentiation. Blade expansion occurs by intercalary growth along and between the major veins. Vessel differentiation occurs away from areas where growth has slowed or ceased. From Fournioux, J.-C., 1972. Distribution et diffe´renciation des tissus conducteurs primaires dans les organes aeriens de Vitis vinifera L. PhD Thesis, University of Dijon, Dijon, France, reproduced by permission.

93

leaf hairs and pearl glands. The latter derive their name from the small, bead-like secretions they produce. Water-secreting hydathodes commonly develop at the pointed tips (teeth) of the blade. The palisade layer consists of cells directly below and elongated perpendicularly to the upper epidermis. When the leaf is young, the cells are tightly packed, but intercellular spaces develop as the leaf matures. In leaves that develop in bright sunlight, the palisade cells are shorter but thicker than those formed in the shade. The palisade layer is the primary site of photosynthesis, although spongy mesophyll cells are also involved. The number of mesophyll layers (typically 5e6) is reduced when the leaf develops under sunny conditions. The cells of the mesophyll are extensively lobed, creating large intercellular spaces in the lower portions of the leaf. The large surface area thus generated, along with the stomata, facilitates the diffusion of water and gases between the inner leaf cells and the surrounding air. Without efficient evapotranspiration, the leaf would rapidly overheat in full sun, suppressing photosynthesis. Effective gas circulation is equally important for the rapid exchange of CO2 and O2. Carbon dioxide is an essential ingredient in photosynthesis, and oxygen (one of its by-products) inhibits the crucial carbonfixing action of ribulose bisphosphate carboxylase. Branching of the vascular tissues into the leaves (traces) is complex. Four to eight traces diverge from the vascular cylinder well below the leaf’s origin. The traces often fuse (anastomose) prior to entering the petiole, giving rise to the main veins. Their subsequent division generates the leaf’s distinctive (often varietal) venation pattern. The vascular tissues translocate

FIGURE 3.14 Transverse (A) and epidermal view (B) of a grape leaf. C, Cuticle; LE, lower epidermis; P, palisade layer; S, stomatal apparatus with guard cells; SM, spongy mesophyll; UE, upper epidermis. After Mounts, B.T., 1932. Development of Foliage Leaves. University Iowa Studies. Nat Hist vol. 14, 1e19, reproduced by permission.

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veins containing a single bundle. Each bundle consists of xylem toward the upper (adaxial) surface and phloem toward the lower (abaxial) surface. At their extremities, they may consist of just a few tracheids. The vascular tissues of larger veins are surrounded by thickened cells, termed the bundle sheath. The latter may extend to the upper and lower epidermis. Xylem translocates water and minerals, such as calcium, manganese, and zinc, whereas the phloem transports organic compounds and minerals such as potassium, phosphorus, sulfur, magnesium, boron, iron, and copper. The main organics transported are sugars (primarily sucrose), amino acids (predominantly arginine), and growth regulators. Because of the divisions created by the bundle sheath extensions, the leaf is subdivided into many relatively distinct compartments with little lateral gas exchange. Under conditions of water deficit, low atmospheric humidity, or saline conditions, each compartment may show distinct differences (patchiness) in its respective rates of transpiration and photosynthesis (Du¨ring and Loveys, 1996). Such leaves are termed heterobaric. In the autumn, abscission layers form at the base of the leaf blade and petiole. Exchanges between the shoot and the leaf cease when a periderm forms between the stem and the petiolar abscission layer. This is preliminary to leaf fall.

Photosynthesis and other light-activated processes

FIGURE 3.15 Density and morphology of stomata of Blanco Legı´timo, 110-Richter, and SO4. Reproduced from Boso, S., Gago, P., Alonso-Villaverde, V., Santiago, J.L., Mendez, J., Pazos, I., et al., 2011. Variability at the electron microscopic level in leaves of members of the genus. Vitis. Sci. Hortic. 128, 228e238. Copyright Elsevier, reproduced by permission.

nutrients to and from the blade and provide some physical support. A similar pattern of vascular development occurs in structures that are developmentally modified leaves (e.g., flower parts). The branching and anastomosis of the vascular system forms a net-like patchwork through the leaf. The network consists of a few large veins containing several vascular bundles and multiple progressively smaller

The production of organic compounds from carbon dioxide and water, energized by sunlight, is the quintessential attribute of most plant life. While sugar is the major organic by-product of photosynthesis, it also provides the intermediates for fatty acid, amino acid, and nitrogen base synthesis. From these, the plant can derive all its organic needs. The process is summarized in Fig. 3.16. Chlorophyll molecules, in a complex called photosystem II (PS II), absorb light energy. This is used to split water molecules into its component elements. The oxygen atoms liberated combine to form molecular oxygen, escaping into intercellular spaces of the leaf and subsequently diffusing out into the surrounding air. In contrast, the liberated hydrogen ions dissolve in the lumen (central fluid) of the chloroplast thylakoidd membrane-enclosed, disk-like structures found in chloroplasts (Fig. 3.16). Energized electrons, removed from the hydrogens during the splitting of water, are passed along an electron-transport chain embedded within the thylakoid membrane. Associated with the shunting of electrons along the transport chain, hydrogen ions are pumped across the thylakoid membrane into the central

Leaves

H 2O O2

ATP

NADPH2 PGA

GAP

RuDP CO2

Pool ATP

RuP

Photosynthates

FIGURE 3.16 Simplified diagram of a chloroplast with an idealized granum containing thylakoids, CO2 fixation, and assimilation via the Calvin cycle. GAP, glyceraldehyde-3-phosphate; PGA, 3phosphoglyceric acid; RuDP, ribulose bisphosphate; RuP, ribulose phosphate. From Larcher, W., 1991. Physiological Plant Physiology, second ed. Springer-Verlag, Berlin, Germany.

fluid (stroma) of the chloroplast. Because the thylakoid membrane is highly impermeable to hydrogen ions, an electrochemical differential develops across the membrane. This drives the movement of hydrogen ions back across the thylakoid membrane via ATPase complexes also embedded in the membrane. This transmembrane movement is associated with the phosphorylation of adenosine diphosphate, generating adenosine triphosphate (ATP). The partially deenergized electrons are subsequently passed along to chlorophylls in photosystem I (PS I), also embedded in the thylakoid membrane. These electrons are reenergized by photon energy absorbed by PS I. The electrons are subsequently passed to a second electron transport chain, finally being transferred to nicotinamide adenine dinucleotide phosphate (NADPþ). The combined result of both photosystems (PSI and PSII) is the production of reducing power (NADPH) and high energy phosphate bonds (ATP). Both NADPH and ATP are required in what is termed the Calvin cycle in the synthesis of sugars during the “dark,” CO2-fixing reactions of photosynthesis. The initial by-product of CO2 fixation that combines with a five-carbon sugar phosphate (RuBP) is extremely unstable and splits almost immediately into two molecules of 3-phosphoglyceric acid. These typically enter the Calvin cycle, regenerating additional molecules of RuBP. As increasing amounts of carbon dioxide are incorporated, enough carbon in incorporated to permit intermediates of the Calvin cycle to be diverted to the synthesis of sucrose and other organic compounds (Fig. 3.16) without disrupting the functioning of the

95

cycle. Quantitatively, sucrose is the most important by-product of photosynthesis to accumulate as well as being the major organic compound translocated in the phloem out of the leaf. Sucrose may also be temporarily stored via conversion into starch but is usually quickly transported out of the leaf to other parts of the plant. It may be subsequently stored at distant sites as starch granules; polymerized into structural components of the cell wall; metabolized in the synthesis of other organic constituents; or respired as an energy source in cellular metabolism. Because of the crucial role of photosynthesis to plant function, providing an optimal environment for its occurrence is an essential goal of canopy management. A favorable light environment also influences other photoactivated processes. These include inflorescence initiation, fruit ripening, and cane maturation. For photosynthesis, radiation in the blue and red regions of the visible spectrum is particularly important. For most other light-activated processes, it is the balance between the red and far-red portions of the electromagnetic spectrum that is crucial, as well as the ultraviolet regions. The differences relate to reaction-energy demands and the pigments involved. In photosynthesis, the important pigments are chlorophylls and carotenoids, whereas in most other lightactivated processes, phytochrome is involved. Both chlorophylls a and b absorb optimally in the red and blue portions of the electromagnetic spectrum, whereas carotenoids absorb primarily in the blue. The splitting of water, the major light-activated process in photosynthesis, is energy-intensive. Nonetheless, for an assortment of reasons, the overall rate of vine photosynthesis is maximally effective at about onethird full sunlight intensity (700e800 mmol/m2/s). This is imposed by rate-limiting factors inherent in the dark (CO2 fixing) reactions of photosynthesis. Due to extensive light absorption by the outer layers of the vine canopy, photosynthesis is minimal in the center of a dense vine canopy. Energy levels may drop to 15e30 mmol/ m2/s. In contrast, phytochrome-activated processes require little energy. For these, shifts between the two states of phytochrome depend on the relative intensities in the red and far-red portions of the spectrum. These regions correspond to the absorption peaks (650e670 nm and 705e740 nm) of the two alternate states of phytochrome, Pr and Pfr. Red light converts Pr to the physiologically active Pfr state, whereas far-red radiation transforms it back into Pr. In sunlight, the natural red/far-red balance (1.1e1.2) generates a 60:40 ratio between Pfr and Pr. However, when light passes through the leaf canopy, the strong red absorbency of chlorophyll shifts the red/far-red balance to about 0.1 (toward far-red). Thus, the relative activities controlled by phytochrome

96

3. Grapevine structure and function

(A)

(B)

Ambient R:FR = 1.19

1.2 Irradiance (W m–2 nm–1)

Irradiance (W m–2 nm–1)

1.2

0.8 Canopy face = 1.07 0.4 Layer 1 = 0.18 Layer 2 = 0.15

0 400

600

800 Wavelength (nm)

1000

Sun 615 W m–2 1.16 R:FR

0.8

0.4

0 400

Overcast 222 W m–2 1.21 R:FR 600

800

1000

Wavelength (nm)

FIGURE 3.17 Spectral distribution of ambient sunlight radiation at the canopy surface and under one- and two-leaf canopies (A), and between sunny and overcast days (B). From Smart, R.E., 1987. Influence of light on composition and quality of grapes. Acta Hortic. (Wagening.). 206, 37e47, reproduced by permission.

can vary depending on the light conditions of the tissue within a vine canopy. Regrettably, the level and actions of phytochrome in grapevine tissues are poorly understood. Because of the negative influence of shade on photosynthesis and modification to phytochrome-induced phenomena, most pruning and training systems are designed to optimize light exposure throughout the canopy. The effects of shading on spectral intensity and the red/far-red balance are illustrated in Fig. 3.17A. In contrast, cloud cover produces little spectral modification in the visible and far-red spectra, although it markedly reduces their intensity (Fig. 3.17B). Cloud cover does, however, significantly reduce the infrared (heat) portion of solar radiation received from the sun. Fig. 3.18 illustrates the daily cyclical effects of shade 330

a Control Single shading Double shading

300

Time radiation (W/m2)

270 240 210

b

180 150 120

c

90 60 30 0 09:00

11:00

13:00 Day time

15:00

FIGURE 3.18 Average total radiation in differently manipulated Sauvignon blanc canopies. From Marais, J., Hunter, J.J., Haasbroeck, P.D., Augustyn, O.P.H., 1996. Effect of canopy microclimate on the composition of Sauvignon blanc grapes. In: Stockley, C.S. (ed.), Proc. 9th Aust. Wine Ind. Tech. Conf. Winetitles, Adelaide, Australia, pp. 72e77, reproduced by permission.

on light conditions within different layers of the canopy. Because photosynthesis is usually maximal at light intensities equivalent to that provided by a slightly overcast sky, cloud cover has its most marked effect on the photosynthesis of shaded leaves. Canopy shading affects pigmentation as well as leaf physiology. In response to shading, pigment content increases (carotenoids and chlorophylls a and b), and the compensation point (the light intensity at which the rates of photosynthesis and respiration are equal) decreases to about 10e30 mmol/m2/s. Nevertheless, the lower respiratory rate of cooler, shaded leaves, their enhanced pigmentation, and the reduced compensation point do not fully redress the poorer spectral quality and diminished intensity of shade light. As a result, shade leaves generally do not photosynthesize sufficiently to export sucrose or contribute significantly to vine growth. This was dramatically shown by Williams et al. (1987), where removal of shade leaves (30% of foliage) had no effect on the timing of berry growth or maturity. This situation might change in windy environments, however. Movement of the exterior canopy could markedly increase sunflecking (periodic exposure to direct sunlight). Although sunflecking is highly variable, average rates of 0.6 s per 2 s interval could significantly improve shadeleaf net photosynthesis (Kriedemann et al., 1973). Sunflecking also appears to delay premature leaf senescence and dehiscence. When young and rapidly unfolding, leaves act as carbohydrate sinks rather than as net sources of photosynthate. Leaves begin to export photosynthates (sugars and other organic by-products) when they reach about 30% of their full size. At this stage they develop their mature green coloration. Nonetheless, leaves continue to import carbohydrates until they have reached 50% e75% of their mature size (Koblet, 1969). Maximal photosynthesis and sugar export occur about 40 days

97

Leaves

after unfolding when the leaves have fully expanded (Fig. 3.19). Old leaves continue to photosynthesize, but their contribution to vine growth declines significantly (Poni et al., 1994). The rate of photosynthetic decline varies considerably depending on the light environment, nitrogen availability, and new leaf development. Leaves cease to be net producers when they lose their dark-green coloration. Leaves formed early in the season may photosynthesize at up to twice the rate of those produced later. The angle of the sun, relative to the leaf blade, also influences photosynthetic rate. Nevertheless, even leaf blades aligned parallel to the sun’s rays may photosynthesize at rates up to 50% those of perpendicularly positioned leaves (Kriedemann et al., 1973). This probably reflects the importance of diffuse sky light, the benefits of a cooler leaf temperature, and maximal photosynthetic rates occurring at well below full sunlight intensity. The effect of temperature on photosynthesis varies slightly throughout the growing season. In the summer, optimal fixation tends to occur between 25 and 30
C, whereas in the autumn, the optimum may decline to between 20 and 25
C (Stoev and Slavtcheva, 1982). Photosynthesis is more sensitive to (suppressed by) temperatures below 15
C than to temperatures well above optimum (>40
C). Fruit load and canopy size affect photosynthesis, presumably through feedback regulation. Within limits, the rate of photosynthesis can adjust itself to demand. For example, fruit removal results in a reduction in the

photosynthetic rate of adjacent leaves (Downton et al., 1987). Conversely, photosynthetic rate increases as a consequence of basal leaf removal (Hunter and Visser, 1988b). Improved photosynthetic efficiency appears to be associated with wider stomatal openings and increased gas exchange. Greater sugar demand could also activate export, thus reducing the concentration of Calvin cycle intermediates that could act as photosynthetic feedback inhibitors. Water conditions also markedly influence the rate of photosynthesis. In particular, water deficit promotes the production of abscisic acid. This in turn enhances the synthesis of other activator molecules, notably hydrogen peroxide and nitric oxide (Patakas et al., 2010). These changes induce stomatal closure, reduce the transpiration rate and gas exchange, increase leaf temperature, suppress photosynthesis, and modify secondary metabolism. Because water status affects the relative incorporation of stable carbon isotopes (13C/12C ratio) (Brugnoli and Farquhar, 2000), this feature has been used to assess the cumulative effects of water status on vine photosynthesis (Gaudille`re et al., 2002). Plate 3.5 illustrates a technique for assessing whole-plant gas exchange. The direction of carbohydrate export from leaves varies with their positions along the shoot and the time of year. Export from young maturing leaves generally is apical, toward new growth. Subsequently, photosynthate from the upper maturing leaves is directed basally toward developing fruit (see Fig. 3.40). By the end of berry ripening, basal leaves are rarely involved in export. This

12 11 10 9

Pn (μmol m–2s–1)

8 7 6

Full leaf expansion

5 4 3 2 1 0 0

20

40

60 80 100 120 Leaf age (10-day intervals)

140

160

180

FIGURE 3.19 Relationship between leaf age, given as days after unfolding, and net photosynthesis (Pn), calculated on a seasonal basis, of Sangiovese vines. Vertical bars represent two SEM. From Poni, S., Intrieri, C., Silverstroni, O., 1994. Interactions of leaf age, fruiting, and exogenous cytokinins in Sangiovese grapevines under non-irrigated conditions, I. Gas exchange. Am. J. Enol. Vitic. 45, 71e78, reproduced by permission.

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3. Grapevine structure and function

function has been taken over by upper, newer leaves. Translocation typically is restricted to the side of the cane and trunk on which leaves are produced. Phytochrome is associated with a wide range of photoperiodic and photomorphogenic responses. Typically, grapevines are relatively insensitive to photoperiod but may show several other phytochrome-induced phenomena. For example, bud dormancy in V. labrusca and V. riparia is induced by short-day photoperiods (Fennell and Hoover, 1991; Wake and Fennell, 2000). Several plant enzymes are partially activated by changes in phytochrome balance. These include those involved in the synthesis and metabolism of malic acid (malic enzyme), the production of phenols and anthocyanins (phenylalanine ammonia lyase and dihydroflavonol reductase) (Gollop et al., 2002), sucrose hydrolysis (invertase), and possibly nitrate reduction and phosphate accumulation (see Kliewer and Smart, 1989). Phytochrome is also suspected of being implicated in the regulation of berry growth (Smart et al., 1988). Nevertheless, the actual importance of the shift in the Pfr/Pr ratio in shaded grapevine leaves is unclear. Also, the significance of sunflecking on phytochrome balance is unknown. A third, light-induced process is activated by ultraviolet (UV) radiation, namely the toughening of the cuticle. Strong ultraviolet absorption by phenolics in the outer leaf canopy results in its almost complete absence in the inner canopy. Consequently, UVinduced hardening of the cuticle in shaded leaves and fruit is absent, resulting in their surfaces being softer than their sun-exposed counterparts. This factor, combined with the slower rate of drying and higher humidity in the understory, may explain the greater sensitivity of shaded tissue to fungal infection. Protection against

PLATE 3.5 Portable whole canopy gas exchange system for several mature field-grown grapevines. From Pen˜a, J.P., Tarara, J., 2004. A portable whole canopy gas exchange system for several mature field-grown grapevines. Vitis 43, 7e14, reproduced by permission.

UV-induced mutation is presumably one of the rationales for young leaves and other tissues possessing anthocyanins. It is suspected, but unconfirmed, that either or both blue/UV-A (315e380 nm) or UV-B (280e315 nm) photoreceptors (such as cryptochrome) are involved in cuticle toughening. Exposure to short-wave ultraviolet radiation (UVeC, 100e280 nm) also appears to activate several metabolic pathways. This is of particular importance in general disease resistance, as the activated pathways include those involved in the synthesis of phytoalexins, chitinases, and pathogenesis-related (PR) proteins (Bonomelli et al., 2004). In addition, exposure to UV-B increases preve´raison flavonol content in the skin of Sauvignon blanc but does not appear to influence amino acid or methoxypyrazine content (Gregan et al., 2012).

Transpiration and stomatal function To optimize photosynthesis, the leaf must act as an efficient light trap. Because of the importance of diffuse (skylight) radiation, most leaves possess a broad surface. Even on clear days, up to 30% of radiation impacting a leaf may come from diffuse skylight. However, the large surface area of the leaf blade also makes it an effective heat trap. Heating is reduced by evaporative cooling, but if unrestricted would place an unacceptably high water demand on the root system. The cuticular coating of the leaf limits water loss but regrettably also retards the gas exchange essential to photosynthesis. These conflicting demands result in a series of complex structural and physiological dynamic compromises. When water supply is adequate, transpirational cooling can effectively minimize leaf heating during direct sun exposuredto about 1
Ce2
C above ambient (Millar, 1972). This, however, places considerable demands on the root system. Even under mild transpiration conditions, water loss can occur at rates of up to 10 mg/cm2/h. Except for the small proportion of water (), and free abscisic acid concentration (-) during the development of Cabernet Sauvignon berries. Standard errors are indicated. From Wheeler, S., Loveys, B., Ford, C., Davies, C., 2009. The relationship between the expression of abscisic acid biosynthesis genes, accumulation of abscisic acid and the promotion of Vitis vinifera L. berry ripening by abscisic acid. Aust. J. Grape Wine Res. 15, 195e204, reproduced by permission.

growth, rising until the onset of ve´raison and then declining (Pe´rez et al., 2000). Ethylene has traditionally been considered to play an insignificant role in grape ripening. However, data from El-Kereamy et al. (2003) suggest it may play a role in initiating anthocyanin synthesis. Its application activates the production of enzymes involved in flavonoid and anthocyanin synthesis, as does the application of brassinosteroids (Luan et al., 2013). In contrast, the joint application of a strigolactone and abscisic acid reduced the latter’s activation sugar and anthocyanin accumulation (Ferrero et al., 2018). In addition, inhibition of its action by the application of 1-methylcyclopropene, a specific inhibitor of ethylene receptors, reduces sugar accumulation postve´raison (Chervin et al., 2006). Ethylene experiences a transient doubling just prior to ve´raison (Chervin et al., 2004). Hilt and Blessis (2003) have also demonstrated ethylene’s action in young fruit abscission, presumably via its promotion of abscisic acid ve´raison. Because of their influence on berry development, growth regulators have been occasionally applied to control fruit growth and spacing within clusters (Plate 3.9). Generally, synthetic growth regulators have been more effective than their natural counterparts. They have the advantage of being less affected, or even unaffected, by natural feedback inhibition pathways in the vine. Artificial auxins, such as benzothiazole-2-oxyacetic acid, can delay ripening up to several weeks when applied to immature fruit. Conversely, the artificial growth retardant methyl-2-(ureidooxy) propionate markedly hastens ripening when applied before ve´raison (Hawker et al., 1981). Ripening can also be shortened marginally by applying growth regulators, such as ethylene or abscisic acid. The discovery that presumptive sites on the UFGT

115

1.2

400

200

0.8

Berry weight (g)

600

IES equivalent μg x 10–2/Tr. S

1.0

Auxin content

Acidity % o

800

°Oe

Berry weight (g)

80

Length of shoot (cm)

Length of shoot (cm)

Berry growth and development

40

110

30

100

20

90

10

80

60 Sugar in °Oe

0.6

Acidity % o

40

0.4 20 0.2

0

0

0 7 VII

1 VII 15 VIII 1 IX 15 IX

1X

0

15 X 28 X

FIGURE 3.36 Auxin content of grape berries in relation to shoot length, berry weight, sugar content, and acidity of the cultivar Riesling. From Alleweldt, G., Hifny, H.A.A., 1972. Zur Stiella¨hme der Reben, II. Kausalanalytische Untersuchungen. Vitis 11, 10e28.

and biochemical changes associated with ripening, direct causal relations are limited. Thus, many of these associations may be due to the combined actions of two or more regulators or be indirect. Precise knowledge of the sequence of steps involved could potentially generate better regulation of berry development and fruit quality.

gene promoter are responsive to abscisic acid, ethylene, light, and sugar (Chervin et al., 2009) is consistent with their role in grape colorationdi.e., augmenting UFGT production (Peppi et al., 2008). UFGT is an essential enzyme in anthocyanin synthesis (see Fig. 3.45). The addition of abscisic acid has been used to accelerate grape development and coloration in cool, wet seasons (Balint and Reynolds, 2013). Although correlations have been found between changes in growth regulators and the physiological

Water uptake

PLATE 3.9 Effect of gibberellin (GA3) application to grapevine flowers on the spacing of “Riesling”: untreated (left); treated (right). Photo courtesy of M. Pour Nikfardjam, Staatl. Lehr-und Versuchsanstalt fuer Wein-und Obstbau, Weinsberg.

During development, structural changes in the skin and vascular tissues influence the types and amount of substances transported to the fruit. Degeneration of stomatal function, disruption of xylem function, and development of a thicker waxy coating can combine to reduce transpirational water loss (Fig. 3.37). This also reduces gas exchange between the berry and its surroundings as well as increasing the likelihood of fruit overheating and diurnal temperature fluctuations. As berries near maturity, water uptake declines (Rogiers et al., 2001). This can produce a 10-fold reduction in hydraulic conductance from ve´raison to ripeness (Tyerman et al., 2004). This has normally been interpreted as due to xylem vessels rupture in the peripheral vascular tissues of the berry (Creasy et al., 1993). However, this view has been challenged (Chatelet et al., 2005). Although vessels may stretch during postve´raison berry enlargement (being annular or spiral thickened) with some rupturing, others remain functionally intact. In addition, some new vessels may develop postve´raison

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3. Grapevine structure and function

120 100

400

cv. Ehrenfelser cv. Riesling cv. Müller-Thurgau cv. 2 x std. dev.

300

80 Contributed volume (mm3)

Transpiration (mg H2O/dm2 h)

500

200

60 40 20 0 –20 –40 –60 –80

Pre-veraison

–100

Post-veraison

–120

100

Xylem

Phloem

Transpiration

FIGURE 3.38 Data for daily water flow for each component of the 1 4 7 10 13 Berry development (weeks after full bloom)

FIGURE 3.37 Transpiration of berry bunches per fruit surface area for three grape varieties. Measurements were performed at 20
C, 52% r.h., 335 vpm CO2, and 800 mmol/m2/s (about 56 kLux). From Blanke, M.M., Leyhe, A., 1987. Stomatal activity of the grape berry cv. Riesling, Mu¨ller Thurgau and Ehrenfelser. J. Plant Physiol. 127, 451e460, reproduced by permission.

(Chatelet et al., 2008). Berry swelling, which can occur during rainy spells near harvest, may result more from osmotic water uptake via the receptacle (Becker and Knoche, 2011) than via microcracks in the skin. Nonetheless, microcracks do weaken the cuticular barrier of the fruit, notably adjacent to the stylar scar (Becker and Knoche, 2012). These could provide sites for nutrient leaching to the surface, facilitating spore germination and successful penetration by pathogens. Data from Bondada et al. (2005) suggest that a decrease in xylem flow results from a reduction in the hydrostatic gradient. This could explain why most water uptake postve´raison is associated with photosynthate transport via the phloem (Fig. 3.38) and why diurnal fluctuations in berry diameter disappear postve´raison (Greenspan et al., 1994). It would also explain why minerals such as calcium, manganese, and zinc, transported primarily in the xylem, become restricted postve´raison (Rogiers et al., 2006b). In contrast, potassium, which is transported equally in the phloem and xylem, continues to accumulate throughout ripening (Hrazdina et al., 1984). The increasing importance of phloem transport for water as well as organic and inorganic nutrients has been equally found in other flesh fruits (e.g., apples and tomatoes). Evidence is contradictory as to whether the xylem acts as a conduit for the movement of water out of the berry (Lang and Thorpe, 1989; Greenspan et al., 1996).

water budget of well-irrigated Cabernet Sauvignon berries. From Greenspan, M.D., Shackel, K.A., Matthews, M.A., 1994. Developmental changes in the diurnal water budget of the grape berry exposed to water deficits. Plant Cell Environ. 17, 811e820, reproduced by permission.

At maturity, the phloem ceases to function due to the formation of a periderm at the base of the berry. Its formation also cuts off xylem connections, facilitating detachment of the fruit from the pedicel. Termination of vascular connections with the fruit probably explains shrinkage of the fruit at this stage (Rogiers et al., 2006a). Cultivars such as Shiraz are particularly sensitive to such shriveling.

Sugars Quantitatively, sucrose is the most significant organic compound translocated into the fruit. Initially though, carbohydrate is photosynthesized in situ. As the berry enlarges and approaches ve´raison, the surrounding leaves become the major carbohydrate source. The trunk and arms of the vine may also provide additional carbohydrate. It has been estimated that under abnormal conditions, up to 40% of the carbohydrate accumulated by fruit may be supplied by the permanent, woody parts of the vine (Kliewer and Antcliff, 1970). Sugar accumulation is particularly marked following ve´raison. This coincides with a pronounced decline in berry glycolysis and a reduction of cell turgor (Matthews et al., 2009). Primary shoot growth also tends to slow dramatically at this time, facilitating a major redirection of photosynthate toward the fruit. Root growth also declines following ve´raison, further reducing a drain on photosynthate. An alternate and seemingly equally plausible interpretation is that decreased shoot and root growth result from carbohydrate drain associated with berry sugar accumulation. An adequate, coordinated explanation of these events must still be provided.

Berry growth and development

Part of the carbohydrate accumulation in berries may also relate to reduced sugar metabolism associated with a shift from glucose to malic acid respiration. Malic acid accumulates in considerable amounts early in fruit development, being respired postve´raison via the tricarboxylic acid (TCA) cycle (see Fig. 7.20). Although potentially involved, malic acid respiration is unlikely to be a major factor in its reduction. Grapes in cool regions accumulate sugars to about the same degree as warmer regions despite retaining much of their malate content during ripening. Another minor factor contributing to an increase in sugar content may be the biosynthesis of glucose from malic acid. Gluconeogenesis may explain the reduction in other TCA intermediates, such as citric and oxaloacetic acids, during maturation. The increase in abscisic acid content associated with the beginning of ve´raison may activate this metabolic shift (Palejwala et al., 1985). Nevertheless, the major factor involved in the marked accumulation of sugar undoubtedly relates to a redirection of phloem sap from leaves to the fruit (Dreier et al., 2000). The most significant factor promoting the redirected flow of sugar appears to be evapotranspiration (Rebucci et al., 1997; Dreier et al., 2000). Because water flow via the xylem slows and essentially stops after ve´raison, water uptake continues via the phloem. Sucrose and other constituents, such as amino acids, are presumably imported in this stream. Conversion of sucrose into glucose and fructose in the berry doubles its osmotic potential, augmenting further sap flow toward the fruit. Chemical modifications of wall constituents facilitate cell enlargement, permitting enhanced water transport and sugar accumulation. Smaller berries have relatively more surface area and are thereby are exposed to higher evapotranspiration rates, and this may explain why smaller berries tend to have higher sugar content than that of larger fruit. On reaching the berry, most sucrose is hydrolyzed by invertase. Although it liberates equal amounts of glucose and fructose, their relative concentrations are seldom identical. In young berries, the proportion of glucose is generally higher. During ripening, the glucose/fructose ratio falls, with fructose retention being slightly higher than that of glucose. Thus, by maturation if not before, fructose is marginally more prevalent than glucose (Kliewer, 1967). The reasons for this disequilibrium are unknown but may originate from differential rates of glucose and fructose metabolism in the berry or selective synthesis of fructose from malic acid. As with other cellular constituents, sugar accumulation varies with the cultivar, maturity, and prevailing environmental conditions. Depending on these factors, sugar content may vary from 12% to 28% at harvest. Further increases during overripening appear to reflect

117

concentration due to water loss rather than additional uptake. For winemaking, optimal sugar content usually ranges between 21% and 25%. Depending on the species or cultivar, some sucrose translocated to the fruit is retained as sucrose. This is most marked in muscadine cultivars, in which 10%e20% of the sugar content may remain as sucrose. In some FrencheAmerican hybrids, sucrose constitutes about 2% of sugar content. In most V. vinifera cultivars, sucrose content averages about 0.4% (Holbach et al., 1998). Small amounts of other sugars are found in mature berries, notably raffinose, stachyose, melibiose, maltose, galactose, arabinose, and xylose. Their presence is disregarded in assessing sugar content because they occur in such limited amounts, are not metabolized by wine yeasts, and do have an impact on perceived sweetness. Sugar storage occurs primarily in the central vacuole of mesophyll cells, with lesser amounts being deposited in the skin (Fig. 3.39). This can vary with the cultivar and stage of ripening. In contrast, the small amount of sucrose that occurs in V. vinifera fruit is restricted to the axial vascular bundles and the skin adjacent to the peripheral vascular strands. During ripening, much of the sugar comes from leaves located on the same side of the shoot as the fruit cluster, directly above the cluster. Carbohydrate supplies come primarily from basal leaves (Hunter and Visser, 1988a) but increasingly come from apically positioned leaves as they mature (Fig. 3.40), with additional amounts from leaves associated with lateral shoots as well as old wood. Estimates of the foliage cover (leaf area/fruit weight) required to fully ripen grape clusters vary considerably, from 6.2 cm2/g (Smart, 1982) to 10 cm2/g (Jackson, 1986). The latter appears to be more typical. The variation may reflect varietal and environmental influences on fruit load and leaf photosynthetic efficiency.

Acids Next to sugar accumulation, reduction in acidity is quantitatively the most marked chemical change during ripening. Tartaric and malic acids account for about 70% e90% of berry acids. The remainder consists of variable amounts of other organic acids (e.g., citric and succinic acids), phenolic acids (e.g., quinic and shikimic acids), amino acids, and fatty acids. Although structurally similar, tartaric and malic acids are synthesized and metabolized differently. Tartaric acid is derived via a complex transformation from vitamin C (ascorbic acid). This appears to involve Lidonic acid as a rate-limiting step (DeBolt et al., 2006). The importance of tartaric acid in grape development and metabolism, if any, is unknown. In contrast, malic acid is an important TCA intermediate. As such, it can be variously synthesized from sugars (via glycolysis

118

3. Grapevine structure and function

Glucose

Locule

Skin bundles

Concentration mg per g fr.wt.

Central bundle

Fructose

100

Inner flesh 0

FIGURE 3.39 Smoothed graph showing glucose and fructose concentrations into the center of a grapedvertical lines in the berry cross-section indicate the position of vascular bundles. From Coombe, B.G., 1990. Research on berry composition and ripening. In: Williams, P.J. (ed.), Proc. 7th Aust. Wine Ind. Tech. Conf. Wine, Adelaide, Australia, pp. 150e152, reproduced by permission.

and the TCA cycle) or via carbon dioxide fixation from phosphoenolpyruvate (PEP) during photosynthesis. Malic acid can be variously respired, decarboxylated to

PEP, or consumed in the gluconeogenesis of sugars. Berry malic acid content can change more strikingly and rapidly during ripening than tartaric acid content. The degree and nature of acid conversions can vary widely depending on the cultivar and environmental conditions. Nevertheless, several major trends are consistent. After an initial intense synthesis of tartaric acid in the ovary, both tartaric and malic acid contents slowly increase up to ve´raison. Subsequently, the amount of tartaric acid tends to stabilize, whereas that of malic acid decreases. It is hypothesized that the initial accumulation of malic acid acts as a nutrient reserve, partially replacing glucose as a postve´raison substrate. This would explain the frequent rapid drop in malic acid content during the latter stages of ripening. However, it leaves unexplained malic acid’s slow decline under cool conditions. This pattern differs in some significant regards in V. rotundifolia. In muscadine cultivars, acidity generated primarily by tartaric acid declines from a maximum at berry set. Succinic acid, insignificant in V. vinifera cultivars, is a major component in very young muscadine fruit (Lamikanra et al., 1995). Its concentration declines rapidly thereafter. Malic acid content tends to increase up to ve´raison, declining thereafter. It has long been known that grapes grown in hot climates often metabolize most of or all their malic acid before harvest. Although exposure to high temperatures activates enzymes that catabolize malic acid, this alone appears insufficient to explain the difference. Reduced

? ?

Separation of translocation flow

Flower bud development

Before flower opening

Flower fertilization

Berry growth

Véraison

Fruit maturation

FIGURE 3.40 Development in the translocation of photosynthate from leaves during shoot growth. After Koblet, W., 1969. Wanderung von Assimilaten in Rebtrieben und Einfluss der Blattfla¨che auf Ertrag und Qualita¨t der Trauben. Wein-Wiss. 24, 277e319, reproduced by permission.

119

Berry growth and development

10

Tartrate

Concentration mg per g fr.wt.

Locule

0

Central bundle

Malate

Skin bundles

synthesis, and possibly heightened gluconeogenesis, may play a role in the decline in malic acid content. The typical decrease in acid concentration during ripening is particularly marked when assessed on a fresh-berry-weight basis. This results from berry enlargement (water uptake) exceeding the accumulation of tartaric acid and the metabolism of malic acid. Shoot vigor is another factor influencing fruit acidity. Vigor (as indicated by an increased leaf surface/fruit ratio) is correlated with reduced grape acidity and higher pH (Jackson, 1986). This connection has usually been ascribed to the leaf and fruit shading associated with increased vigor. Nevertheless, even where shading was prevented, greater vine vigor reduced indicators of fruit quality (higher pH, decreased acidity, and lower
Brix) (Jackson, 1986). In addition to environmental influences, hereditary factors affect berry acid content. Some varieties, such as Zinfandel, Cabernet Franc, Chenin blanc, Syrah, and Pinot noir are proportionally high in malic acid, whereas others, such as Riesling, Se´millon, Merlot, Grenache, and Palomino are inherently higher in tartaric acid content (Kliewer et al., 1967). Tartaric acid tends to accumulate in the skin, with lower amounts relatively evenly distributed throughout the flesh (Fig. 3.41). By comparison, malic acid deposition is highest in the epidermis, low in the hypodermis, and increases again to a maximum near the berry locule. As ripening advances, these differences become less marked. By maturity, malic acid concentration in the skin, although low, may surpass that in the flesh. This appears to result from malate metabolism initiating around the axial vascular bundles and progressing outward. The balance between free and salt states of tartaric acid generally changes throughout maturation. Initially, most of the tartaric acid exists as free acid. During ripening, progressively more tartaric acid combines with cations, predominantly Kþ. Salt formation with Ca2þ usually results in the deposition of calcium tartrate crystals in vacuoles of the skin (Ruffner, 1982). In contrast, malic acid generally remains as a free acid. The combination of the changing acid concentrations and their salt states results in the lowest titratable acidity occurring next to the skin. The highest titratable acidity normally exists next to the seeds. The factors regulating tartaric and malic acid synthesis are poorly understood despite the effect of temperature on malic acid degradation already noted. Nutritional factors can also considerably influence berry acidity and acid content. Increased potassium availability results in slight increases in the levels of both acids. The amplification is probably driven by the need to produce additional acids to maintain ionic balance associated with potassium uptake. Transport of Kþ into cells requires the simultaneous

Inner flesh

FIGURE 3.41 Smoothed graph showing tartaric and malic acid concentrations into the center of a grapedvertical lines in the berry cross-section indicate the position of vascular bundles. From Coombe, B.G., 1990. Research on berry composition and ripening. In: Williams, P.J. (ed.), Proc. 7th Aust. Wine Ind. Tech. Conf. Wine, Adelaide, Australia, pp. 150e152, reproduced with permission.

export of Hþ. Although potassium uptake may enhance acid synthesis, it also results in a rise in pH due to its association with free acids generating acid salts. This effect may be far from simple. For example, the proportion of free tartaric acid in skin cells may rise during maturation, even though up to 40% of potassium accumulation occurs in the skin. This apparent anomaly may result from a differential deposition of potassium and acids in vacuoles of the skin (Iland and Coombe, 1988). Nitrogen fertilization enhances malic acid synthesis but may induce a reduction in tartaric acid content. The increase in malic acid synthesis may involve cytoplasmic pH stability. Nitrate reduction (to ammonia) tends to raise cytoplasmic pH by releasing OHþ ions. The synthesis of malic acid could neutralize this effect. However, because of the low level of malic acid ionization in grape cells and its metabolism via respiration during ripening, the early synthesis of malic acid does not permanently prevent a rise in pH during ripening. Although both malic and tartaric acids appear to be involved in maintaining a favorable cellular ionic and osmotic balance, they tend to be stored in vacuoles to avoid excessive cytoplasmic acidity. Here, the differential

120

3. Grapevine structure and function

permeability and distinctive transport characteristics of vacuolar membranes isolate the acids from the cytoplasm.

Phenolics In red grapes, flavonoid phenolics constitute the third most significant group of organic compounds. They accumulate primarily in the seeds and skins. By comparison, white grapes have lower total phenolic contents, partially because they do not synthesize anthocyanins (at least in significant amounts). Despite these major differences, the proportion, amounts, and types found in seeds, skins, and pulp can vary significantly among cultivars (Bourzeix et al., 1983). Of these, seed phenolics are the most slowly extracted and correspondingly become a significant source of phenolics in wine only during extended fermenting with the pomace. Anthocyanins are primarily synthesized and accumulate in epidermal and hypodermal vacuoles of the skin (see Fig. 3.31). They accumulate along with various mono- and polymeric flavonoids. Polymeric flavonoids (tannins) typically amass as granules that adhere to the vacuolar membrane or cell wall and are not easily extracted. Hydroxycinnamic acid esters of tartaric acid form the predominant phenolic components found in

K Inorganic anions

Locule

Central bundle

Phenolics 0

Skin bundles

Potassium uptake during maturation affects both acid synthesis and ionization. Regrettably, the factors controlling potassium accumulation are poorly understood. It is transported in both the xylem and phloem and can be redistributed from older to younger tissues. Its uptake increases after ve´raison, especially in the skin (Fig. 3.42). The skin, which constitutes about 10%e15% of berry weight, may contain up to 30%e40% of the potassium content. The high correlation between potassium and sugar accumulation in the skin and flesh, respectively, suggests that potassium acts as an important osmoticum in skin cells, whereas sugar is the principal osmoticum in the flesh. Nonetheless, potassium accumulates at only about 40% the rate of sugar from ve´raison to harvest (Coetzee et al., 2017). Potassium accumulation also correlates with berry softening, associated with apoplast acidification. Hþ ion export compensates for the import of Kþ ions. Because potassium accumulation in vacuoles affects membrane permeability, potassium uptake may influence the release of malic acid, favoring its cytoplasmic metabolism. Potassium is also a well-known enzyme activator. However, the reasons for the marked but nonuniform accumulation of potassium in berries (Storey, 1987) remain a mystery. Concerning other minerals, boron tends to be located in the skin and pulp, whereas calcium, manganese, phosphorus, sulfur, and zinc are primarily deposited in the seeds (Rogiers et al., 2006b).

10

Concentration mg per g fr.wt.

Potassium and other minerals

Inner flesh

FIGURE 3.42 Smoothed graph showing potassium, inorganic ions and phenolic concentration into the center of a grapedvertical lines in the berry cross-section indicate the position of vascular bundles. From Coombe, B.G., 1990. Research on berry composition and ripening. In: Williams, P.J. (ed.), Proc. 7th Aust. Wine Ind. Tech. Conf. Wine, Adelaide, Australia, pp. 150e152, reproduced by permission.

the flesh. Their concentration tends to decline during ripening, whereas benzoic acid derivatives may increase (Ferna´ndez de Simo´n et al., 1992). In contrast, the color of white grapes comes from the presence of carotenoids, xanthophylls, and flavonols, such as quercetin. Carotenoids accumulate predominantly in plastids, whereas flavonoids are deposited in cell vacuoles. Similar pigments occur in red grapes, but their presence is masked by anthocyanins. Anthocyanin synthesis is regulated in maturing grapes by two homologous MYB transcription factors (VvMYBA1 and VvMYBA2). Activation of either generates a regulator protein that initiates anthocyanin synthesis. The protein activates UFGT, a critical gene involved in anthocyanin synthesis. Typically these transcription factors become activated only in the skin, except for teinturier varieties. Two other closely related genes (VvMYBA3 and VvMYBA4), also on chromosome 2, are inactive in skin cells. VvMYBA4 acts in repressing the synthesis of small phenolics in some tissues (Cavallini et al., 2015). In general, wild Vitis species have higher concentrations of anthocyanins than domesticated

Berry growth and development

cultivars, while wine varieties tend to possess higher concentrations than table grapes. Allelic (or epistatic) versions of the VvMYBA1 gene likely are the prime source of the various colors expressed by grapes. Other MYB genes (e.g., MYBC2), which regulate other aspects of the phenylpropanoid pathway, are also probably involved, some being repressors of anthocyanin precursors (Cavallini et al., 2015). Some of these genes, such as VvMYBF1, are light activated, favoring the production of UV-protectant flavonols (Czemmel et al., 2009). Transgenic alteration in the expression of anthocyanin VvMYBA genes can affect up- and downregulation of multiple other genes, notably those affecting phenylpropanoid synthesis and transport (Rinaldo, 2014). These alterations indirectly affect various aspects of primary and secondary metabolism (Fig. 3.43). Their effect on terpenoid (e.g., E-beta-ocimene synthase) and fatty acid synthesis appears to induce unsuspected sensorially consequences. Probably they result from the redirection of synthesis pathways as a result of blocked anthocyanin synthesis. V. vinifera cultivars typically possess at least one nonfunctional VvMYBA1 allele (Mitani et al., 2009). Insertion of the retrotransposon Gret1 in the promotor region prevents the gene, designated VvMYBA1a, from being activated (Walker et al., 2007). The functional VvMYBA1c allele lacks the retrotransposon. White cultivars typically are homozygous for the nonfunctional VvMYBA1a allele as well as possess inactive alleles of a second regulator gene (VvMYBA2). The nonfunctional status of this second set of alleles may be due to the presence of both a substitution and a deletion mutant (Walker et al., 2007). Pigmented cultivars (gray, pink, red to black) are more variable in allelic composition but possess at least one functional MYB allele (Walker et al., 2007; This et al., 2007). Because the alleles function in a dominant/recessive manner, the presence of one

121

functional allele can activate production of the enzyme UFGT, essential for anthocyanin synthesis (Fig. 3.44). The vast majority of white-berried cultivars possess identical versions of the VvMYBA1a allele, while the majority of red cultivars are heterozygous for the same allele (Walker et al., 2007). This has been interpreted as indicating that Gret1 inactivation occurred early in domestication, as white strains of V. vinifera spp. sylvestris have not be found. Color is important in attracting birds for seed dispersal. In addition to lacking anthocyanins, white cultivars are deficient in several flavonols, notably trihydroxylated forms (e.g., myricetin, laricitrin, and syringetin) (Flamini et al., 2013). This may explain the presence of higher concentrations of certain aromatics (Rinaldo, 2014). Exceptions to these trends include Brown Frontignac and Black Frontignac, members of the normally whitecolored Muscat family, rose´, and red forms of Italia, and rare, rose´-colored clones of Chardonnay and Gewu¨rztraminer. With colored Frontignac cultivars, recombination may have reestablished a functional allele (Walker et al., 2007), whereas with Chardonnay and Gewu¨rztraminer, excision of the Gret1 retrotransposon may be the source (This et al., 2007). White variants of red cultivars, such as Pinot Blanc, appear to have lost the functional VvMYBA1c allele found in Pinot noir but retain the inactive VvMYBA1a allele (Yakushiji et al., 2006). Brown and white versions of Cabernet Sauvignon (Plate 3.10) possess other genetic modification in their promoter genes, as does a white variant of Rhoditis (Lijavetzky et al., 2006). For other examples of somatic color variants see Migliaro et al. (2014). Additional sources of color variation can involve allelic variants of genes leading to and directly involved in anthocyanin synthesis (Fig. 3.45). Several enzymes are directly involved in anthocyanin synthesis, while others regulate earlier steps of flavonoid synthesis (Fig. 3.45). These are regulated by other VvMYB transcription (regulator) factors. Most are

FIGURE 3.43 Comparison of the number of genes with altered transcription in response to transgenic expression of VvMYB genes in whole Chardonnay and Shiraz berries. Chardonnay containing the 35S:VvMYBA1 construct were “red” whereas Shiraz containing the VvMYBAsi silencing construct appeared “white.” From Rinaldo, A., 2014. An Investigation of the Role of the Regulatory Gene VvMYBA1 in Colour, Flavour and Aroma Metabolism Using Transgenic Grapevines. PhD Thesis conducted under the supervision of Dr. M. Walker, School of Agriculture, Food and Wine, University of Adelaide, Waite Campus, reproduced with permission.

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3. Grapevine structure and function

FIGURE 3.44 Schematic representation of the flavonoid pathway in grape skins. CHI, chalcone isomerase; F3H, flavanone-3-hydroxylase; F3 H, flavonoid three hydroxylase; F3 5 H, flavonoid 3,5 -hydroxylase; FLS, flavonol synthase; DFR, dihydroflavonol 4-reductase; LAR, leucoanthocyanidin reductase; LDOX, leucoanthocyanidin dioxygenase; ANR, anthocyanidin reductase; UFGT, UDP-glucose-flavonoid 3-O-glucosyl transferase. The products in the solid-line boxes are members of cyanidin-based anthocyanins (left) and delphinidin-based anthocyanins (right). The products in the dotted-line boxes are members of flavan-3-ols. The products in the broken-line boxes are members of flavonols. The mechanisms of the condensation reactions of proanthocyanidins, shown by the dotted arrows, are not yet fully understood. From Koyama, K., Goto-Yamamoto, N., 2008. Bunch shading during different developmental states affects the phenolic biosynthesis in berry skins of ‘Cabernet Sauvignon’ grapes. J. Am. Soc. Hort. Sci. 133, 743e753, reproduced by permission.

expressed throughout the plant, unlike the localized activation of VvMYBA. The relative transcription of other genes (F30 50 H and F30 H) in the fruit influences the degree of anthocyanin hydroxylation and correspondingly the proportion of trihydroxylated anthocyanins (malvidin, delphinidin, petunidin) and berry color depth. Lighter colored cultivars tend to possess relatively more dihydroxylated anthocyanins (cyanidin and peonidin) (Castellarin and Di Gaspero, 2007). The pathways appear to be similar in all grapevine species studied: V. vinifera, Vitis aestivalis, and V. rotundifolia (Samuelian et al., 2009). Expression of flavonoid synthesis genes, such as chalcone synthase and flavanone 3-hydroxylase and those associated with anthocyanin synthesis, are enhanced by abscisic acid (Jeong et al., 2004; Peppi et al., 2008). Because high temperatures reduce abscisic acid content (Yamane et al., 2006), this probably explains the reduced anthocyanin synthesis in hot weather. UFGT, a crucial

gene in anthocyanin synthesis, can be activated by the application of 2-chloroethylphosphonic acid, an ethylene-releasing compound (El-Kereamy et al., 2003). Thus, activation of ethylene production by spraying grapes with a dilute solution of ethanol or methanol (Chervin et al., 2001) presumably explains its effect on enhancing grape coloration. In contrast, application of auxins (naphthaleneacetic acid) and shading suppressed the anthocyanin pathway regulator VvMYBA1 (Jeong et al., 2004) and thereby anthocyanin synthesis. The most intensely pigmented layers of the skin are typically the epidermis and first hypodermal layer. The next two layers contain smaller amounts of anthocyanins, and subsequent hypodermal layers tend to be sporadically and weakly pigmented (see Plate 3.10). Pigmentation seldom occurs deeper than the sixth hypodermal layer except in teinturier varieties. These possess uniformly but weakly pigmented flesh (Hrazdina and Moskowitz, 1982). However, as skin cells senescence

123

Berry growth and development

(A)

(B)

(D)

(C)

(E)

(F)

(G)

PLATE 3.10 Examination of berry cell layers. (A, (B, C) Oblique angle hand sections cut on an arc through berry to show epidermis (marked with a trapezoid) and internal cells, constituting the skin and flesh. (A Cabernet Sauvignon (arrows indicate large subepidermal colored cells); (B) Malian; (C) Shalistin; D, (E, F) Thin surface section looking through skin (photo taken with Axioplan microscope); (D) Cabernet Sauvignon; (E) Malian; (F) Shalistin. (G) Diagrammatic representation of cross-section of Cabernet Sauvignon, Malian, and Shalistin berries. Bar in AeC is 1 mm; DeF is 100 lm. Reproduced from Walker, A.R., Lee, E., Robinson, S.P., 2006. Two new grape cultivars, bud sports of Cabernet Sauvignon bearing palecoloured berries, as the result of deletion of two regulatory genes of the berry colour locus. Plant Mol. Biol. 62, 623e635 with kind permission from Springer Science and Business Media.

during overripening, pigmentation may diffuse from the skin into the flesh. Anthocyanin synthesis and glycosylation is localized to the endoplasmic reticulum. Anthocyanins are subsequently transported for storage to adjacent vacuoles. The anthocyanin content in the outer hypodermal layer(s) soon approaches saturation. Anthocyanins subsequently combine in self-association or copigment complexes. The decline in anthocyanin content, occasionally observed in some cultivars as they approach maturity or overmature, is probably caused by b-glycosidases and peroxidases. These enzymes are normally sequestered in vacuoles of the skin (Caldero´n et al., 1992) but may be released and become active as grapes mature. In addition to anthocyanins, all cultivars synthesize and accumulate variable amounts of catechins, their polymers (proanthocyanidins), flavonols, benzoic and cinnamic acids, their aldehyde derivatives, and hydroxycinnamic acid tartrate esters. Most of these flavonoids occur in small amounts in the grape skin and at even lower concentrations in the flesh. The predominant flavonol in V. vinifera tends to be kaempferol, whereas

quercetin is the principal form in V. labrusca cultivars. Like anthocyanins, most flavonols are glycosidically linked, but to rhamnose and glucuronic acid in addition to or instead of glucose. The predominant hydroxycinnamic acid ester in grapes is caffeoyl tartrate, with smaller amounts of coumaroyl tartrate and feruloyl tartrate. As with most other chemical constituents, the specific concentrations can vary widely from season to season and from cultivar to cultivar. Flavonoid synthesis begins with the condensation of erythrose derived from the pentose phosphate pathway with phosphoenolpyruvate generated via glycolysis. The phenylalanine so produced is converted to the first of a series of phenylpropanoidsd cinnamic acid. It is subsequently metabolized to coumaric acid and then 4-coumaroyl-CoA. It contributes what is designated the B ring of flavonoids (see Illustration 6.10). Its binding with three acetates (derived from three malonyl CoA moieties) forms the A ring. The product, naringenin chalone, is the progenitor of grape flavonoids (Hrazdina et al., 1984). Subsequent alterations give rise to the full range of flavonoid phenolics (Fig. 3.45).

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3. Grapevine structure and function

FIGURE 3.45 General structure for the proanthocyanidin oligomers found in seeds (top panel) and skins (bottom panel) of grapes. From Mattivi, F., Vrhovsek, U., Masuero, D., Trainotti, D., 2009. Differences in the amount and structure of extractable skin and seed tannins amongst red grape varieties. Aust. J. Grape Wine Res. 15, 27e35, reproduced by permission.

Some may become anthocyanins, while others give rise to flavonols as well as catechins and their polymers, proanthocyanidins, and condensed tannins (Boss et al., 1996; Dixon et al., 2005). The latter are under the control of another MYB transcription factor, VvMYBPA1 (Bogs et al., 2007). The synthesis of phenolics begins shortly after berry development commences. Some anthocyanins are synthesized early, but most production involves nonflavonoid and other flavonoid phenolics. Anthocyanin synthesis, if it occurs, becomes pronounced only after ve´raison. The timing and degree of anthocyanin synthesis depend on a variety of factors, such as the temperature, light exposure, water status, sugar accumulation, and genetic factors. After reaching a maximum (usually at grape maturity), anthocyanin concentration tends to decline. Tannin content tends to mirror changes in anthocyanin content, especially for those more readily extracted from the skin. A decline in solubility

associated with increasing polymer size contributes to a reduction in fruit astringency. Although the anthocyanin content of red grapes increases during ripening, the proportion of the various anthocyanins and their acylated derivatives can change dramatically (Gonza´lez-SanJose´ et al., 1990). As the final combination can significantly influence the hue, intensity, and color stability of wine, these changes have great enological significance. Methylation of oxidationsensitive o-diphenol sites (adjacent hydroxyl groups) improves anthocyanin stability. Thus, the monophenolic anthocyanins, peonin and malvin, are the least sensitive to oxidation. Oxidative susceptibility is also decreased by bonding with sugars and acyl groups (Robinson et al., 1966). Thus, the activity of anthocyanin acyltransferase in grapes also influences potential wine color intensity and stability. Other factors can also affect color developmentdfor example, mild water deficit activates abscisic acid synthesis, thereby enhancing flavonol

Berry growth and development

synthesis. Other flavonols that accumulate during ripening can contribute to the development of oxidation-resistant anthocyanin copigments. The precise conditions that initiate anthocyanin synthesis during ve´raison are unknown. One hypothesis suggests that sugar accumulation provides the substrate needed for synthesis. The marked correlation between the accumulation of sugars in the skin and anthocyanin synthesis is consistent with this view. Sugar accumulation beyond the immediate metabolic needs of a tissue often favors the synthesis of secondary metabolites. Nevertheless, sugar accumulation may act indirectly through its effect on the osmotic potential of skin cells. In tissue culture, high osmotic potentials can induce anthocyanin synthesis and subsequent methylation (Do and Cormier, 1991). However, as abscisic acid content correlates with sugar accumulation up to ve´raison, and its application at ve´raison enhances UFGT gene expression (Peppi et al., 2006), sugar accumulation may act indirectly through enhanced abscisic acid production (Ban et al., 2003). Alternatively, anthocyanin synthesis may be activated by changes in the potassium and calcium content of skin cells. Potassium accumulation in skin cells following ve´raison is as nonuniform in cellular distribution as is initial anthocyanin synthesis. Conversely, a decline in calcium content is inversely correlated with anthocyanin synthesis. Which of these alone or in combination, if any, ultimately triggers anthocyanin synthesis remains to be established. During development, changes similar in scope to those involving anthocyanin composition affect skin tannins (Downey et al., 2003). There is often good correlation between sugar accumulation in the skin (but not the flesh) and phenolic content (Pirie and Mullins, 1977). Phenol accumulation occurs in both cellular vacuoles (where polymerization appears less common) and cell walls (Gagne´ et al., 2006). The degree of polymerization increases during ripening, as does the proportion of epigallocatechin subunits. This may also involve the inclusion of anthocyanin monomers in proanthocyanidin polymers (Kennedy et al., 2001). The average number of catechin subunits in skin tannins is about 25 in Shiraz, between 24 and 33 in Cabernet Sauvignon, and may reach up to 80 in Merlot. As polymer size increases, tannin interaction with cell-wall constituents tends to decrease, at least in red grapes (Bindon and Kennedy, 2011). This change may facilitate tannin extraction during fermentation. The binding potential of cell-wall polysaccharides (notably their rhamnogalacturonans) (see Hanlin et al., 2010) remains undiminished in the must, a feature that could reduce extraction during fermentation. The principal extension subunits of skin tannins are ()-epicatechin and ()-epigallocatechin, with the terminal units being primarily (þ)-catechin (Fig. 3.45), although this varies among cultivars (Mattivi et al.,

125

2009). Epicatechins (cis isomers) are biosynthesized from anthocyanidins, whereas catechins (trans isomers) are derived from leucocyanidins (flavan-3,4-diols) (Fig. 3.45). Tannin monomers usually reach their peak content prior to ve´raison, probably because the precursors are subsequently redirected toward anthocyanin accumulation in red cultivars as well as downregulation of abscisic acid synthesis, progressive polymerization, and dilution associated with water uptake. Variation in tannin concentration among cultivars, between different tissues, in degrees of bonding with cell-wall constituents, and with climatic conditions presumably relates to the relative expression of multiple flavonoid synthesis enzymes (Jeong et al., 2008). Multiple and distinct gene variants as well as their regulators occur in the grape genome. For example, three variants of chalone synthase are presently known as are two forms of chalone isomerase and flavanone 3-hydroxylase. Because of the extensive number of potential proanthocyanidins, discrepancies among the findings of different studies may have as much to do with the analytic techniques used for extraction and assessment as with legitimate variation. The synthesis of nonflavonoid phenolics tends to decline or cease following ve´raison, possibly explaining the decline in their esters. Nonetheless, some lowmolecular-weight phenolic compounds (e.g., benzoic and cinnamic acid derivatives) may increase during ripening (Ferna´ndez de Simo´n et al., 1992). In white grapes, the primary phenolic changes during ripening involve hydroxycinnamic tartrates and catechins. Although the dynamics of hydroxycinnamic tartrate metabolism are unclear, their levels tend to decline strikingly during ripening (Lee and Jaworski, 1989, Fig. 3.46). As the degree and nature of this decline are influenced by genetic and environmental factors, their role in the oxidative browning of wine can differ markedly from variety to variety and from year to year. In contrast, catechin levels may rise following ve´raison, subsequently declining to low levels by maturity. Catechin monomers show a lower tendency to polymerize into large tannins in white than red cultivars. This may involve the partial inactivity of VvMYBPA transcription factor genes in white cultivars, but this is currently just speculation. The predominant phenolics in seeds are flavan-3-ols (catechin, epicatechin, and their proanthocyanidin polymers). The proportion of catechin to epicatechin in proanthocyanidins varies considerably from cultivar to cultivar (Mattivi et al., 2009). Those in the seed wall are more polymerized and contain a higher proportion of epicatechin gallate than those in inner parts of the seed (Geny et al., 2003). Extension subunits are principally epicatechin and epicatechin gallate, with the terminal moiety being about equally epicatechin, epicatechin gallate, and catechin (Downey et al., 2003; see Fig. 3.45).

Hydroxycinnamic acid-tartaric acid esters (calfeoyl-tartrate + teruloyl-tartrate + p-coumaroyl-tartrate) (μ moles / g of freeze-dried grape)

126

3. Grapevine structure and function

After a pronounced increase during early berry development, the content of individual flavanols in seeds declines as they polymerize into proanthocyanidins and condensed tannins. This can be more marked in vines with low vigor (Pen˜a-Neira et al., 2004) and may partially explain why wines made from these vines tend to be less bitter and astringent than equivalent wines made from grapes harvested from vigorous vines. Reduced astringency may also result from the association of pectins with proanthocyanidins (Kennedy et al., 2001). A group of stilbene phenolic compounds (phytoalexins) have attracted considerable attention recently.

GRENACHE

60

MACCABEO UGNI BLANC CLAIRETTE CARIGNANE

45

30

(A)

700 600 Content (mg/L)

15

5

500 400 300 200 100 0

1/07

1/08

1/09

1/10

(B) Content (mg/g)

Evolution of the overall hydroxycinnamic ester content of five (2 red, three white) grape varieties during development and maturation. From Romeyer, F.M., Macheix, J.M., Goiffon, C.P., Reminiac, C.C., Sapis, J.C., 1983. The browning capacity of grapes. 3. Changes and importance of hydroxycinnamic acid-tartaric acid esters during development and maturation of the fruit. J. Agric. Food Chem. 31, 346e349. Copyright, 1983, American Chemical Society, reproduced by permission.

CABERNET S.

TEMPRANILLO

GRACIANO

CABERNET S.

TEMPRANILLO

GRACIANO

CABERNET S.

Monomers

Oligomers

60 50 40 30 20 10 0

(C) Content (mg/g)

Correspondingly, the degree of galloylation in seed tannins is much higher than in skin tannins (13%e29% vs. 3%e6%, respectively) (Prieur et al., 1994; Souquet et al., 1996). In Shiraz, the average number of subunits in proanthocyanidin polymers is about 5. Although they constitute the major source of phenols in grapes (about 60% vs. 20% each in the skins and stalks), they dissolve more slowly and contribute less to the phenolic composition of wine except when maceration during fermentation is prolonged. In the latter situation, their galloylation is likely to contribute to the initial coarseness of the wine’s astringency (Vidal et al., 2003). Extraction of seed tannin is reduced during wine production due to galloylation and the lipid content of the seed coats, at least until the alcohol content of the ferment facilitates their solubilization. The proportion of monomeric, oligomeric, and polymeric flavonols in seeds, skins, and wine from three cultivars is illustrated in Fig. 3.47.

GRACIANO

80 70

Date

FIGURE 3.46

TEMPRANILLO

18 16 14 12 10 8 6 4 2 –1

Polymers

FIGURE 3.47 Flavan-3-ol content of the monomeric, oligomeric, and polymeric fractions of (A) wine, (B) seeds, and (C) skins from Tempranillo, Graciano, and Cabernet Sauvignon. Bars represent standard deviation. From Monagas, M., Go´mez-Cordove´s, C., Bartolome´, B., Laureano, O., and Ricardo da Silva, J. M. (2003) Monomeric, oligomeric, and polymeric flavan-3-ol composition of wines and grapes from Vitis vinifera L. cv. Graciano, Tempranillo, and Cabernet Sauvignon. J. Agric. Food Chem. 51, 6475e6481, reproduced by permission. Copyright, 2003 American Chemical Society.

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Berry growth and development

Pectins

They are predominantly synthesized in response to localized stress, either biotic or abiotic. They are involved in the vine’s phytoalexin defense system. The major phytoalexin is resveratrol (as well as its glycoside, piceid). Smaller amounts of several related compounds, derived from resveratrol (pterostilbene and viniferin), may also accumulate. Although activation of phytoalexin synthesis occurs in response to stress, phytoalexins may also show constitutive synthesis and accumulation during ripening (Gatto et al., 2008). In this regard, there is marked variation between cultivars (Fig. 3.48), some being high producers, such as Pinot noir, whereas others are low producers, such as Nebbiolo. White cultivars generally produce less resveratrol. In a few cultivars, reduction in resveratrol content after ve´raison has been detected, coinciding with increased anthocyanin synthesis. Because the synthesis of anthocyanins and stilbenes has the same precursor (coumaroyl-CoA), it has been hypothesized that the synthesis of anthocyanins may divert precursors away from resveratrol synthesis (Jeandet et al., 1995). However, this seems inconsistent with the greater synthesis of stilbene phytoalexins in red than in white grapes.

(A)

One of the more obvious changes during berry ripening is softening. It facilitates juice release from the flesh and the extraction of phenolic and flavor components from the skin. The increase in water-soluble pectins and decrease in wall-bound pectins (Silacci and Morrison, 1990) result in loosening of the bonds that hold plant cells together. These changes probably result from a combination of enzymatic degradation and decline in pectin calcium content. The latter is associated with acidification of the apoplast. Calcium is important in maintaining the solid pectin matrix that characterizes most plant-cell walls. Calcium uptake essentially ceases following ve´raison. The involvement of cellulases and hemicellulases, in addition to that of pectinases, in softening is unknown.

Lipids The lipid content of grapes consists of skin cuticular and epicuticular waxes, cutin fatty acids, and membrane phospho- and glycolipids as well as seed oils. Seed oils are important as an energy source during seed germination (a potential commercial by-product of wine production) but seldom found in juice or wine. They consist primarily of polyunsaturated fatty acids

25

Trans-resveratrol Cis-piceid

20

Trans-piceid

15 10 5 ND

0 v

r

p

Pinot Noir

(B)

v

r

p

v

Tarrango

r

p

Malvasia Puntinata

v

r

p

v

r

p

v

Pinot Tete de Roussanne Negre

r

p

Marsanne

v

r

p

Franconia

v

r

p

v

r

p

v

Schioppettino Pinot Gris

r

p

Xarello

v

r

p

Refosco

6 5 4 3 2 1 ND

0 v

r

p

Primitivo di Gioia

v

r

p

Aglianico

v

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FIGURE 3.48 Stilbene content of V. vinifera grape varieties (vintage 2003) classified as high (A) and low (B) resveratrol producers. The grape varieties were collected at ve´raison (v), ripening (r), and postripening (p) phases. The height of the bars refers to the average of the total resveratrol content as a sum of trans- and cis-piceid and trans-resveratrol expressed as mg/kg of fresh weight of total grape berry. ND, not determined. From Gatto, P., Vrhovsek, U., Muth, J., Segala, C., Romualdi, C., Fontana, P., et al., 2008. Ripening and genotype control stilbene accumulation in healthy grapes. J. Agric. Food Chem. 56, 11773e11785. Copyright, 2008, American Chemical Society, reproduced by permission.

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(notably linoleic and oleic acids) as well as palmitic and stearic saturated fatty acids (Currle et al., 1983). Their presence in wine in significant amounts could generate rancid odors. The lipid concentration of the berry changes little during growth and maturation. Thus, synthesis appears to be equivalent to berry growth. Membrane phospholipids constitute the most abundant grape lipid fraction followed by neutral lipids. Glycolipids and phytosterols (primarily b-sitosterol) constitute the least abundant groups (Le Fur et al., 1994). The predominant fatty components are the longchain fatty acids: linoleic, linolenic, oleic, and palmitic acids (Roufet et al., 1987). Most of the cuticular layer is deposited in folds by anthesis and expand during berry growth (see Fig. 3.30B). In contrast, epicuticular wax plates are deposited throughout berry growth, commencing at about anthesis (Rosenquist and Morrison, 1988). Most of the epicuticular coating consists of hard wax composed of oleanolic acid dimers attached to the edges of plates consisting of C24eC26 alcohols, notably n-hexacosanol (Casado and Heredia, 1999). The content seems to be cultivar-dependent. For example, Palomino cuticular wax showed about 30% oleanolic acid (Casado and Heredia, 1999) vs. nearly 65% in Sultana (Fig. 3.49). The softer, underlying cuticle contains the fatty acid polymer cutin. It consists primarily of C16 and C18 fatty acids, such as palmitic, linoleic, oleic, and stearic acids and their derivatives. Cutin is embedded in a complex mixture of relatively nonpolar waxy compounds. About 30%e40% of the long-chain fatty acids of the berry are located in the skin. As such, they could potentially supply unsaturated fatty acids used in the synthesis of yeast cell membranes during vinification. Regrettably,

high concentrations of these phytosterols disrupt yeast cell membrane function (Luparia et al., 2004). The relative concentration of fatty acids changes little during maturation, but the concentration of individual components can vary considerably. The most significant change involves a decline in linolenic acid during ripening (Roufet et al., 1987). This could have sensory impact by reducing the likelihood of the development of a herbaceous odor in the wine. Carotenoids are another important class of grape lipids. In red varieties, the concentration of both major (b-carotene and lutein) and minor (5,6-epoxylutein and neoxanthin) carotenoids falls markedly after ve´raison (Razungles et al., 1988). The decline appears to be partially the result of dilution during stage III berry growth, following cessation of synthesis. Because of their insolubility, most carotenoids remain with the skins and pulp following crushing. Nevertheless, enzymatic and acidic hydrolysis during maceration may release water-soluble carotenoid derivatives. These can include important aromatic compounds such as damascenone and b-ionone. They tend to occur primarily in white wines left on the skins sufficiently long for enzymatic carotenoid degradation. Carotenoid breakdown during ripening can also lead to an increasing norisoprenoid content (Razungles et al., 1993).

Nitrogen-containing compounds Most information concerning grape proteins relates to enzymes and soluble proteins associated with maturation. The most studied are those involved in anthocyanin synthesis and other phenolics of vitivinicultural interest and polyphenol oxidases sequestered in cytoplasmic lysosomes. Other extensively studied proteins are PR proteins. These have direct enological interest due to their Hydrocarbons Aldehydes Acids

CH3(CH2)nCH3 CH3(CH2)nCHO CH3(CH2)nCOOH

Esters CH3(CH2)nCOO(CH2)nCH3

COOH

Oleanolic acid

CH3(CH2)nCH2OH

Alcohols

HO Unknown

FIGURE 3.49 Cuticular wax content of Sultana grapes. From Grncarevic, M., Radler, F., 1971. A review of the surface lipids of grapes and their importance in the drying process. Am. J. Enol. Vitic. 22, 80e86, reproduced by permission.

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nitrogen content may remain with the pomace as nonsoluble components of the fruit. Changes during ripening also occur in the level of inorganic nitrogen, notably ammonia. Ammonia may show either an increase (Gu et al., 1991) or decline (Solari et al., 1988) during ripening. Although a decline might appear to negatively affect fermentation, it probably is more than compensated for by a simultaneous increase in amino acid content. Amino acids are incorporated by yeasts at up to 5e10 times that of the rate of ammonia. Their incorporation also alleviates the requirement of diverting metabolic intermediates to amino acid synthesis. However, as individual amino acids are accumulated by yeast cells at different rates, the specific amino acid content of the juice can significantly affect fermentability. Some changes in nitrogen grape content are summarized in Fig. 3.50.

Aromatic compounds Until comparatively recently, most research focused on the major constituents of grapes. Studies of its aromatic components lagged behind due to a lack of analytic equipment possessing sufficient resolving power. Long-held views about the synthesis and location of aromatic compounds thus remained unsubstantiated. Developments and refinement in gas chromatography and other analytic procedures have opened the floodgates for an unabated flow of new information. Eventually, these data may permit the scheduling of harvest to achieve predefined wine aromatic profiles. Initially, research centered on very aromatic cultivarsdthose easier to analyze because their aroma 300 Variety: Cabernet sauvignon N mg per liter Gr.sugar per liter

resistance to proteolytic breakdown and solubility at low pH values. Correspondingly, they favor tannin precipitation during winemaking as well as lead to protein instability (haze induction) in bottled wine. Grape-soluble protein content increases markedly during maturation, reaching levels from 200 to 800 mg/L. In addition to changes during maturation, cultivars differ considerably in protein content (Tyson et al., 1982). This diversity is highest in nascent berries, becoming less variable as the fruit ripens. The composition also changes, with PR proteins becoming increasingly abundant after ve´raison. Their synthesis in healthy berries is seemingly activated by sugar accumulation (Tattersall et al., 1997). They may constitute 50% e75% of the soluble protein content at maturity (Monteiro et al., 2007). PR proteins are produced in all plants and are highly diverse, being grouped into 14 structurally and functionally distinct categories. Those found in grapes include PR-5 (thaumatin-like proteins and osmotins), PR-2 (b-1,3-glucanases), and PR-3, -4, -8, and -11 (chitinases). They assist in defending against microbial invasion by creating transmembrane pores in the pathogen or degrading their cell-wall constituents (Ferreira et al., 2004). Although produced constitutively, various environment stresses as well as wounding can increase their synthesis. The most common PR proteins in ripe grapes are thaumatin-like and chitinase proteins (Robinson and Davies, 2000). The simplest common peptide found in ripe grapes is glutathione, a tripeptide consisting of glutamine, cysteine, and glycine. Its accumulation begins at the onset of ve´raison and closely follows the rise in berry sugar content (Adams and Liyanage, 1993). It functions as an important antioxidant by maintaining ascorbic acid in its reduced form. As such, glutathione is a significant grape juice constituent. In must, it reacts rapidly with oxidized caftaric acid and related phenolics, forming S-glutathionyl complexes. The free amino acid content of grapes rises at the end of ripening, consisting primarily of proline and arginine. The latter is the principal form of nitrogen transported in the phloem. The relative concentrations of these two amino acids can vary up to 20-fold depending on the variety and fruit maturity (Sponholz, 1991; Stines et al., 2000). Their ratio may shift from an early preponderance of arginine to proline at maturity (Polo et al., 1983), or the reverse (Bath et al., 1991). In many plants, proline acts as a cytoplasmic osmoticum (Aspinall and Paleg, 1981), similar to that of potassium. Whether proline accumulation has the same role in grapes is unclear. Amino acids usually constitute the principal group of soluble organic nitrogen compounds released during crushing and pressing. Nevertheless, up to 80% of berry

250 Total nitrogen 200

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FIGURE 3.50 Changes in the nitrogen fraction of grapes during maturation. Data from Peynaud, E., Maurie´, A., 1953. Sur l’e´volution de L’azote dans les diffe´rentes parties du raisin au cours de la maturation. Ann. Technol. Agric. (Paris) 1, 15e25, from Amerine, M.A., Berg, H.W., Kunkee, R.E., Ough, C.S., Singleton, V.L., Webb, A.D., 1980. The Technology of Wine Making. Avi Publ., Westport, CT. reproduced by permission.

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was based primarily on monoterpenes. Although these compounds accumulate during ripening, they are progressively converted to nonvolatile forms. Most bonded with sugars to form glycosides, whereas others were converted into their corresponding oxides or polymerized into polyols (Wilson et al., 1986). Thus, mature grapes typically exhibited less aromatic character than their total terpene content would suggest. Similar trends have also been noted with fragrant norisoprenoids and aromatic phenols (Strauss et al., 1987a). Glycoside formation often increases solubility of the aglycone (nonsugar) component, facilitating terpene accumulation in cellular vacuoles. Similar complexes may also form at even higher concentrations in leaves (Skouroumounis and Winterhalter, 1994). Those synthesized in leaves appear not to be translocated to maturing fruit (Gholami et al., 1995). Details on the accumulation of free and bound terpenoids in Muscat of Alexandria are given in Fig. 3.51. The accumulation of most varietal flavorants (terpenes, norisoprenoids, volatile phenolics, etc.) tends to coincide with the beginning of ve´raison. For some constituents, this may occur rapidly (Coombe and McCarthy, 1997, Fig. 3.52), followed by subsequent declines (Coelho et al., 2007). Although most terpenes accumulate in the skin, notably geraniol and nerol, not all do. For example, free linalool and diendiol I occur more frequently in the flesh. In addition, the proportion of free and glycosidically bound terpenes often differs between the skin and the flesh. In contrast, norisoprenoids, such as 1,1,6-trimethyl-1,2dihydronaphthalene (TDN), vitispirane, and damascenone, are localized predominantly in the flesh. They occur primarily in their more soluble, glycosidically bound forms, with only trace amounts of damascenone occurring in a free, volatile state. As with monoterpenes, their concentrations increase during ripening (Strauss et al., 1987b). In addition to monoterpenes and norisoprenoids, other aromatic compounds are present in nonvolatile, conjugated (glycosidically bound) forms. One group consists of low-molecular-weight aromatic phenols. Examples in Riesling include vanillin, propiovanillone, methyl vanillate, zingerone, and coniferyl alcohol. Even phenolic alcohols, such as 2-phenylethanol and benzyl alcohol, may become glycosylated during ripening. Conjugated aromatic phenols have also been isolated from Sauvignon blanc, Chardonnay, and Muscat of Alexandria (Strauss et al., 1987a). Other nonvolatile precursors of varietal significance are thiols, typically bound to glutathione. They appear to be the precursors of both free and S-cysteine thiol conjugates (Grant-Preece et al., 2010). The latter are metabolized by yeasts during fermentation, liberating their volatile thiol moiety. These play significant roles in the aroma of several Carmenet cultivars, notably Sauvignon blanc, and to a lesser extent cultivars such as Pinot

Grigio and Riesling. Most precursors are localized in the skin, such as the precursors of 3-mercaptohexan-1ol. In contrast, others are equally distributed in the skin and pulp (Peyrot des Gachons et al., 2002). Their concentrations can increase rapidly, by up to 10-fold during the latter stages of ripening (Capone et al., 2011). Unlike the compounds noted above, others may remain in a free, volatile form through to maturity. An example is methyl anthranilate (Robinson et al., 1949). In other instances, such as the methoxypyrazines, the concentrations may decline during maturation (Fig. 3.53). In this instance, the reduction is considered desirable. Methoxypyrazines can easily overpower a wine’s flavor, suppressing the perception of other compounds and thus aromatic complexity. Studies comparable with those on white grapes have not been conducted on red grapes. In some varieties, however, aroma development has been associated with ripeness and sun exposure. In Cabernet Sauvignon, the total volatile component of the fruit was considered to be about equally derived from the skin and flesh (Bayonove et al., 1974). However, because the skin constituted between 5% and 12% of the fruit mass, the aromatic concentration in the skin must be considerably higher than that in the flesh. The synthesis of secondary metabolites tends to be correlated with accumulation of sugars. In relation to aromatic compounds, their synthesis and accumulation have tended to attract herbivores, favoring seed dispersal. Most mammals possess only two colorreceptive pigments and cannot visually recognize ripe from unripe fruit, as can primates or birds. In contrast, odor acuity is often highly developed in most mammals. Thus, changes in fragrance tend to be more useful to most mammals in recognizing ripeness.

Cultural and climatic influences on berry maturation Any factor affecting grapevine growth and health has the potential to influence the ability of the vine to nourish and ripen its crop. Consequently, most viticultural practices are directed at regulating these factors to achieve the maximum yield, consistent with grape quality and long-term vine health and productivity. Although macro- and mesoclimatic factors directly affect berry maturation, they are generally beyond the control of the grape grower. Thus, most attention is directed toward microclimatic factors.

Yield Because of the obvious importance of yield to commercial success, much research has focused on

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FIGURE 3.51 Changes in juice concentration of free (solid lines) and glycosidically bound (dashed lines) monoterpenes in developing Muscat of Alexandria grapes. Juice pH and sugar levels are shown in the bottom right graph. Berry set was complete by the first week of December and ve´raison occurred between day 47 and day 54 of the survey. Reprinted by permission from Wilson, B., Strauss, C.R., Williams, P.J., 1984. Changes in free and glycosidically bound monoterpenes in developing Muscat grapes. J. Agric. Food Chem. 32, 919e924. Copyright, 1984. American Chemical Society.

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0.10 Methyl furaneol (ng/g)

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FIGURE 3.52 Measurement of American character impact compounds during ripening of Concord grapes. From Shure, K.B., Acree, T.E., 1994. Changes in the odor-active compounds in Vitis labruscana cv. Concord during growth and development. J. Agric. Food Chem. 42, 350e353. Copyright, 1994, American Chemical Society, reproduced by permission.

Methoxypyrazine concentration (ng/L)

increasing fruit production. However, yield increases can reduce the ability of the vine to mature the fruit or its potential to produce subsequent crops. This has led to an ongoing debate over the appropriate balance between grape yield, fruit quality, long-term vine health, and fiscal solvency. In France, yield is viewed as being so directly associated with grape and wine quality that theoretical maximum crop yields have been set for Appellation Control regions. This is partially justified by the negative correlation between increased yield and sugar accumulation (Fig. 3.54; see Fig. 4.4). Nevertheless, focusing attention simply on yield deflects attention from other

80 - Cabernet Sauvignon - Sauvignon blanc V - Veraison

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FIGURE 3.53 Typical concentration of methoxypyrazines of Cabernet Sauvignon grape juice and Sauvignon blanc wine compared to their aroma threshold. From Allen, M.S., Lacey, M.J., Boyd, S.J., 1996. Methoxypyrazines of grapes and wines e differences of origin and behaviour. In: Stockley, C.S. (ed.), Proc. 9th Aust. Wine Ind. Tech. Conf. Winetitles, Adelaide, Australia, pp. 83e86, reproduced by permission.

factors of equal or greater importance, such as improved nutrition or light exposure (Reynolds et al., 1996). Elimination of viral infection often improves the vine’s capacity to produce and ripen fruit. The yield quadrupling in German vineyards in the twentieth century (Fig. 3.55) is a dramatic but not isolated example of yield increases without comparable changes in ripeness. Clonal selection has also identified clones able to produce more and better-quality fruit (see Fig. 2.28 (upper) and 2.31). Correcting for nutrient imbalances and water deficits, improving weed and pest control, and optimizing canopy management can often further improve the capacity of the vine to produce more and fully ripened fruit (see the “Management of Vine Growth” section in Chapter 4). Although serious overcropping (>110e150 hL/ha) can reduce grape (and wine) quality, the specific cause-and-effect relationships are poorly understood. Factors frequently involved include delayed ripening, higher acidity, enhanced potassium content, reduced anthocyanin synthesis, diminished sugar accumulation, and limited flavor development. Achieving ideal yield is one of the most perplexing and probably quixotic demands of grape growing. It depends not only on grape variety, soil, and climatic conditions but also on the type of wine desired. Although overcropping reduces grape quality and negatively affects subsequent yield and vine health, low yield does not necessarily equate with improved quality. Undercropping can prolong shoot growth and leaf production, increase shading, depress fruit acidity, and undesirably influence berry nitrogen and inorganic nutrient contents. Reduced yield also tends to induce the vine to produce larger berries. The reduced skin/flesh may negatively affect attributes derived primarily from the skin. This may partially explain the disjunct between enhanced “good” wine aroma and aggressive taste intensity associated with reduced yield (Fig. 3.56). In addition, an overly intense aroma may compromise subtlety, masking subtlety and diminishing complexity. Attributes associated with the skin do not necessarily change in direct proportion to the surface area/volume ratio when comparing various berry-size categories. The interaction can be complex and diverse depending on the constituents assessed, how easily they are extracted, whether they are volatile or bound, and how they are measureddchemically (e.g., mg/berry, mg/g skin, mg/kg grape, and mg/cm skin) (Barbagallo et al., 2011) or sensorially (tasting methodology). In a study by Chapman et al. (2004), reduced yield was associated with a slight increase in bell pepper odor (2-methoxy-3-isobutylpyrazine), bitterness, and astringency in Cabernet Sauvignon wines. In contrast, higher yield generated wine moderately higher in red/ black berry aromas, jammy flavors, and fruitiness. The effects were less marked than those found with

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FIGURE 3.54 Relationship between crop load and sugar accumulation in the variety Gewu¨rztraminer. From Huglin, P., Balthazard, J., 1976. Donne´es relatives a l’influence du rendement sur le taux de sucre des raisins. Connaiss. Vigne Vin 10, 175e191, reproduced by permission.

(A) 120 110

Yield (hl/ha) – Smoothed

100 90 Yield (hl/ha)

80 70 60 50 40 30 20 10 0 1893 1903 1913 1923 1933 1943 1953 1963 1973 1983 1993

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100 90 80 70 60 50 1893 1903 1913 1923 1933 1943 1953 1963 1973 1983 1993

FIGURE 3.55

Fruit production (A) and soluble solids (B) from Riesling at Johannesburg from 1885 to 1991 and 1890 to 1991, respectively. From Hoppmann, D., Hu¨ster, H., 1993. Trends in the development in must quality of ‘White Riesling’ as dependent on climatic conditions. Wein-Wiss. 48, 76e80, reproduced by permission.

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(A) 140

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FIGURE 3.56 Relationship between crop level of Zinfandel vines and (A) good wine aroma and (B) taste intensity. From Sinton, T.H., Ough, C.S., Kissler, J.J., Kasimatis, A.N., 1978. Grape juice indicators for prediction of potential wine quality. I. Relationship between crop level, juice and wine composition, and wine sensory ratings and scores. Am. J. Enol. Vitic. 29, 267e271, reproduced by permission.

Zinfandel (Fig. 3.56). Of particular interest was the observation that how yield variation was achieved was important (Chapman et al., 2004). Winter pruning, which had less effect on yield manipulation, had a greater effect on sensory characteristics than did the more marked yield variations generated by cluster thinning. It has generally been considered that wines produced from vines bearing light to intermediate crops are preferred (Cordner and Ough, 1978; Gallander, 1983; Ough and Nagaoka, 1984). Nevertheless, the yield/quality ratio is not necessarily constant within the midyield range (Sinton et al., 1978). Excessive yield reduction, at least on a hectarage basis, can also jeopardize the financial solvency of a vineyard/winery. In contrast, reduced yield per vine induced by higher-density planting has often been considered to enhance grape quality. This presumed advantage does come at considerable up-front costs associated with vineyard establishment as well as increased maintenance expenses. An improved understanding of the complexities of yield/quality relationships presumably will come with a better grasp of the factors that lead to grape and wine quality (see the “Criteria for Harvest Timing” section in Chapter 4). In addition, it is difficult to separate the indirect effects of vigorous growth, such as excessive canopy shading, from its more directs effects on yield and grape maturity. Canopy shading can influence fruit quality even when the grapes themselves are not shaded (Schneider et al., 1990). An excellent example of how confounding factors were reduced is provided by Cortell et al. (2005). Their

study was conducted in a commercial vineyard planted with vines of the same age, clone, and rootstock. Georeferencing permitted sampling and wine preparation from sites showing differing levels of vigor. Vine vigor had a more pronounced influence on proanthocyanin concentration and skin vs. seed composition. It is also essential that studies on yield/quality relationships investigate more than just their sensory effects on young wines. The effects of yield on wine-aging potential also requires verification. Regrettably, such long-term studies do not mesh well with the time limits of funding agencies, the need to publish frequently, or the duration of graduate or postdoctoral studies. Another commonly held belief is that “stressing” vines increases their ability to produce fine-quality wine. This view presumably arose from the reduced vigor/improved grape quality association of some low-nutrient status, renowned European vineyards. Nevertheless, balancing the leaf area/fruit ratio can improve the microclimate within and around the vine, increase photosynthetic efficiency, and promote fruit maturation and flavor development (see the “Yield/ Quality Ratio” section in Chapter 4). Judicious balancing the vegetative and reproductive functions of the vine can also favorably affect fruit acidity and pH.

Sunlight As the energy source for photosynthesis, sun exposure is the single most important climatic factor affecting berry development. Because sunlight contains ultraviolet, visible, and infrared (heat) radiation, and most

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Chlorophyll 667 nm 40 Transmission of grape leaf and skin of grape berry [%]

absorbed radiation is released as heat, it is often difficult to separate light from its temperature modifying effects. Because berry photosynthesis typically declines rapidly postve´raison, most of the direct effects of sunlight on berry maturation are either thermal or phytochromeinduced. Because of the selective absorption of light by chlorophyll, there is a marked increase in the proportion of farred light within vine canopies. This shifts the proportion of physiologically active phytochrome (Pfr) from 60% in full sun to below 20% in shade (Smith and Holmes, 1977). Evidence suggests that this may delay the initiation of anthocyanin synthesis, decrease sugar accumulation, and increase the ammonia and nitrate content in shaded fruit (Smart et al., 1988). The red/far-red balance of sunlight and ultraviolet radiation is known to influence flavonoid biosynthesis in many plants (Hahlbrock, 1981) including grapevines (Gregan et al., 2012). Whether sunflecks can offset the effects of shading is unknown but is theoretically possible. Pr is more efficiently converted to Pfr by red light than is the reverse reaction (exposure to far-red radiation). However, because some plants also show high-intensity blue and far-red light responses, the effects of leaf shading may be more complex than the red/far-red balance alone might suggest. Berry pigmentation and its change following ve´raison (Fig. 3.57) can also influence the Pr/Pfr ratio in maturing fruit (Blanke, 1990), especially in sun-exposed fruit. Whether this significantly influences fruit ripening is unknown, but it could affect the activity of lightactivated enzymes such as phosphoenolpyruvate carboxylaseda critical enzyme in the metabolism of malic acid (Lakso and Kliewer, 1975). Light exposure is generally essential for flavonol synthesis and may promote anthocyanin synthesis, inducing deep coloration in red cultivars (Rojas-Lara and Morrison, 1989). Anthocyanin synthesis seems most sensitive to light induction from fruit set to preve´raison (Li et al., 2013). Sun exposure may also reduce pigment extraction from grapes during winemaking (Rustioni et al., 2011). Light exposure can also increase the relative proportion of skin to seed tannins. In this regard, the ratio has been proposed as an indicator of wine flavonoid composition, wine color, and quality (Ristic et al., 2010). Nevertheless, this finding is not universal, seemingly being varietally dependent. For example, Weaver and McCune (1960) found little difference in the pigmentation of berries covered (to prevent directsunlight exposure) and that of sun-exposed fruit. Downey et al. (2004) have confirmed this observation with Shiraz (Plate 3.11). High-intensity light may actually decrease coloration in varieties such as Pinot noir (Dokoozlian, 1990). Berry phenol content tends to reflect those of light exposure on anthocyanin synthesis (Morrison and

green berry

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FIGURE 3.57 Relative transmission of light through the skin of white (Mu¨ller-Thurgau) and red (Blauer Spa¨tburgunder) grapes at ve´raison, compared with that of a Mu¨ller-Thurgau leaf. From Blanke, M.M., 1990. Carbon economy of the grape inflorescence, 4. Light transmission into grape berries. Wein-Wiss. 45, 21e23, reproduced by permission.

Noble, 1990) but again not consistently (Price et al., 1995). Some of these differences may relate to macroclimatic factors, where increased exposure (and temperature) favors anthocyanin and phenolic synthesis in cool climes but can have adverse effects in hot climates. The influence of light exposure on the anthocyanin composition of grapes is largely unknown. In Nebbiolo, shading not only reduced anthocyanin accumulation but increased the relative proportion of trihydroxylated forms (malvidin, delphinidin, petunidin) (Chorti et al., 2010). Because anthocyanins differ in their susceptibility to oxidation, changes in composition can be more important to wine color stability than total anthocyanin content. The involvement of light intensity and spectral quality on catechin synthesis appears not to have been studied despite their involvement in color stability (see Chapter 6). Sun-exposed fruit may show higher titratable acidity and tartaric acid contents than shaded fruit (Smith et al., 1988; DeBolt et al., 2008). The increase in acidity may or may not be reflected in a decrease in pH and potassium accumulation. Shading also correlates with increased magnesium and calcium accumulation (Smart et al., 1988). The lower malic acid content occasionally observed in sun-exposed fruit may result from the

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PLATE 3.11 (A) Light-proof boxes in the field at the time of application at flowering; (B) ’Shiraz’ berries 2 weeks preve´raison showing differences in chlorophyll accumulation between treatments; (C) ’Shiraz’ berries 6 weeks postve´raison, showing no obvious difference in coloration between treatments. The shaded bunch is on the left in both pictures. From Downey, M.O., Harvey, J.S., Robinson, S.P., 2004. The effect of bunch shading on berry development and flavonoid accumulation in Shiraz grapes. Aust. J. Grape Wine Res. 10, 55e73, reproduced with permission.

associated heat-induced increase in malic enzyme activity. The lower sugar concentration in fruit, associated with leaf shading, is thought to result from dilution during berry enlargement (Rojas-Lara and Morrison, 1989). Berry size generally correlates with the degree of canopy shading, but is little affected when only the fruit is shaded (Morrison, 1988; DeBolt et al., 2008). These differences may result from reduced transpiration under shade conditions. Sun exposure can also hasten the processes of ripening. There are several reports of sun exposure affecting grape aroma. Usually the level of grassy or herbaceous odors in Sauvignon blanc (Arnold and Bledsoe, 1990) and Se´millon (Pszczolkowski et al., 1985) are reduced by sun exposure. In contrast, other fruit flavors may be unaffected or augmented. Sun exposure enhances the synthesis of carotenoids in young grapes, notably the UV-B portion (Steel and Keller, 2000). Following ve´raison, however, carotenoid level declines, probably as a partial consequence of conversion into aromatic norisoprenoidsdfor example, b-damascenone and vitispirane (Marais et al., 1992a; Baumes et al., 2002)das well as by dilution. There is also a correlation between monoterpene content, activation of genes associated with monoterpene synthesis, and sun exposure (Smith et al., 1988; Reynolds and

Wardle, 1989). The resulting enhanced terpene content may help mask the vegetative odors produced by methoxypyrazines in Se´millon and Sauvignon blanc wines (Reynolds and Wardle, 1991). In addition, the methoxypyrazine content in Cabernet Sauvignon may be up to 2.5 times lower in sun-exposed fruit than in shaded fruit (Allen, 1993). Similar findings have also been obtained for Sauvignon blanc (Marais et al., 1996; Gregan et al., 2012). These effects can also be affected by the degree and timing of leaf removal around clusters. For Sauvignon blanc, the effect was most marked when leaves around the cluster were removed several weeks before ve´raison (Arnold and Bledsoe, 1990), and for Cabernet Franc and Merlot several weeks after anthesis (Scheiner et al., 2010). The effect of shading appears to influence preve´raison synthesis, not postve´raison degeneration (Ryona et al., 2008). In addition to direct light-activated influences, sun exposure indirectly influences fruit maturation through associated heating effects. As berries mature, their stomata cease to open, and the cooling induced by transpiration decreases. Although the grape skin is more water-permeable than those of many other fleshy fruits (Nobel, 1975) and therefore could show enhanced evaporative cooling, sun exposure can generate temperatures considerably above ambient. This influence is dependent on factors

137

Cultural and climatic influences on berry maturation

such as skin color, berry shape, cluster density, duration and intensity of the sun exposure, angle of incidence, and wind velocity. Correspondingly, densely packed clusters of dark fruit show greater heating than loose clusters of light-colored fruit. Temperatures can reach up to 15
C above ambient (Millar, 1972; Smart and Sinclair, 1976). Shaded fruit are often cooler than ambient due to heat reflection by the canopy (see Fig. 5.36).

Temperature is known to markedly influence enzyme function and cell-membrane permeability. What is poorly understood is how these molecular influences generate some of their known effects (Fig. 3.58). This is compounded, as with all environmental factors, by difficulties in designing rigorously controlled experiments, one factor often indirectly affecting other critical factors and making definitive statements about any one somewhat dubious (Bonada and Sadras, 2015). Although regrettable, some trends are better than none at all. The effect of temperature on malate respiration is well known, but its precise mode of action still remains unestablished. Although temperature enhances the action of malic enzymes, especially above 30
C (Lakso and Kliewer, 1978), this influence appears inadequate to fully explain its pronounced affect. High temperatures might provoke malic acid leakage from vacuoles favoring breakdown in the cytoplasm, but this is unknown. In contrast, tartaric acid content is little affected by seasonal temperature changes. Temperature conditions also have a pronounced effect on anthocyanin synthesis (Kliewer, 1977b) (and their subsequent stability in wine). In several varieties, synthesis is favored by warm daytime temperatures and cool nights (20
C e25
C and 10
C e15
C, respectively). However, in varieties such as Kyoho, optimal anthocyanin synthesis may occur at temperatures as low as 15
C. Temperatures above 35
C often suppress or inhibit anthocyanin synthesis (Kliewer, 1977a; Spayd et al., 2002). Disruption of the typical correlation between sugar accumulation and anthocyanin synthesis, which occurs at the onset of ve´raison, may be absent when water deficit develops shortly before ve´raison (Sadras and Moran, 2012). This may be due to the negative effects of water deficit on abscisic acid levels, primarily just prior to ve´raison (Yamane et al., 2006). Abscisic acid is considered partially involved in the activation of genes directly involved in anthocyanin synthesis. There is some evidence that could be interpreted as suggesting the synthesis of other phenolic compounds (caffeoyl tartrate) may be augmented in warm climates (Herrick and Nagel, 1985), diverting precursors from anthocyanin synthesis. In white cultivars, such as Riesling, this can be undesirable, leading to excessive bitterness in their wines.

Mal Relative concentration

Temperature

Sugar

ate

Tartrate Potassiu

Proli

m

ne

other amino acids cv K

cv

15

yoho

be Ca

rne

Anth

t

20

ocya

nin

25 Temperature °C

30

35

FIGURE 3.58 Summary of some of the effects of temperature on the concentration of chemical compounds in grapes. Data from Buttrose, M.S., Hale, C.R., Kliewer, W.M., 1971. Effect of temperature on the composition of Cabernet Sauvignon berries. Am. J. Enol. Vitic. 22, 71e75; Hale, C.R., 1981. Interaction between temperature and potassium in grape acids. In: CSIRO Division of Horticulture Research Report for 1979e81, Glen Osmond, Australia, pp. 87e88; Hale, C.R., Buttrose, M.S., 1973. Effect of temperature on anthocyanin content of Cabernet Sauvignon berries. In: CSIRO Division of Horticulture Report for 1971e73, Glen Osmond, Australia, pp. 98e99, Hale, C.R., Buttrose, M.S., 1974. Effect of temperature on ontogeny of berries of Vitis vinifera L. cv. Cabernet Sauvignon. J. Am. Soc. Hortic. Sci. 99, 390e394; Kliewer, W.M., 1971. Effect of day temperature and light intensity on concentration of malic and tartaric acids in Vitis vinifera L. grapes. J. Am. Sci. Hortic. Sci. 96, 372e377; Kliewer, W.M., Lider, L.A., 1970. Effect of day temperature and light intensity on growth and composition of Vitis vinifera L. fruits. J. Am. Soc. Hortic. Sci. 95, 766e769; Kobayashi, A., Yukinaga, H., Itano, T., 1965a. Studies on the thermal conditions of grapes, III. Effects of night temperature at the ripening stage on the fruit maturity and quality of Delaware grapes. J. Jpn. Soc. Hortic. Sci. 34, 26e32, Kobayashi, A., Yukinaga, H., Matsunaga, E., 1965b. Studies on the thermal conditions of grapes. V. Berry growth, yield, quality of Muscat of Alexandria as affected by night temperature. J. Jpn. Soc. Hortic. Sci. 34, 152e158; Lavee, S., 1977. The response of vine growth and bunch development to elevated winter, spring and summer day temperature. In: International Symposium on Quality in the Vintage, Oenology and Viticultural Research Institute, Stellenbosch, South Africa, pp. 209e226; Radler, F., 1965. The effect of temperature on the ripening of ‘Sultana’ grapes. Am. J. Enol. Vitic. 16, 38e41; Tomana, T., Utsunomiya, N., Kataoka, I., 1979a. The effect of environmental temperatures on fruit ripening on the tree, II. The effect of temperatures around whole vines and clusters on the colouration of ‘Kyoho’ grapes. J. Jpn. Soc. Hortic. Sci. 48, 261e266; graphed by Coombe, B.G., 1987b. Influence of temperature on composition and quality of grapes. Acta Hortic. (Wagening.). 206, 23e35, reproduced by permission.

3. Grapevine structure and function

The high temperatures that can develop in sun-exposed fruit (see Fig. 5.36) can lead to color loss in some varieties. This is probably more important in varieties that terminate anthocyanin synthesis early in berry development. Although the proportional composition of anthocyanins changes during ripening (Gonza´lez-SanJose´ et al., 1990), and its corresponding influence on a wine’s color stability, the influence of temperature on this feature of anthocyanin synthesis has been little investigated. Increasing temperatures generally has a beneficial influence on sugar accumulation during ripening, except above 32
C e35
C. The upper limit may result from the concomitant disruption of photosynthesis (Kriedemann, 1968). High temperatures also temporarily increase the rate of respiration. Nonetheless, relative sugar content may increase during overripening due to the concentrating effect of heat-enhanced water loss. Warm conditions can increase the amino acid content of developing fruit, notably in the accumulation of proline, arginine, and amino acid amides (Sponholz, 1991). Potassium accumulation usually increases with warm temperatures (Hale, 1981). Whether this is driven by increased transpiration, associated with sugar accumulation, or some other factor is unclear. Not surprisingly, the increase in potassium content is equally associated with a rise in pH, especially in association with warm night temperatures (Kliewer, 1973). The effect of temperature on the accumulation of aromatic compounds has drawn little attention. The common view is that cool conditions (in warm climates) enhances flavor development. This may arise from reduced vegetative growth and slower respiration, which in turn may favor the redirection of nutrients toward secondary metabolic pathways. These pathways generate most aromatic constituents. For example, the synthesis of aromatic compounds in Pinot noir is reported to be higher in grapes grown in long, cool seasons than in short, hot seasons (Watson et al., 1991). However, if conditions are too cool or short, the expression of berry aromas and fruit flavors may be reduced and the development (or retention) of less desirable vegetable odors enhanced (Heymann and Noble, 1987). Gladstones (1992) has suggested that this may arise from enhanced unsaturated fatty acid synthesis, and their subsequent metabolism to methoxypyrazines as well as various leaf aldehydes and leaf alcohols. Methoxypyrazine content is typically higher in grapes that mature in shade, or cool conditions (Fig. 3.59). Light suppresses VvOMT3, the gene activating production 3isobutyl-2-hydroxypyrazine, the precursor of methoxypyrazines (Dunlevy et al., 2013). Cool climatic conditions also reduce carotenoid production. This might explain the reduced production and accumulation of TDN in Riesling grapes typically grown in hot climates (Marais et al., 1992b). Cooler conditions are also generally

Hot Methoxypyrazine (ng/L)

138

18 16 14 12 10 8 6 4 2 0 15.0

17.0

Cool

19.0 Maturity (°Brix)

21.0

23.0

FIGURE 3.59 Influence of hot and cool climates on the content of methoxypyrazine in Sauvignon blanc grapes. From Allen, M.S., Lacey, M.J., 1993. Methoxypyrazine grape flavor: influence of climate, cultivar and viticulture. Wein-Wiss. 48, 211e213, reproduced by permission.

favorable to varieties characterized by the production of monoterpene flavorants (Ewart, 1987). The distinctiveness of Marlborough Sauvignon blanc wines is suspected to partially arise from the cool evenings that characterize the growing season. High temperatures during or shortly after pollination negatively affect fertility and fruit development. Reduced fertility may arise from direct effects on inflorescence suppression, or indirectly from increasing vine water stress (Matthews and Anderson, 1988). Heat stress early in fruit development appears to irreversibly restrict berry enlargement (Hale and Buttrose, 1974), leading to smaller fruit as well as delayed maturation.

Inorganic nutrients Low soil nitrogen content appears to favor anthocyanin synthesis, whereas high nitrogen levels negatively influence production. These effects are probably indirect, acting through influences on vegetative growth and canopy shading, and their impacts on fruit maturation and sugar accumulation. Increasing nitrogen availability also increases free amino acid accumulation, notably arginine. Phosphorus and sulfur deficiencies are known to increase anthocyanin content in several plants. In grapevines, phosphorus deficiency induces interveinal reddening in leaves, but how it may affect berry anthocyanin synthesis is obscure. In contrast, high potassium contents can reduce berry pH and thereby lower fruit color and negatively influence color stability in red wines.

Water It is often thought that mild water deficit following ve´raison is beneficial to grape quality. This may result from suppressing further vegetative growth, leading to

139

References

2004 26 Anthocyanins (mg g1 of skin)

24

CT WS

2005 14

22

CT WS

12

20 18

10

16 8

14 12

6

10 8

4

6 4

2

2 Jul10

Jul20

Jul30 Aug09 Aug19 Aug29 Sep08 Sep18 Sep28

Jul10

Jul20

Jul30 Aug09 Aug19 Aug29 Sep08 Sep18 Sep28

FIGURE 3.60 Accumulation of anthocyanins in berry skin of water stressed (WS) and control (CT) vines in two seasons. White background represents pre-ve´raison; light shading represents ve´raison (from 10 to 100% of colored berries); deep shading represents post-ve´raison (maturation until harvest). From Castellarin, S.D., Pfeiffer, A., Sivilotti, P., Degan, M., Peterlunger, E., Di Gaspero, G., 2007. Transcriptional regulation of anthocyanin biosynthesis in ripening fruits of grapevine under seasonal water deficit. Plant Cell Environ. 30, 1381e1399, reproduced by permission.

a more balanced leaf area/fruit ratio. These may indirectly enhance anthocyanin synthesis and limits berry enlargement. A direct acceleration of ripening by postve´raison mild water deficit also appears possible (Freeman and Kliewer, 1983). In contrast, early season water deficit can lead to reduced berry set and irreversibly limit berry enlargement (McCarthy, 1997; Ojeda et al., 2001). Whether this results from a marked increase in early fruit abscisic acid levels (Okamoto et al., 2004) is unestablished. Thus, water deficit should be minimized, by irrigation if available and permissible, at least up to ve´raison. In excess, however, irrigation can more than double fruit yield, increase berry size, and delay maturitydall detrimental to fruit quality. The effect of irrigation on sugar content depends on its application relative to need. Although potentially resulting in a decline, irrigation can increase grape sugar content under certain conditions. Commonly, irrigation results in an initial increase in grape acidity, possibly by permitting sufficient evaporative cooling to reduce malic acid respiration. However, there is seldom an accompanying drop in pH. This anomaly may partially be explained by the dilution associated with fruit enlargement. When harvest is sufficiently delayed, an associated high fruit acidity may dissipate, presumably from the slower rate of malate respiration being compensated by a longer maturation period. The poor wine pigmentation often attributed to excessive irrigation may result from reduced anthocyanin as well as reduced flavonol and proanthocyanin synthesis (Kennedy et al., 2002). However, poor wine color may also arise from dilution, associated with increased berry volume. Poor coloration in red cultivars is one of the prime reasons why irrigation is generally restricted after ve´raison. In addition, limited water deficit is often correlated with enhanced anthocyanin synthesis (Fig. 3.60). This is reflected in the activity of genes involved in anthocyanin synthesis (UFGT) and selective production of

trihydroxylated anthocyanins (F30 50 H) as well as the production of flavonoid precursors (Castellarin et al., 2007). Little is known about the effects of irrigation on aroma development. It has been noted, however, that irrigation can diminish the rate of monoterpene accumulation and the development of juice aroma in Riesling grapes (McCarthy and Coombe, 1985). However, this and other negative associations typically ascribed to irrigation may relate as much to the hot climates where it is needed.

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Suggested reading

Vasconcelos, M.C., Castagnoli, S., 2000. Leaf canopy structure and vine performance. Am. J. Enol. Vitic. 51, 390e396. Vergara, R., Pe´rez, F.J., 2010. Similarities between natural and chemically induced bud-endodormancy release in grapevine Vitis vinifera L. Sci. Hortic. 125, 648e653. Vidal, S., Francis, L., Guyot, S., Marnet, N., Kwiatkowski, M., Gawel, R., et al., 2003. The mouth-feel properties of grape and apple proanthocyanidins in a wine-like medium. J. Sci. Food Agric. 83, 564e573. Volder, A., Smart, D.R., Bloom, A.J., Eissenstat, D.M., 2005. Rapid decline in nitrate uptake and respiration with age in fine lateral roots of grape: implications for root efficiency and competitive effectiveness. New Phytol. 165, 493e502. Wake, C.M.F., Fennell, A., 2000. Morphological, physiological and dormancy responses of three Vitis genotypes to short photoperiod. Physiol. Plant. 109, 203e210. Walker, A.R., Lee, E., Robinson, S.P., 2006. Two new grape cultivars, bud sports of Cabernet Sauvignon bearing pale-coloured berries, as the result of deletion of two regulatory genes of the berry colour locus. Plant Mol. Biol. 62, 623e635. Walker, A.R., Lee, E., Bogs, J., McDavid, D.A.J., Thomas, M.R., Robinson, S.P., 2007. White grapes arose through the mutation of two similar and adjacent regulatory genes. Plant J. 49, 772e785. Waschkies, C., Schropp, A., Marschner, H., 1994. Relations between grapevine replant disease and root colonization of grapevine (Vitis sp.) by fluorescent pseudomonads and endomycorrhizal fungi. Plant Soil 162, 219e227. Watson, B.T., McDaniel, M., Miranda-Lopez, R., Michaels, N., Price, S., Yorgey, B., 1991. Fruit maturation and flavor development in Pinot noir (abstract). Am. J. Enol. Vitic. 42, 88e89. Watt, A.M., Dunn, G.M., May, P.B., Crawford, S.A., Barlow, E.W.R., 2008. Development of inflorescence primordia in Vitis vinifera L. cv. Chardonnay from hot and cool climates. Aust. J. Grape Wine Res. 14, 46e53. Weaver, R.J., McCune, S.B., 1960. Influence of light on color development in Vitis vinifera grapes. Am. J. Enol. Vitic. 11, 179e184. West, D.W., Taylor, J.A., 1984. Response of six grape cultivars to the combined effects of high salinity and rootzone waterlogging. J. Am. Soc. Hortic. Sci. 109, 844e851. Westfall, C.S., Zubieta, C., Hermann, J., Kapp, U., Nanao, M.H., Jez, J.M., 2012. Structural basis for prereceptor modulation of plant hormones by GH3 proteins. Science 336, 1708e1711. Wheeler, S., Loveys, B., Ford, C., Davies, C., 2009. The relationship between the expression of abscisic acid biosynthesis genes, accumulation of abscisic acid and the promotion of Vitis vinifera L. berry ripening by abscisic acid. Aust. J. Grape Wine Res. 15, 195e204. Williams, L.E., Biscay, P.J., Smith, R.J., 1987. Effect of interior canopy defoliation on berry composition and potassium distribution in Thompson Seedless grapevines. Am. J. Enol. Vitic. 38, 287e292. Wilson, B., Strauss, C.R., Williams, P.J., 1984. Changes in free and glycosidically bound monoterpenes in developing Muscat grapes. J. Agric. Food Chem. 32, 919e924. Wilson, B., Strauss, C.R., Williams, P.J., 1986. The distribution of free and glycosidically-bound monoterpenes among skin, juice, and pulp fractions of some white grape varieties. Am. J. Enol. Vitic. 37, 107e114. Winkler, A.J., Cook, J.A., Kliewer, W.M., Lider, L.A., 1974. General Viticulture. University of California Press, Berkeley, CA. Yakushiji, H., Kobayashi, S., Goto-Yamamoto, N., Tae Jeong, S., Sueta, T., Mitani, N., et al., 2006. A skin color mutation of grapevine, from black-skinned Pinot noir to white-skinned Pinot blanc, is caused by deletion of the functional VvmybA1 allele. Biosci. Biotechnol. Biochem. 70, 1506e1508. Yamane, T., Jeong, S.T., Goto-Yamamoto, N., Koshita, Y., Kobayashi, S., 2006. Effects of temperature on anthocyanin biosynthesis in grape berry skins. Am. J. Enol. Vitic. 57, 54e59.

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Yang, Y., Hori, I., Ogata, R., 1980. Studies on retranslocation of accumulated assimilates in Delaware grape vines, II and III. Tokyo J. Agric. Res. 31, 109e129. Zapata, C., Magne´, C., Dele´ens, E., Brun, O., Audran, J.C., Chaillou, S., 2001. Grapevine culture in trenches: root growth and dry matter partitioning. J. Grape Wine Res. 7, 127e131. Zapriagaeva, V.I., 1964. Grapevine e Vitis L. Wild Growing Fruits in Tadzhikistgan (Russian, with English Summary). Nauka, Moscow, pp. 542e559. Zhang, Y., Keller, M., 2015. Grape berry transpiration is determined by vapor pressure deficit, cultivar conductance, and berry size. Am. J. Enol. Vitic. 66, 454e462. Zhao, C.-X., Shao, H.-B., Chu, L.-Y., 2008. Aquaporin structure-function relationships: water flow through plant living cells. Colloids Surfaces B Biointerfaces 62, 163e172. Zion, B., Halaly, T., Regev, R., Barak, M., Lidor, E., Weissblum, A., et al., 2012. A heat shock generator for dormancy release grapevines in the vineyard. Trans. ASABE (Am. Soc. Agric. Biol. Eng.) 55, 753e758. van Zyl, J.L., van Huyssteen, L., 1987. Root pruning. Decid. Fruit Growth 37 (1), 20e25.

Suggested reading General References Huglin, P., Schneider, C., 1998. Biologie et e´cologie de la Vigne, 2eme ed. Tec & Doc Lavoisier, Paris. Keller, M., 2015. The Science of Grapevines: Anatomy and Physiology, second ed. Academic Press, New York, NY. Iland, P., Dry, P., Proffitt, T., Tyerman, S., 2011. The Grapevine: From the Science to the Practice of Growing Vines for Wine. Patrick Iland Wine Promotions Pty. Ltd., Adelaide. Australia. Pongra´cz, D.P., 1978. Practical Viticulture. David Philip, Cape Town, South Africa. Winkler, A.J., Cook, J.A., Kliewer, W.M., Lider, L.A., 1974. General Viticulture. University of California Press, Berkeley, CA.

Root System Archer, E., Saayman, D., 2018. Vine Roots. The Institute for Grape and Wine Sciences (IGWS), Stellenbosch University, South Africa. Linderman, R.G., 1988. Mycorrhizal interactions with the rhizosphere microflora: the mycorrhizosphere effect. Phytopathology 78, 366e371. Menge, J.A., Raski, D.J., Lider, L.A., Johnson, E.L.V., Jones, N.O., Kissler, J.J., et al., 1983. Interaction between mycorrhizal fungi, soil fumigation, and growth of grapes in California. Am. J. Enol. Vitic. 34, 117e121. Pratt, C., 1974. Vegetative anatomy of cultivated grapes, a review. Am. J. Enol. Vitic. 25, 131e148. Richards, D., 1983. The grape root system. Hortic. Rev. 5, 127e168. Van Zyl, J.L. (Ed.), 1988. The Grapevine Root and its Environment. Department of Agricultural Water Supply Technical Communication, Pretoria, South Africa. No. 215.

Shoot System Gerrath, J.M., Posluszny, U., 1988. Morphological and anatomical development in the Vitaceae, I. Vegetative development in Vitis riparia. Can. J. Bot. 66, 209e224. Koblet, W., Perret, P., 1982. The role of old vine wood on yield and quality of grapes. In: Grape and Wine Cent. Symp. Proc. University of California, Davis, CA, pp. 164e169. Lavee, S., May, P., 1997. Dormancy of grapevine buds e facts and speculation. Aust. J. Grape Wine Res. 3, 31e46.

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Morrison, J.C., 1991. Bud development in Vitis vinifera. Bot. Gaz. 152, 304e315. Or, E., 2009. Grape bud dormancy release e the molecular aspect. In: Roubelakis-Angelakis, K.A. (Ed.), Grapevine Molecular Physiology and Biotechnology, second ed. Springer Science þ Business Media B.V., Dordrecht, The Netherlands, pp. 1e29. Pratt, C., 1974. Vegetative anatomy of cultivated grapes, a review. Am. J. Enol. Vitic. 25, 131e148.

Photosynthesis and Transpiration Du¨ring, H., 1988. CO2 assimilation and photorespiration of grapevine leaves: responses to light and drought. Vitis 27, 199e208. Du¨ring, H., Dry, P.R., Loveys, B.R., 1996. Root signals affect water use efficiency and shoot growth. Acta Hortic. (Wagening.) 427, 1e13. Intrieri, C., Zerbi, G., Marchiol, L., Poni, S., Caiado, T., 1995. Physiological response of grapevine leaves to light flecks. Sci. Hortic. 61, 47e60. Smart, R.E., 1983. Water relations of grapevines. In: Kozlowski, T.T. (Ed.), Water Deficits and Plant Growth, Additional Woody Crop Plants, vol. 7. Academic Press, New York, NY, pp. 137e196.

Reproductive System Carmona, J.J., Chaı¨b, J., Martı´nez-Zapater, J.M., Thomas, M.R., 2008. A molecular genetic perspective of reproductive develop-ment in grapevine. J. Exp. Bot. 59, 2576e2596. Fasoli, M., Richter, C.L., Zenoni, S., Bertini, E., Vitulo, N., Santo, S.D., et al., 2018. Timing and order of the molecular events marking the onset of berry ripening in grapevine. Plant Physiol. 178, 1187e1206. Meneghetti, S., Gardiman, M., Calo`, A., 2006. Flower biology of grapevine. A review. Adv. Hortic. Sci. 20, 317e325. May, P., 2004. Flowering and Fruitset of Grapevines. Winetitles, Adelaide, Australia. Pratt, C., 1971. Reproductive anatomy of cultivated grapes, a review. Am. J. Enol. Vitic. 21, 92e109. Ristic, R., Iland, P.K., 2005. Relationships between seed and berry development of Vitis vinifera L. cv Shiraz: developmental changes in seed morphology and phenolic composition. Aust. J. Grape Wine Res. 11, 43e58.

Vasconcelos, M.C., Greven, M., Winefield, C.S., Trought, M.C.T., Raw, V., 2009. The flowering process in Vitis vinifera: a review. Am. J. Enol. Vitic. 60, 411e432.

Berry Maturation Coombe, B.G., McCarthy, M.G., 2000. Dynamics of grape berry growth and physiology of ripening. Aust. J. Grape Wine Res. 3, 131e135. Dai, Z.W., Ollat, N., Gome`s, E., Decroocq, S., Tandonnet, J.-P., Bordenave, L., et al., 2011. Ecophysiological, genetic, and molecular causes of variation in grape berry weight and composition: a review. Am. J. Enol. Vitic. 62, 413e425. Ferrer, J.-L., Austin, M.B., Stewart Jr., C., Noel, J.P., 2008. Structure and function of enzymes involved in the biosynthesis of phenylpropanoids. Plant Physiol. Biochem. 46, 356e370. Hardie, W.J., O’Brien, T.P., Jaudzems, V.G., 1996. Morphology, anatomy and development of the pericarp after anthesis in grape, Vitis vinifera L. Aust. J. Grape Wine Res. 2, 97e142. Matthews, M.A., Shackel, K.A., 2005. Growth and water transport in fleshy fruit. In: Holbrook, N.M., Zwieniecki, M.A. (Eds.), Vascular Transport in Plants. Academic Press, London, UK, pp. 181e197. May, P., 2004. Flowering and Fruitset in Grapevines. Winetitles, Adelaide, Australia. Robinson, S., Davies, C., 2000. Molecular biology of grape berry ripening. Aust. J. Grape Wine Res. 6, 175e188.

Factors Affecting Berry Maturation Coombe, B.G., 1987. Influence of temperature on composition and quality of grapes. Acta Hortic. (Wagening.) 206, 23e35. Ferrandino, A., Lovisoli, C., 2014. Abiotic stress effects on grapevine (Vitis vinifera L.): focus on abscisic acid-mediated consequences on secondary metabolism and berry quality. Environ. Exp. Bot. 103, 138e147. Lavee, S., 1987. Usefulness of growth regulators for controlling vine growth and improving grape quality in intensive vineyards. Acta Hortic. (Wagening.) 206, 89e108. Lei, Y., Xic, S., Guan, X., Song, C., Zhang, Z., Meng, J., 2018. Methoxypyrazines biosynthesis and metabolism in grape: a review. Food Chem. 245, 1141e1147. Smart, R.E., Robinson, M., 1991. Sunlight into Wine: A Handbook for Winegrape Canopy Management. Winetitles, Adelaide, Australia.

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4 Vineyard practice O U T L I N E Vine cycle and vineyard activity

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Management of vine growth Yield/quality ratio Physiological effects of pruning Pruning options Pruning level and timing Bearing-wood selection Pruning procedures Training options and systems Selected training systems Ancient Roman example Vigor regulation (devigoration)

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In Chapter 3, details were given concerning grapevine anatomy and physiology. In this chapter, vineyard practice and how it influences grape yield and quality are the foci. Because vineyard practice is so closely tied to the vine’s growth cycle, an introductory overview is given. Many of its aspects are illustrated in Fig. 4.1.

Wine Science, Fifth Edition https://doi.org/10.1016/B978-0-12-816118-0.00004-0

Assessment of nutrient need Nutrient requirements Organic fertilizers

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Disease, pest, and weed management Pathogen control Consequences of pathogenesis for fruit quality Fungal diseases Bacterial diseases Viruses, virus-like, and viroid diseases Nematode pathogens Insect and mite pests Mammalian and bird damage Physiological disorders Air pollution Weed control

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Harvesting Criteria for harvest timing Sampling Harvest mechanisms Measurement of vineyard variability

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Vine cycle and vineyard activity The termination of one growth cycle and preparation for another coincide, in temperate regions, with the onset of winter dormancy. It provides grape growers the opportunity to do many of the less urgent vineyard activities, for which there is often insufficient time during the

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FIGURE 4.1

Growth rate of various organs of Colombar grapevines grown in South Africa throughout the season. After Van Zyl, J.L., 1984. Response of Colombar grapevines to irrigation as regards quality aspects and growth. S. Afr. J. Enol. Vitic. 5, 19e28, from Williams, L.E., Matthews, M.A., 1990. Grapevines. In: Stewart, B.A., Nielsen, D.R. (Eds.), Irrigation of Agricultural Crops. American Society of Agronomy, Madison, WI, pp. 1019e1055. (Agronomy Series Monograph No. 30), reproduced by permission.

growing season. In addition, pruning is conducted more conveniently when the vine is dormant, the absence of foliage, permitting easier wood selection and cane tying. With the return of warm weather, both the metabolic activity of the vine and the pace of vineyard endeavors quicken. Usually, the first sign of renewed activity is the “bleeding” of sap from cut ends of canes or spurs. Hydrolysis of stored starch in the roots generates the turgor pressure that induces the early upward flow of sap. In addition to water, the sap contains sugars and amino acids (Campbell and Strother, 1996), as well as trace amounts of other organic compounds, growth regulators, and inorganic nutrients. These are critical to bud reactivation and initial growth. Reactivation of sap flow is reflected in a marked decrease in root and trunk carbohydrate reserves (Bennett et al., 2005). Between bud burst and 50% bloom, root starch and sugar reserves may decline by about 42% and 72%, respectively. Soluble sugars in the trunk can decline by 92% during the same period. These levels usually return to near winter values by leaf fall. When temperatures rise above a critical value, depending on the variety, buds begin to burst. Bud activation progresses downward from the tips of canes and spurs, as the phloem regains its functionality. The cambium subsequently resumes its meristematic activity, producing new vascular tissues. Typically, only the primary bud in the overwintered bud develops. The secondary and tertiary buds remain inactive, unless the primary bud has been killed or severely damaged. Once initiated, shoot growth rapidly reaches its maximum, as the climate continues to warm

(Fig. 4.1). Development and enlargement of the primordial leaves, tendrils, and inflorescence clusters recommence (Fig. 4.2). As growth continues, the shoot differentiates new leaves and tendrils. Simultaneously, new buds begin to form in the leaf axils. Those that form early may become active, giving rise to lateral shoots. Root growth typically lags behind shoot growth, often coinciding with flowering (five- to eight-leaf stage). Peak root development commonly occurs between the end of flowering and the initiation of fruit coloration (ve´raison). Subsequently, root development slows, with a possible second growth phase in the autumn. Despite these trends, details can vary considerably with the rootstock, water availability, and climatic conditions. Flower development progresses from the outermost ring of flower parts (the sepals) inward to the pistil. By the time the anthers mature and split open (anthesis), the cap of fused petals (calyptra) has usually separated from the ovary base and been shed. In the process, selfpollination typically results, as pollen, liberated during calyptra rupture, falls on the stigma. If followed by fertilization, embryo and berry development commences. Concurrent with flowering, inflorescence induction may begin in the nascent primary buds of leaf axils. These typically become dormant by midseason, completing their development only in the subsequent season. Several weeks after bloom, many small berries dehisce (shatter). This is a normal process in all varieties, reflecting flowers in which fertilization was unsuccessful, or the embryos aborted. Except in seedless cultivars, at least one seed is required for complete berry development.

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FIGURE 4.2 Phenological (growth) stages of the grapevine: the modified E-L system. From Coombe, B.G., 1995. Adoption of a system for identifying grapevine growth stages. Aust. J. Grape Wine Res. 1, 100e110, reproduced by permission.

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Once shoots have developed sufficiently, they are normally tied to, or restrained by, a support system. Shoots developing from “old wood” on the trunk are removed. Application of disease and pest control measures may commence, if it has not already begun. Irrigation may begin if required and legislatively permissible. It is usually restricted between ve´raison and full maturity. This tends to avoid promoting continued vegetative growth after ve´raison, which can negatively affect fruit maturity and quality. If necessary, irrigation may be reinitiated after harvest, to avoid stressing the vine and to favor optimal cane maturation. By ve´raison, primary shoot growth has usually ceased. Shoot topping may be practiced, if primary or lateral shoot growth continues, or to facilitate machinery movement in dense plantings. Fruit cluster thinning may be performed when fruit yield is considered (correctly or not) excessive and likely to negatively affect fruit (and wine) quality. Flower cluster thinning may be used for the same reason. If unnecessary, though, it results only in financial loss (Preszler et al., 2013). As the fruit approaches maturity, vineyard activity shifts toward preparing for harvest. Fruit samples are taken throughout the vineyard for sensory and chemical analysis. Based on the results, environmental conditions, and desires of the winemaker, a tentative harvest date is set. In cool climates, measures are put in place for frost protection, similar to those that may have been required in the spring to protect the vines from late frost. Once harvest is complete, vineyard activity is usually directed at preparing the vines for winter. In particularly cold climates, this may vary from mounding soil up around the shooterootstock union to removing the whole shoot system from its support system for burial. In warmer climates, it entails protection of the foliage until abscission. Leaves may remain functional for several months after harvest, or continuously, in tropical regions.

Management of vine growth Vineyard practice is primarily directed at obtaining the maximum yield relative to quality. One of the major techniques used in achieving this involves training and pruning. Both have been exhaustively studied for decades, and empirically assessed for millennia. The result is a bewildering array of systems. Although a full discussion of this diversity is beyond the scope of this text, the following provides an overview of the sources of the heterogeneity. For a discussion and description of local training and pruning systems, the reader is directed to regional governmental publications, universities, and research stations. However, before discussing vine management, it is advisable to first define several commonly used terms.

Training refers to the development of the permanent vine structure and the location of renewal wood (that which will generate the next year’s shoots). Its purpose is to position each year’s growth (shoots) in an environmentally favorable location. Renewal wood consists of lengths of the previous year’s growthdshorter sections being termed spurs (two to four buds) and longer sections (with many buds) being called canes. These sections of last year’s growth possess the buds from which the bearing wood (shoots) of the current season develop (see Figs. 4.11 and 4.13). Spurs may also be retained to position (or reposition) bearing wood close to the head (a central zone at the top of a trunk) or cordon (a horizontally positioned section of trunk). Most training systems are also associated with a support system (trellis). Training ideally takes into consideration multiple factors, such as the prevailing climate, desired harvesting practices, and cultivar’s fruiting characteristics. By comparison, canopy management is concerned with the positioning and maintenance of the growing shoot and fruit in a microclimate optimal for inflorescence initiation, fruit ripening, and cane maturation. Pruning involves the selective removal of canes, shoots, wood, and leaves, or the severing of roots to obtain the goals of training and canopy management. Thinning is similar, but refers to the removal of whole or parts of flower and fruit clusters to improve the berry microclimate and leaf area/fruit (LA/F) balance. Typically, though, pruning refers to the removal of unnecessary shoot growth at the end of the season. Two other important viticultural terms are vigor and capacity. Vigor refers to the rate and extent of vegetative growth, whereas capacity denotes the amount of growth and the vine’s ability to mature its fruit.

Yield/quality ratio Central to all vine management systems is regulation of the yield/quality ratio. This is inherently a function of the photosynthetic surface area (cm2) to fruit mass (g). It provides an indicator of the vine’s capacity to ripen fruit. The LA/F ratio is related to the more familiar concept of yield per hectare. However, the LA/F ratio focuses directly on the fundamental photosynthate source/sink relationship involved in fruit maturation. The first to publish extensively on the relationship was Winkler (1958). Regrettably, application of the LA/F concept was limited by the absence of a simple means of obtaining the requisite data (Tregoat et al., 2001). Suggestions by Lopes and Pinto (2005), Blom and Tarara (2007), Tsialtas et al. (2008), Beslic et al. (2010), and Guisard et al. (2010) are attempts to provide a more effective means of assessing leaf area.

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Management of vine growth

200 180 160 Grape Color (P.T.A Units)

Multispectral remote sensing (Johnson et al., 2003) has potential, as it can be readily correlated with field data. In addition, Costanza et al. (2004) and Siegfried et al. (2007) have shown a strong correlation between shoot length and leaf area. Obtaining the requisite data in a timely and convenient manner was an issue in the past. However, the ready availability of cameras and algorithms capable of rapidly analyzing the images are permitting accurate, real-time data on leaf area and fruit load to be collected (e.g., Fuentes et al., 2014; Ballesteros et al., 2015; Klodt et al., 2015). Representative, vineyard-wide analysis is being further aided by cameras attached to unmanned airborne vehicles, or drones (Siva balan, 2016). The images may also be used to reduce if not eliminate most, labor- and time-intensive, manual assessments of features such as flower number/inflorescence, vine vigor and health, water deficit, and pruning weight (Caruso et al., 2017), as well as microclimatic data (e.g., details of potentially dangerous temperature inversions and other weather data in 3D). Some applications now come with computer- or smartphone-based apps to speed interpretation (De Bei et al., 2016; Diago et al., 2016a). Still at issue, though, is the rapid and practical interpretation of the data, relative to the nutrient and water status of the soil, temperature and light conditions, disease pressures, and effects of the pruning and training systems on photosynthetic efficiency and carbohydrate shunting through the vine. For example, LA/F values can vary from 8 to 12 cm2/g and from 5 to 8 cm2/g for single and divided canopy systems (Kliewer and Dokoozlian, 2005). Increasing shoot numbers can decrease LA/F values (Myers et al., 2008). Calculated ratios considered adequate to fully ripen the fruit vary from w7 to 14 cm2/g. In addition, the relationship tends to be nonlinear, becoming increasingly negative as the ratio falls below a broad acceptable range (Kliewer and Dokoozlian, 2005). Another source of variability involves cultivar response (Fig. 4.3). In addition, the ratios should be developed using only leaves that significantly contribute to vine growth (i.e., disregarding young, old, and heavily shaded leaves). When data from different canopy configurations are compared, variations in the proportion of exterior to interior leaf canopy must also be taken into consideration. A simplistic view would suggest that increased leaf area should correlate with an increased ability to produce additional, fully ripened fruit. However, the grapevine is an uncommonly complex and adaptive plant. In addition, the potential of the vine to produce fruit in 1 year is largely predetermined, inflorescence primordia

s

Shiraz

140

ss

120

ss s ss

100

ss

s s s

s

Pinot Noir

80

P P

60 P P

40

P PP PP P

P

P

P P P

P

P P

P

20 0

0

5

10

15

20

25

Leaf Area/Fruit Weight

30

35

40

(cm2/g)

FIGURE 4.3 The relationship between grape color and leaf area/ fruit weight for Shiraz and Pinot noir vines. From Iland, P.G., Gawel, R., Coombe, B.G., Henschke, P.M., 1993. Viticultural parameters for sustaining wine style. In: Stockley, C.S. (Ed.), Proc. 8th Aust. Wine Ind. Tech. Conf. Winetitles, Adelaide, Australia, 167e169, reproduced by permission.

being initiated in the preceding year. This is particularly marked in cool climatic regions, where seasonal variations are often pronounced. Nutrient availability and growth may also be markedly influenced by vine health and its ability to store nutrients during the previous year. Another problem with straightforward yield/quality associations is the potential of some cultivars to produce several shoot sets per year. Lateral shoots, and any fruit they generate, not only retard ripening of the primary fruit, but also reduce overall uniformity at harvest. Induction of a second (or third) crop can be caused by overly zealous pruning, especially on fertile, moist soils. In contrast, pruning of vines grown on comparatively nutrient-poor dry soils can direct photosynthetic capacity to fully ripen a restricted fruit crop. Fig. 4.4 illustrates how different training systems affect the yield/
Brix ratio. There can also be marked variation between individual vines (Fig. 4.5), sites, or vintages (Plan et al., 1976). Sensory quality may actually decrease with reduced yield, by enhancing the presence of undesirable flavorants (Fig. 4.6) and diminishing varietal aroma (Chapman et al., 2004a). Thus, a universal relationship between vine yield and grape (wine) quality does not exist. Vine growth does not submit to a simple formula. A universal yield/quality ratio is especially untenable when pruning techniques developed for dry, lownutrient, hillside sites are applied to vines grown on moist,

156

4. Vineyard practice

25

28

(Average 1971–1975)

r2 = 0.410 r = 0.1683

26 24

°Brix

TSS (°Brix)

20

15

GDC Hedgerow Gobelet

y = –0.49x + 31.7 r2 = 0.81 y = –0.35x + 24.6 r2 = 0.95 y = –0.36x + 25.8 r2 = 0.64

22 20 18 16

10 10

15

20

25

30

r2 0.1118 r = –0.334

35

t/ha Color (mg/g FW)

FIGURE 4.4 Relationship between yield and soluble solids (3-year average) using three distinct training systems. GDC, Geneva double curtain. From Intrieri, C., Poni, S., 1995. Integrated evolution of trellis training systems and machines to improve grape quality and vintage quality of mechanized Italian vineyards. Am. J. Enol. Vitic. 46, 116e127, reproduced by permission.

1.00

0.75

0.50

0.25

0.00 0

10

20 30 Yield (t/ha)

40

50

1.25

1.00

r = 0.644 Color (mg/g FW)

nutrient-rich, lowland soils. Severe pruning under such conditions may spur excess vegetative growth, rather than limiting it. Nutrients are directed to shoot growth and dormant buds activated, increasing shading, enhancing disease incidence, delaying ripening, and generating nonuniform fruit maturation. Furthermore, cool climatic conditions provide less opportunity for the vine to compensate for temporary poor growth conditions, in contrast to the extended growing season and higher light intensities that typify warmer Mediterranean climates (Howell, 2001). The important question is not whether there might be some ideal yield/hectare ratio, but what is the optimal canopy size and placement to adequately nourish the crop to the desired state of ripeness under existing conditions. Insufficient leaf surface can be as detrimental to quality as excessive cover. A centuries-old view holds that in vines grown on relatively nutrient-poor soils, and that experience moderate water limitation, the potential for undesirable vigorous growth is restrained and fruit ripens optimally. Although reasonable, under these conditions, scientific validation for the benefits of restricted fruit production has been difficult to obtain. Cortell et al. (2005) compared data from two commercial vineyards under near identical conditions (similar clones, rootstocks, vine age, and management practices). Sites expressing reduced vine vigor showed proanthocyanidin, epigallocatechin, and pigmented polymer contents significantly higher than vines in blocks showing higher vigor. Variation in vine vigor was correlated to differences in soil depth and water-holding capacity. The results also indicate that, for Pinot noir, the relative contribution of skin

r2 0.4143 0.75

0.50

0.25

0.00 16

18

20

22

24

26

28

TSS (°Brix)

FIGURE 4.5

Relationship between total soluble solids (TSS), yield, and color in Shiraz showing the correlation coefficient (r) and the fitted line. From Holzapfel, B.P., Rogiers, S., Degaris, K., Small, G., 1999. Ripening grapes to specification: effect of yield on colour development of Shiraz grapes in the Riverina. Aust. Grapegrow. Winemak. 428 (24), 26e28, reproduced by permission.

to seed proanthocyanidins increases with reduced vine vigor (Fig. 4.7). Although enhancing potential wine color, this is not necessarily associated with improved wine flavor (Chapman et al., 2004a). In addition, data from Chapman et al. (2004a, 2005) indicate that the manner of yield reduction (pruning vs. water deficit) may be more significant than yield reduction itself. In addition, the means by which low yield is obtained

157

Management of vine growth

20

(A) 5000

MIBP (ng/L)

Seed Procyanidin Concentration

2000 2001

15 2

R = 0.7385***

10 5 0

2

R = 0.7007***

–5 20

25 30 35 Buds/Vine

40

45

4.6 Concentration

of 2-methoxy-3-isobutylpyrazine (MIBP; bell pepper odor) of Cabernet Sauvignon wines in relation to pruning treatment. From Chapman, D.M., Thorngate, J.H., Matthews, M.A., Guinard, J.X., Ebeler, S.E., 2004b. Yield effects on 2-methoxy-3isobutylpyrazine concentration in Cabernet Sauvignon using a solid phase microextraction gas chromatography/mass spectrometry method. J. Agric. Food Chem. 52, 5431e5435, reproduced by permission.

can markedly influence its beneficial/detrimental effects on the relative vegetative vs. fruity character of the wines so produced (Roby and Matthews, 2004). Clearly more needs to be known about how vineyard practice influences fruit physiology and the development of desirable attributes. One of the techniques historically used to reduce yield has been dense planting. Intervine competition tends to promote the production of many fine roots. This, in turn, may enhance nutrient extraction from the soil. Roots may also grow deep enough to reach subsurface supplies of water. The severe pruning associated with dense planting, however, can delay budbreak, as well as limit fruiting potential (by removing fruit buds). In the New World, growers have often had the luxury of planting grapevines on rich, loamy soil, with ample moisture supplies. Wide vine spacing, appropriate for standard farm equipment, further encouraged vigorous growth. Under such conditions, it is essential that the increased capacity be directed toward fruit production, not pruned away, potentially causing dormant bud activation and spurring vegetative growth. The modern view is that when an appropriate functional LA/F ratio is developed, both fruit yield and wine quality can be enhanced. The development of training systems based partially on achieving an optimal LA/F ratio is one of the major success stories of modern viticulture. The first practical application of the concept of balancing photosynthetic capacity to yield was the Geneva double curtain (GDC). This has been followed by others, more appropriate for Vitis vinifera cultivars. Examples are vertical

4000

3000

2000 200

Wine, mg/L y = 16(x)+117 r2=0.10

150 100

50

(B) Skin Proanthocyanidin Concentration

FIGURE

15

1600 1400 1200

Grape, mg/kg y = –1017(x)+1539 r2=0.88

1000 800 600 400

Wine, mg/L y = –324(x)+436 r2=0.90

200 0

(C) Total Proanthocyanidin Concentration

10

Grape, mg/kg y = –1725(x)+4091 r2=0.82

5000

Grape, mg/kg y = –2714(x)+5591 r2=0.87

4000

3000 600 500

Wine, mg/L y = –308(x)+554 r2=0.87

400 300 200 0.0

0.2

0.4

0.6

0.8

1.0

Vigor Index

FIGURE 4.7 Concentration of proanthocyanidins in (A) seed, (B) skin, and (C) skin plus seed in grapes at harvest and in the corresponding wine. Error bars, SEM (n ¼ 3). From Cortell, J.M., Halbleib, M., Gallagher, A.V., Righetti, T.J., Kennedy, J.A., 2005. Influence of vine vigor on grape (Vitis vinifera L. cv. Pinot noir) and wine proanthocyanidins. J. Agric. Food Chem. 53, 5798e5808, reproduced by permission. Copyright 2005 American Chemical Society.

shoot positioning (VSP), the Scott Henry, the Smarte Dyson, the Lyre, and the Ruakura twin two tier (RT2T; see later). These training systems, in addition to favoring an optimal LA/F ratio, provide better fruit exposure to light and air. These features typically promote inflorescence initiation, fruit health, and good berry coloration and flavor development (Iland et al., 1993). Optimal LA/F ratios tend to equate to five or six mature main leaves per medium-sized grape cluster (Jackson, 1986). LA/F

158

4. Vineyard practice

ratios lower than 6e10 cm2/g (0.6e1.0 m2/kg) are generally insufficient to fully ripen fruitddepending on the cultivar and growing conditions (Kliewer and Dokoozlian, 2005). Significantly higher values often indicate excessive shading, reduced anthocyanin content, delayed ripening, and inadequate
Brix values. However, as shown in Fig. 4.3, this relationship must be determined for each cultivar. Generalities are just that, always needing to be confirmed under local conditions.

Physiological effects of pruning Pruning is often used for several separate but related reasons. It permits the grape grower to establish a particular training system and manipulate vine yield. It also provides the opportunity to select bearing wood (spurs and canes), thereby influencing the spacing, location, and development of the canopy. This, in itself, can affect grape yield, health, and maturation, as well as pruning and harvesting costs. These influences should theoretically also affect wine quality, but until recently experimental evidence was lacking. A fascinating comparison of three pruning systems (as well as irrigation treatments) has been conducted by Holt et al. (2008a,b). Their data confirm anecdotal evidence of slight, but statistically significant differences in fruit quality. Interestingly, small berry size and higher anthocyanin contents did not translate into darker-colored wines or higher-quality scores, possibly due to some aspect associated with higher tannin levels. Like most temperate-zone perennial fruit crops, grapevines initiate inflorescence primordia a year in advance of their development. However, unlike most orchard crops, inflorescence initiation occurs opposite leaves on vegetative shoots. In contrast, fruit development on pomaceous and stone-fruit crops occurs on specialized, short, determinant spurs. Inflorescence initiation, a year in advance of flower production, allows the grape grower to limit fruiting capacity by bud removal long before flowering. Winter pruning is the primary means by which grapevine yield is managed. However, as a perennial crop, the vine stores considerable energy reserves in its woody parts. Thus, pruning removes nutrients that can limit the initiation of rapid spring growth, notably in young vines. Vines with little mature wood are less able to fully ripen their crop in poor years than vines possessing large cordons or trunks. Some of these influences may also result from the balance between growth regulators, such as cytokinins and gibberellins (from the roots) and auxins (derived primarily from the shoot system). When performed judiciously, pruning can limit shoot vigor, permitting the vine to fully ripen its fruit load. Both excessive pruning and excessive overcropping have the potential to suppress vine capacity (Fig. 4.8).

The reduced capacity associated with overcropping has long been known. It has been part of the rationale for the annual pruning of 85%e90% of any year’s shoot growth. However, as noted, severe pruning can lead to both reduced yield and reduced grape quality, as well as delayed budbreak, leaf production, and potentially activate dormant buds. While beneficial in some climates, and with certain cultivars, delayed bud activation can result in leaf production continuing well into the fruiting period. This can be a drain on photosynthate available to ripen fruit. Without pruning, shoot growth tends to develop rapidly and promote earlier fruit development. However, the extensive shoot development so permitted can delay berry maturation. Yields can be up to 2.5 times that of pruned vines. This can also suppress fruit production in subsequent years, by reducing inflorescence initiation and diminishing the nutrient reserves in old wood. However, if left unpruned for several years, the vine usually self-regulates itself, and good- to excellent-quality fruit can be obtained (see “Minimal pruning”). Because both inadequate and excessive pruning can compromise fruit quality, and diminish long-term fruitfulness, it is crucial that pruning be matched to vine capacity. The general relationship of shoot to berry growth and the concentration of carbohydrate in the shoot and fruit are illustrated in Fig. 4.9. Winter pruning is the most practical, but least discriminatory. Capacity can be only imprecisely predicted. By contrast, fruit thinning is the most precise, but the most labor intensive and difficult, due to clusters being partially obscured by foliage. The typical compromise is judicious winter pruning, followed by remedial flower cluster thinning in the spring if deemed necessary. This solution limits potential fruit production, while delaying final adjustment until a more accurate estimate of grape yield is likely. It also favors early budbreak, maximizing carbohydrate production over the year, while minimizing competition between foliage and fruit during berry ripening. Where advisable, budbreak can be delayed by performing pruning in late winter/early spring. Additional augmentation of vine capacity and fruit quality may be obtained with canopy management, improving the microclimate in and around the grape cluster. Positioning the basal region of shoots in a favorable light environment also improves inflorescence induction and helps maintain fruitfulness from year to year. Clonal selection, the elimination of debilitating viruses, improved disease and pest control, irrigation, and fertilization have significantly amplified potential vine vigor. Thus, one of the challenges of modern viticulture is to channel this potential into enhanced fruit and wine quality, and away from excessive, undesirable

159

Management of vine growth

Fruitfulness–clusters to a shoot the following season

Lack of vigor because of: Overcropping Drought Poor soil Disease or insect injury

Area of normal vigor with good commercial practice

Too much vigor because of: Too severe pruning Over thinning Early frost that takes crop Overfertilization and irrigation

3.0

2.0 1.0 3.0

4.0

5.0

6.0

7.0

Vigor–length of seasonal shoot growth, in feet

A

Growth of shoots, inches

B

Starch and sugars, percent dry weight

C

Berry size increase, ml

D

Degree Brix of the fruit

22 20

1.4 1.2

B C

18

1.0

16

0.8

14

0.6

12

0.4 0.2 0.0

D A

10 8

Soluble solids in degree Balling and usable carbohydrates in %

Shoot growth in 0.1 per day and berry volume in cc

FIGURE 4.8 The relationship of vigor (length) of shoot growth to fruitfulness of buds of Muscat of Alexandria and Alicante Bouschet at Davis, California. From Winkler, A.J., Cook, J.A., Kliewer, W.M., Lider, L.A., 1974. General Viticulture. University of California Press, Berkeley, reproduced by permission.

6 11 21 31 10 20 30 10 20 30 10 20 30 10 20 30 10 20 30 10 20 30 10 20 Mar Apr May June July Aug Sept Oct

FIGURE 4.9 The annual cycle of the vine, showing shoot and fruit development as well as seasonal changes in the levels of usable carbohydrates. From Winkler, A.J., Cook, J.A., Kliewer, W.M., Lider, L.A., 1974. General Viticulture. University of California Press, Berkeley, reproduced by permission.

vegetative growth. Interest has been shown in minimal pruning, based on the potential of the vine to selfregulate its fruit production. However, as the applicability of this technique to many cultivars and climates is unestablished, most of the following discussion deals with the fundamentals of traditional pruning. In general, the principles of pruning enunciated by Winkler et al. (1974) are as valid today as ever: 1. Pruning reduces vine capacity by removing both buds and stored nutrients in canes. Thus, pruning should be kept to the minimum necessary, to permit the vine to fully ripen its fruit.

2. Excessive or inadequate pruning depresses vine capacity for several years. To avoid this, pruning should attempt to match bud removal to vine capacity. 3. Capacity partially depends on the ability of the vine to rapidly generate a leaf canopy. This usually requires light pruning, followed by cluster thinning to balance crop production to the existing canopy. 4. Increased crop load and shoot number depress shoot elongation and leaf production during fruit development and, up to a point, favor full ripening. Moderately vigorous shoot growth is most consistent with vine fruitfulness.

160

4. Vineyard practice

5. In establishing a training system, the retention of one main vigorous shoot enhances growth and suppresses early fruit production. Both speed development of the permanent vine structure. With established vines, balanced pruning augments yield potential. 6. Cane thickness is a good indicator of bearing capacity. Thus, to balance growth throughout the vine, the level of bud retention should reflect the diameter of the cane. Alternatively, if bud numbers should remain constant, the canes or spurs retained should be relatively uniform in thickness. 7. Optimal capacity refers to the maximal fruit load that the vine can ripen fully within the normal growing season. Reduced fruit load has no effect on the rate of ripening. In contrast, overcropping delays maturation, increases fruit shatter, and decreases berry quality. Capacity is a function of the current environmental conditions, those of the past few years, and the genetic potential of the cultivar.

Pruning options When considering specific pruning options, features such as the prevailing climate, genetic attributes of both rootstock and scion, soil fertility, and training system need to be involved. Several of these factors are considered next. The prevailing climate imposes constraints on where and how grapevines can be grown. In cool continental climates, severe winter temperatures may kill most buds. Equally, late-spring frosts can damage or destroy newly emerged shoots and inflorescences. Where such losses occur frequently, more buds need to be retained than theoretically ideal, to compensate for potential bud kill. Subsequent disbudding, or flower cluster thinning, can adjust the potential yield downward, as deemed appropriate. Alternatively, varieties may be pruned in late winter or very early spring to permit the pruning level to reflect actual bud viability and/or delay bud burst. Another possibility, with spur-pruned wines, is to leave a few canes as insurance (Kirk, 1999). Because buds at the base of canes usually break late, they may survive and replace emerging shoots killed by late frosts. There may also be some apical dominance delay in the buds on spurs, imposed by those on the canes. If no frost damage occurs, the canes can be easily removed before significantly affecting spur growth. As a deep fertile soil enhances vigor, moderate pruning is often employed to permit sufficient early shoot growth to restrict late vegetative growth during fruit ripening. Vines on poorer soils usually benefit from more extensive pruning. This channels nutrient reserves into the

remaining buds, ensuring sufficient capacity for canopy development, fruit ripening, and inflorescence initiation. Nutrients discarded during pruning are compensated for by the higher proportion of old wood in established vines. Varietal fruiting characteristics, such as bunches per shoot, flowers per cluster, berry weight, and bud position along the cane, can influence the extent and type of appropriate pruning. Some cultivars, such as Sultana and Nebbiolo, produce sterile buds near the base of the cane. As spur pruning would leave few if any fruit buds, these varieties need to be cane pruned. Varieties showing strong apical dominance may also fail to bud out from the base to the middle of the cane (Fig. 4.10). Arching and inclining the cane downward can reduce apical dominance in varieties where this is a problem, balancing budbreak along the cane. Alternatively, spur pruning, where applicable, limits the expression of apical dominance. Varieties tending to produce small clusters are left with more buds than those bearing large clusters, to adjust pruning degree to varietal fruiting characteristics. Many of the more renowned cultivars produce small clusters, for example, Riesling, Pinot noir, Chardonnay, Cabernet Sauvignon, and Sauvignon blanc. Largeclustered varieties include Chenin blanc, Grenache, and Carignan. The susceptibility of varieties such as Gewu¨rztraminer to inflorescence necrosis (coulure) also demands adjustment of how many buds should be retained. In addition, the ideal number of buds retained depends on the tendency of the variety to produce fertile buds along the cane. Examples of cultivars having a high percentage of fertile buds are Muscat of Alexandria and Rubired. Harvesting method (manual vs. mechanical) can also influence the choice of pruning and training. Spur pruning (along a cordon) is generally more adapted to mechanical harvesting than cane pruning. Spur pruning is also easier for inexperienced pruners to learn than cane pruning, and more suited to mechanical (or robotic) pruning. Not least among grower concerns is the expense of pruning. Although winter pruning is the most economical (not competing with other vineyard activities), it is the most difficult to employ skillfully due to the unpredictability of bud viability. This can periodically be assessed with selective bud dissection. However, to reflect actual potential, the procedure must be conducted near the end of winter, which would delay pruning more than is usually desired (relative to other essential vineyard activities), or preferred (vineyardist philosophy). Cluster and fruit thinning permit precise yield regulation, but is costly (in terms of labor and lost yield) and may not yield a crop of better or more uniform quality (Calderon-Orellana et al., 2014). Pragmatism generally favors winter pruning as the method of choice.

161

Management of vine growth

120 30 8 150 10

30

70

5

2

0

150 250

105 10

30 170

10

15 115 20

20

20 10

5

5 15 0

0 0

0

0

3

0

0

5

4

4 0

10

2 2

2

1 0

0 10

0 0

100

30

0 0

80

2 5

10 0

50

8

2 3

20

20

0

0

5

5 10

0

10

2

70

3

170

30

20

90 0

10

15

30

10

0

153

30

80

5 15

65

5 5

35

10

10

270

0

35

7

30

0

140

10

0 15

0

120 0

0 4

45

15 0

25

0

FIGURE 4.10 Apical dominance of shoot growth on unpruned vertically positioned canes. From Huglin, P., 1958 Recherches sur les bourgeons de la vigne. Initiation Florale et De´ve´loppement Ve´ge´tatif. Doctoral Thesis, University of Strasbourg, France, reproduced by permission.

Pruning level and timing Widely spaced vines of average vigor and pruning degree may produce up to 25 canes, possessing about 30 buds each by the end of the season. Thus, before pruning, the vine may possess upward of 750 buds. Even without pruning, only a small number of these buds (about 100e150) are likely to burst the following spring. Despite this, they would generate a fruit load considerably in excess of the wine’s capacity to mature fully. Thus, pruning is traditionally required. However, as noted, excessive removal can have an undesirable impact on fruit maturity and subsequent vine fruitfulness. Thus, determining the appropriate number of buds to retain is one of the critical yearly vineyard tasks. The job is made all the more complex because actual fruitfulness is known only in the spring, after winter pruning is complete. This has led to several attempts to improve yieldpotential assessment. One method involves direct observation of sectioned buds just prior to pruning. Although reliable, it can be a significant labor expensedas of this

writing there is no electronic device that assesses bud viability. Nonetheless, it can be worth the effort in cold climatic regions. Another option involves “hedging one’s bets,” termed “balanced pruning.” It estimates the vine’s capacity, based on the weight of cane cuttings removed during pruning. From this, an optimal number of buds for retention is established. Depending on experience, additional buds may be retained to account for possible bud mortality during the winter. Based on bud burst, any excess is removed. Only buds separated by obvious internodes are counteddbase (noncount) buds usually remain dormant. In balanced pruning, the number of buds retained is noted in increments (weight of prunings) above a minimum, established empirically for each region and cultivar (e.g., Table 4.1). Pruning recommendations are usually given in parentheses, for example (30 þ 10). The first value indicates the number of buds (count nodes) retained for the first pound of prunings, and the second value suggests the number of buds to retain for each additional pound of prunings. “Pound of

162 TABLE 4.1

4. Vineyard practice

Suggested pruning severity for balanced pruning of mature vines of several American (Vitis labrusca) and French-American varieties in New York State. Number of nodes to retain per pound (0.45 kg) of cane prunings

Grape variety

Each extra pound (0.45 kg)

First pound (0.45 kg)

American cultivars Concord

30

þ

10

Fredonia

40

þ

10

Niagara, Delaware, Catawba

25

þ

10

20

þ

10

Small-clustered varieties (Mare´chal Foch, Leon Millot, etc.)

20

þ

10

Medium-clustered varieties (Aurore, Cascade, Chelois, etc.)

10

þ

10

Large-clustered varieties (Seyval, de Chaunac, Chancellor, etc.)

20

þ

10

Ives, Elvira, Dutchess a

French-American hybrid cultivars

a

All require suckering of the trunk, head, and cordon during the spring and early summer. After Jordan, T.D., Pool, R.M., Zabadal, T.J., Tomkins, J.P., 1981. Cultural Practices for Commercial Vineyards. New York State College of Agriculture, Cornell University, Ithaca, NY, (Miscellaneous Bulletin 111), reproduced by permission.

prunings” (assessed by a direct weighing of bundling cuttings from individual vines) is taken as an indicator of the current year’s capacity, and a predictor of subsequent potential. Values for vinifera cultivars, under Australian conditions, are in the range of 30e40 buds/ kg of prunings (Smart and Robinson, 1991). Vines producing less than a minimum weight of prunings are pruned to less than the suggested basic number of buds. Balanced pruning was initially developed for labrusca varieties grown in eastern North America. Its application to other varieties has been less successful. Regardless, pruning recommendations must account for cultivar fertility and prevailing climatic conditions. For example, varieties with low fertility need more buds to improve yield potential, whereas bud retention must be adjusted downward in regions with short growing seasons to permit full crop ripening. In addition, the tendency of buds to produce more than the standard two clusters per bud (up to four), and the production of flower clusters from shoots derived from noncount (base) buds, has limited the usefulness of balanced pruning with most French-American hybrids (Morris et al., 1984). Preliminary evidence indicates that flower cluster and shoot thinning may be more effective means of limiting overcropping for hybrids (Morris et al., 2004), though results can vary from cultivar to cultivar. Balanced pruning has found particular value in research, where objective values are required (values of 0.3e0.6 kg/m prunings are frequently considered optimal). Taking measurements has found less use

under commercial practice. Good pruners intuitively learn to adjust bud number to vine capacity by visual assessment, without the need for quantification. Alternative measures for assessing pruning level employ data collected over several years. This involves programming in bud retention data to obtain a formula appropriate for a specific target. The yield per vine is divided by anticipated (desired) bunch weight to determine the number of bunches per vine. This is, in turn, divided by the number of bunches typical per shoot. This supplies the number of buds to retain per vine. Final adjustments are based on the capacity (relative size) of individual vines, cultivar characteristics, information on bud viability prior to harvesting, and data on actual/likely frost and freeze damage subsequent to pruning. Another method calculates pruning based on nodes per meter row. Based on Australian conditions (and 3-m row spacing), severe, moderate, and light pruning are 75 nodes per meter row (Tassie and Freeman, 1992). With the development of mechanical prepruners, it is far more economical to have the initial pruning done by machine, with manual pruning limited to fine-tuning bud number and distribution to match individual vine capacity (see Tassie and Freeman, 1992). Prepruners were initially designed for spur-pruned vines, but advances have adapted machines for cane-pruned vines, but require an initial manual tying of the desired canes. The machine sequentially cuts off the excess, pulls out the cut canes, and mulches them as it moves down the row. Pruners can follow, removing cane remnants and

Management of vine growth

supplying any fine-tuning required. Recent developments with robotics combined with artificial intelligence and real-time, stereoscopic, camera feedback may eliminate even the need for a manual cleanup pruning (Smith, 2009). Machines may soon be global positioning (GPS) directed and programmed to automatically prune and trim based on individual vine need (e.g., https:// efficientvineyard.com/blog/scheid-vineyards). Winter pruning may be conducted from leaf fall until after budbreak. The earliest start depends on when the phloem has sealed itself with callose for the winter. This avoids nutrient loss and the activation of partially dormant buds. Winter pruning has many advantages, in addition to a dearth of other vineyard activities. Bud counting and cane selection are easier to conduct with bare vines. Equally, prunings can be more easily disposed of, either by chopping for soil incorporation or, as common in the past, burning. Wet periods should be avoided, both for human comfort and as a precaution against infection by xylem-infecting pathogens, notably Eutypa lata. For tender varieties, pruning is normally performed in early spring, when the danger of killing frost has passed. The proportion of dead buds can then be assessed directly by cutting a sample. Winter-killed buds show blackened primary buds (Plate 5.1). In severe cases, secondary buds may also be moribund. An alternative method already noted is double pruning, with most pruning and cleanup occurring during the winter and a final removal delayed until budbreak indicates survival. The slight delay in bud burst induced by this technique has occasionally been used as frost protection. It can retard budbreak by one to several weeks in precocious varieties. Late pruning has also been used to delay budbreak in regions experiencing serious early season storms, such as the Margaret River in Western Australia. Friend and Trought (2007) also found that late pruning improved fruit set, presumably due to flowering occurring under warmer conditions, favorable to fertilization. Because the delay induced by late pruning often persists through berry maturity, harvesting may be equally retarded. Thus, the advantages of delayed budbreak must be weighed against the potential disadvantages of deferred ripening. For some cool-climate cultivars, delayed maturation can be desirable when they are grown in warm regions. It can produce fruit higher in acidity, enriched in color, and augmented in flavor (Dry, 1987). Regrettably, it can also reduce yield, although this could also be an advantage, depending on the conditions. Late pruning can also be used as a means to reduce the incidence of Eutypa dieback. In contrast, early budbreak may be desired in hot climates. This adjusts flowering and fruit set to a period when temperature conditions are more likely to be conducive to their occurrence.

163

Pruning and thinning in late spring and early summer are designed as much to improve vine microclimate or minimize wind damage as to limit fruit production. These activities may involve a range of activities termed disbudding, pinching, suckering, topping, and thinning of flower or fruit clusters. However, due to their cost and increasing complexity, it is preferable to limit their use as much as possible by appropriate winter pruning. Disbudding is commonly used in training young vines to a desired mature form. It is also employed to remove unwanted basal or latent buds that may have become active. Early removal economizes nutrient reserves and favors strong growth of the remaining shoots. Repeat activation of dormant buds (e.g., suckers and water sprouts) (Fig. 4.11) throughout the growing season is often a sign of overpruning and insufficient energy being directed to fruit production. Their removal limits excessive foliage density that enhances disease severity, impedes disease and weed control, complicates mechanical harvesting, and reduces inflorescence induction, fruit yield, and grape quality. Alternatively, application of naphthalene acetic acid (NAA), mixed with a latex paint or asphalt, can control suckers for 2e3 years. The incorporation of hexyl 2-naphthyloxyacetate in the mix increases NAA solubility, reduces the required concentration, and extends the effective application period (Dolci et al., 2004). Trunk application is best conducted after budbreak on the bearing wood. Although the growth of suckers is undesirable (often originating from the rootstock of grafted vines), some water sprouts can occasionally be of use. If favorably positioned, they can act as replacement spurs, in repositioning the vine’s growing point(s) (Fig. 4.11). As the vine grows, the location of renewal wood tends to move outward. Replacement spurs reestablish, and thus can maintain the vine’s shape and training system. The need for renewal and replacement spurs has been long known, having been enunciated by Roman authors such as Columella (De Re Rustica, 4.21). Depending on their position, water sprouts may also supply nutrients for fruit development. Shoot thinning at flowering can correct imperfections in the position of shoots, or remove weak or nonbearing shoots. If the vine is in balance, and winter’s cold or late frosts have not caused extensive bud or shoot kill, the need for shoot thinning should be minimal. Partial shoot removal (trimming) can vary widely in degree and timing. Pinching refers to the removal of the uppermost few centimeters. More extensive trimming, respectively called tipping, topping, or hedging, depends on the timing and relative amount removed. In general, trimming removes the bare minimum. When performed periodically and moderately, it is less physiologically disruptive.

164

4. Vineyard practice

FIGURE 4.11

Illustration showing a head-trained vine, spur pruned (left) and cane pruned (right). Shaded areas refer to water sprouts, suckers, and portions of canes removed at pruning. From Weaver, R.J., 1976. Grape Growing. John Wiley, New York, NY. Copyright © John Wiley and Sons Inc. Reprinted by permission of John Wiley and Sons Inc.

Pinching, if used, is usually conducted in early season. When performed during flowering, fruit set may be enhanced (Collins and Dry, 2006). The procedure may reduce inflorescence necrosis in varieties predisposed to this disorder, presumably by reducing carbohydrate competition between the developing leaves and embryonic fruit at this critical period. Pinching can also be used to maintain shoots in an upright position. The activation of limited lateral shoot growth may provide desirable fruit shading in hot sunny climates. Tipping (topping) occurs later and may be repeated periodically throughout the growing season. It usually removes only the shoot tip and associated young leaves, leaving at least 15 or more mature leaves per shoot. Depending on when implemented, tipping can reduce competition between developing flowers/fruit and young leaves for photosynthate (Quinlan and Weaver, 1970). It can also improve canopy microclimate by removing undesirable draping leaf cover, thereby reducing grape cluster compactness and improving bunch rot control (Molitor et al., 2015). In windy environments, tipping produces shorter, more sturdy, upright shoots, less susceptible to wind damage. However, tipping can reduce cane and pruning weights and the number of shoots and grape clusters in the succeeding year (Vasconcelos and Castagnoli, 2000). Hedging (trimming of the vine to produce vertical canopies resembling a hedge) removes entangling vegetation that can impede machinery movement through the vineyard. This is often necessary with dense plantings (rows 2 m wide). Like tipping, it reduces carbohydrate competition between new, expanding leaves and the fruit, but also lowers vine capacity. The latter may be the goal for vines where overcropping is a varietal

tendency or normal fruit loads have traditionally been considered detrimental to wine quality (e.g., Pinot noir) (Parker et al., 2016). Hedging increases the number of shoots, but reduces their relative length, tending to increase light and atmospheric exposure of the leaves and fruit. Trimming shoots to fewer than 15 leaves is generally undesirable. If performed during or before fruit set, it can stimulate undesirable lateral bud activation. The extent to which this occurs is partially dependent on the variety and the form of training (upright vs. downward shoot growth) (Poni and Intrieri, 1996). In contrast, late trimming (after ve´raison) seldom activates lateral growth. However, a deficiency in photosynthetic potential may persist, delaying both fruit and cane maturation, as well as decreasing cold hardiness. Physiological compensation by the remaining leaves, through delayed leaf senescence and higher photosynthetic rates, is usually inadequate. The degree to which these problems manifest themselves depends on trimming severity. Trimming can variously affect fruit composition, depending on its timing, severity, and vine capacity. Many of the effects resemble those of basal leaf removal discussed later. The potassium content of the fruit increases more slowly and peaks at a lower value (Solari et al., 1988). The rise in berry pH, and decline in malic acid content associated with ripening, may be less marked. Total soluble solids may be little influenced, or reduced, in trimmed vines. Anthocyanin synthesis may be adversely affected in varieties such as de Chaunac (Reynolds and Wardle, 1988). This tendency presumably is not found in Bordeaux, where hedging is common. Improvement in amino acid and ammonia nitrogen levels associated with light trimming shortly

165

Management of vine growth

12 Titratable acidity (g/liter)

after fruit set (Solari et al., 1988) may be one of the more desirable features induced by trimming. Its effect in delaying ripening and reducing sugar accumulation is being considered as a means of moderating the negative effects of climate change (Martı´nez De Toda and Balda, 2013). Considerable interest has been shown in single, or repeat, partial leaf removal (and associated lateral shoots), notably around and slightly above fruit clusters (basal leaf removal). The procedure selectively improves air and light exposure around the clusters. This can modify fruit attributes as well as yield (Palliotti et al., 2012). It also facilitates the more effective application of protective/control agents to the fruit. The reduced incidence of bunch rot was one of the first benefits detected with basal leaf removal. This has been attributed to the increased evaporative potential, wind speed, higher temperature, and improved light exposure in and around the fruit (Thomas et al., 1988). The desirability of basal leaf removal depends on its perceived benefits, timing, cultivar characteristics, climatic conditions (Moss, 2013; Frioni et al., 2017), and expense of its application. If the vine canopy is open and the fruit adequately exposed to light and air (e.g., with divided canopy systems), leaf removal is probably unnecessary and undesirable. It removes photosynthetic potential, reducing both fruit yield (and compactness) in the current season, and potentially the next year (by limiting fruit bud initiation). In addition, in hot, sunny climates the procedure may expose the fruit to sunburn, delay maturity, reduce acidity, and undesirably modify wine quality (Greer et al., 2006). An effect generally associated with basal leaf removal is a decline in titratable fruit acidity (Fig. 4.12). This is associated with a reduction in potassium uptake and enhanced malic acid degradation. Tartaric and citric acid levels are seldom affected. The amino acid content tends to be modified, being particularly noticeable in a reduced proline content. Anthocyanin levels generally are enhanced, as is the flavonol content, whereas total phenol content may rise or remain unchanged. Berry sugar content is seldom affected significantly. Basal leaf removal tends to diminish grassy, herbaceous, or vegetable odors, as well as the bell pepper character derived from methoxypyrazines (Gregan and Jordan, 2016). Fruity aromas may rise or remain unaffected. Depending on the cultivar, terpene (Table 4.2), norisoprenoid (Song et al., 2015), and fruity-smelling thiol precursor contents (Murat and Dumeau, 2005) may increase. Although usually beneficial, excessive production of some norisoprenoids (e.g., 1,1,6trimethyl-1,2-dihydronaphthalene), as well as their precursors, can generate undesirable kerosene-like fragrances in Riesling wines (Marais et al., 1992). Basal leaf removal may increase the glycosyl-glucose (G-G)

r2 = 0.86 11

10

9 0

20 40 60 80 % Fruit exposure (véraison)

100

FIGURE 4.12 Relationship between fruit exposure at ve´raison and titratable acidity at harvest of the cultivar Sauvignon blanc. From Smith, S., Codrington, I.C., Robertson, M., Smart, R., 1988. Viticultural and oenological implications of leaf removal for New Zealand vineyards. In: Smart, R.E. (Ed.), Proc. 2nd Int. Symp. Cool Climate Vitic. Oenol. New Zealand Society of Viticulture and Oenology, Auckland, New Zealand, pp. 127e133, reproduced by permission.

Aroma profile analysis and protein content of Sauvignon blanc fruit with and without basal leaf removal.

TABLE 4.2

Control

Basal leaf removal

Free aroma constituents (mg/L) Geraniol

1.6

9.5

trans-2-Octen-1-al

0.3

3.1

1-Octen-3-ol

2.4

6.6

trans-2-Octen-1-ol

6.5

11.7

trans-2-Penten-1-al

4.6

12.7

a-Terpineol

1.9

4.1

Potential volatiles (mg/L) Linalool

23

49

trans-2-Hexen-1-ol

29

61

cis-3-Hexen-1-ol trans-2-Octen-1-ol

5.1 321

6.3 830

2-Phenylethanol

17.9

50

b-Ionone

26

66

Molecular weight >66,000

32

33

Molecular weight 5

0.8 (B) Depth below emitter (m)

214

0

K, mg/kg >250

0.2

>200

0.4

>150 0.6

1 summer's fertigation

>100

0.8

(C) 0

P, mg/kg >0.5

0.2

>0.3

0.4

>0.2 Apple orchard, Clay, Avignon

0.6 0.8

0

>0.1

0.2 0.4 0.6 0.8 Distance from emitter (m)

FIGURE 4.37 Measured patterns of (A) nitrogen, (B) potassium, and (C) phosphorus in soil after one summer’s application of soluble fertilizer from a drip emitter. Redrawn from Guennelon, R., Habib, R., Cockborn, A.M. 1979 Aspects particuliers concernant la disponibilite´ de N, P et K en irrigation localise´e fertilisante sur arbres fruitiers. In Se´minaires sur l’Irrigation Localise´e I, pp. 21e34. L’Institut d’Agronomie de l’Universite´ de Bologne, Italy, in Elrick, D.E., Clothier, B.E., 1990. Solute transport and leaching. In: Stewart, B.A., Nielsen, D.R. (Eds.), Irrigation of Agronomic Crops American Society of Agronomy, Crop Science Society of America, and Soil Science Society of America Publication, Madison, WI, pp. 93e126 (Agronomy Monograph 30), reproduced by permission.

forms help maintain a relatively stable supply of nutrient cations. Roots may gain access to nutrients by several means. Usually, nutrients are directly assimilated from dissolved ions in the soil solution. This shifts the equilibrium between free and sorbed forms, replenishing the supply in the soil solution. In addition, the release of Hþ ions, carbon dioxide, and organic acids from roots makes Hþ ions available for cation exchange with sorbed cations. This further releases nutrient cations into the soil solution. Hydrogen ions are also involved in converting insoluble ferric ions (Fe3þ) into the more soluble, ferrous (Fe2þ) state. A somewhat similar release of negatively

Fertilization

charged nutrients occurs in alkaline soils by anion exchange, but is largely restricted to phosphate salts. Microbial enzymes are another significant factor in nutrient release. By degrading organic compounds, their mineral components are released. For example, organic phosphates are liberated through the action of extracellular phosphatases. Finally, chelating and reducing compounds, secreted by roots and microbes, help keep metallic ions, such as iron and zinc, in readily available forms. Most metallic cations tend to be in limited supply in neutral and alkaline soils, due to the formation of insoluble oxides, sulfides, silicates, and carbonates. In addition to their incorporation into organic molecules, uptake of nutrients, such as potassium, helps maintain cytoplasmic electric and osmotic balance. This may be required to counter the negative charges associated with 3 the uptake of the major nutrient anions, NO 3 and PO4 , or the accumulation of organic acids in cellular vacuoles. Despite the processes releasing nutrients into the soil solution, plant demand often outstrips the soil’s capacity to replenish nutrients adjacent to feeder roots. Consequently, root extension is usually vital to maintaining an adequate nutrient supply. This is especially important for nutrients such as phosphates, zinc, and copper, which do not migrate significantly in the soil. The extension of feeder roots into new sites permits the slow reestablishment of an equilibrium between dissolved and bound nutrients in regions where most available nutrients have been extracted. Although nutrient uptake activates the liberation of sorbed ions, fertilizer addition can reverse the process, resulting in the precipitation of soluble nutrients. This, combined with significant nutrient storage in the vine, and the extensive and deep penetration of vine roots (Conradie, 1988), helps explain why grapevines often respond slowly and marginally to fertilizer application. Other than texture (Table 4.8), soil pH probably has the greatest influence on nutrient availability TABLE 4.8 Cation exchange capacity of a range of soil textural types. Soil type

Exchange capacity

Sand

2.0e3.5

Sandy loam

2.3e17.1

Loam

7.5e15.9

Silty loam

9.4e26.3

Clay and clay loam

4.0e57.5

The increasing variation in exchange capacity from sand to clayey soils partially reflects the size range of its sand, silt, and clay constituents and the nature of the clay component. For example, the surface area/volume ratio of clay can vary from as low as 5 m2/g for kaolinite to 750 m2/g for sodium montmorillonite. Data from Lyons, T.L., Buckman, H.O., Brady, N.C., 1952. The Nature and Properties of Soils. Macmillan, New York, NY.

215

(Fig. 4.38). Depending on the chemical nature of the parental substrate, the degree of weathering, and the organic content of the soil, most soils are buffered within a narrow pH range. In calcareous (lime) soils, the primary buffering salts are CaCO3 and Ca(HCO3)2. Because of this buffering, the availability of Fe2þ, Mn2þ, Zn2þ, Cuþ, Cu2þ, and PO3 4 is restricted. Through prolonged leaching, most soils in high-rainfall areas become acidic. Such soils are often deficient in available Ca2þ, Mg2þ, 2 Kþ, PO3 4 , and MoO4 . In contrast, arid regions tend to generate saline or sodic soils, especially in the subsoil, where leachates often accumulate. Soil pH also influences the activity of microbes, especially bacteria, most of which are unable to grow under acidic conditions. The consequential slowing of microbiþ al activity retards the oxidation of NHþ 4 to NO3 and the liberation of organically bound phosphates. These influences are compounded under cool and wet conditions, both of which retard microbial activity. Thus, if fertilizer is applied in the spring, nitrate and inorganic phosphate should be used. Soil microbial activity is insufficient to effectively oxidize ammonia or liberate organically bound phosphates in the early spring. Soil pH also indirectly affects nutrient availability by affecting root growth. Fig. 4.39 illustrates the effect of soil acidity on a range of rootstocks. Acidic soils are often treated with crushed limestone, preferably before planting. Alternatively, surface and subsurface soil horizons may be mixed with lime and gypsum in a procedure called soil slotting. Slots 80 cm deep, 15 cm wide, and 1 m apart can significantly increase root penetration in acidic soils (Kirchhof et al., 1990). The amount required depends on the neutralizing power of the lime source, as well as the soil’s pH, organic content, and texture (Fig. 4.40). Acid-tolerant rootstocks, such as 140 Ruggeri, 110 Richter, or Gravesac, can minimize the detrimental effects of acidic soils, notably toxicity resulting from the increased solubility of aluminum, copper, and manganese. Lime-tolerant rootstocks are extensively used on calcareous soils. In addition, elemental sulfur or gypsum (calcium sulfate) may be added to alkaline and sodic soils. Elemental sulfur is microbially oxidized to sulfuric acid, neutralizing hydroxides, whereas calcium sulfate both neutralizes hydroxides and permits sodium leaching (by displacing it from soil colloids and carbonates). An alternative or additional procedure involves midrow ripping and planting of vines on mounds of soil. This is particularly valuable where the water table is high (Cass et al., 2004). Rootstock choice can also differentially affect nutrient uptake. Although rootstocks differ little in their accumulation of nitrogen, phosphorus, and zinc, they vary widely in their absorption of potassium, calcium, magnesium, and chlorine (Fig. 4.41A). Such variation may

216

4. Vineyard practice

4.0 pH

4.5

Extreme acidity

5.0 Very strong acidity

5.5 Strong acidity

6.0

6.5 Slight acidity

Medium acidity

7.0

7.5

Very slight acidity

8.0

8.5

Moderate alkalinity

Slight alkalinity

9.0 Strong alkalinity

9.5

pH 10

Very strong alkalinity

Nitrogen Phosphorus Potassium Sulfur Calcium Acidity H-ION concentration

Alkalinity OH-ION concentration Magnesium

Iron Manganese Boron Copper and zinc

FIGURE 4.38 General relationship between soil pH (and associated factors) and availability of plant nutrients. The width of each band representing an element indicates the potential availability of the nutrient at a particular pH value. The width of the striped area between the curved lines is proportional to the relative excess of hydrogen ions (left) or hydroxyl ions (right) at the corresponding pH. From Truog, E., 1946. Soil reaction influence on availability of plant nutrients. Proc. Soil Sci. Soc. Am. 11, 305e308, reproduced by permission.

200 b

Unlimed soil (pH-4.1)

Dry mass of roots (g per vine)

180 160 140

c

Soil limed to pH 5.0 c

Soil limed to pH 6.0 a a a

c

b b a a

120

b a

b

a

100

b

b

a

a 80

a

a

a a

60 a 40 140 Rug

FIGURE 4.39

110 R

89 R

R. du Lot

S04

44–53 Malegue

101–14

Ramsey

Effect of soil acidity on root growth of eight rootstock cultivars. Mean separation for individual rootstocks by Duncan’s multiple range test, %. From Conradie, W.J., 1988. Effect of Soil Acidity on Grapevine Root Growth and the Role of Roots as a Source of Nutrient Reserves the Grapevine Root and its Environment. Department of Agricultural Water Supply, Pretoria, South Africa, van Zyl, J.L., pp. 16e29. (Technical Communication No. 215), reproduced by permission.

Fertilization

OM (%) CEC 7.0

(a) Sands (b) Sandy loams (c) Loams and silt loams (d) Silty caly loams

Soil pH

6.5 6.0

2.5 3 4 5

5 12 18 25

5.5 5.0 (a)

(b)

(c)

(d)

4.5 1 2 3 4 5 6 7 8 9 Ground limestone required to raise soil pH to 7.0 (tons/acre)

FIGURE 4.40 Relationship between soil texture and amount of limestone required to raise the soil pH to 7.0. CEC, cation exchange capacity; OM, organic matter. From Peech, M., 1961. Lime Requirements vs Soil pH Curves for Soils of New York State. Mimeograph. Department of Agronomy, Cornell University, Ithaca, NY, reproduced by permission.

arise from the selective activity of transport systems in their cell membranes. Differences in nutrient storage in grapevines may also develop because of differential accumulation by the scion (Fig. 4.41B). Although nutrient uptake and transport may be initially passive and apoplasmic (via the cell wall and intercellular spaces), all nutrients must pass into the cytoplasm on, or before, reaching the endodermis. Direct access to the vascular system is restricted by the Casparian strip, which makes the endodermal cell wall essentially water impermeable. Thus, transport into the vascular tissues is under the metabolic control of one or more transport systems of the endodermal plasma membrane. Unloading into the xylem and phloem is also likely to be under metabolic, and thus genetic, control. Accumulation within different grapevine tissues is undoubtedly under genetic regulation. Further variation in mineral uptake may arise from variability in rootstock sensitivity to mycorrhizal symbiosis. This is especially likely with phosphate, where soils low in this nutrient favor the development of mycorrhizal associations. Nonetheless, leaf phosphorus content appears to be correlated more with scion characteristics than with phosphorus availability, or the degree of mycorrhizal colonization (Karagiannidis et al., 1997). In addition to phosphorus, mycorrhizae can improve zinc and copper availability. Due to the complexities of soil micronutrient availability, deficiency problems may be circumvented with foliar application. When properly formulated to avoid leaf “burning” and precipitation, spraying has the advantage of rapidly, effectively, and efficiently supplying nutrients precisely when, and as, needed. One of the latest innovations in fertilizer formulation involves nanotechnology (production of products in the size range of 1e100 nm). Nanofertilizers are not yet

217

commonly used, but possess unique properties (Achari and Kowshik, 2018). Possessing very high surface-tovolume ratios, they diffuse well and remain in suspension, taking on novel catalytic and quantum attributes. These can be adjusted by their coatings and thickness. Nanofertilizers also improve uptake through leaf and fruit cuticular pores (particles 2 mm) calcium carbonate. This could complicate conditions that limit root growth, such as anaerobiosis following heavy rains, increased ethylene synthesis in the soil (Perret and Koblet, 1984), and enhanced risk of heavy-metal toxicity. Overcropping has also been correlated with increased incidence of chlorosis in the subsequent year (Murisier and Aerny, 1994). Other factors implicated in the complex relationship between iron and lime-induced chlorosis include the rootstock cultivar, mycorrhizal symbiosis, and the presence of endophytic bacteria (Bavaresco and Fogher, 1996). Young roots are the primary site of iron acquisition. In a study by Bavaresco et al. (1991), iron uptake and resistance to chlorosis were closely correlated with root diameter and root-hair development. Resistance has also been closely associated with acidification of the rhizosphere, production of Fe3þ reductase, increased membrane redox potential, and augmented synthesis of organic acids (Brancadoro et al., 1995). Acidification increases the solubility of the ferric (Fe3þ) ion, and favors its reduction to the more soluble ferrous (Fe2þ) state, the form in which iron is incorporated. Nevertheless, the mechanism of resistance or tolerance varies with the species (Bavaresco et al., 1995). Once in the plant, ferrous ion is reoxidized, chelated primarily with citrate, and transported in the xylem stream. In the leaf, it may be stored in a protein complex called phytoferritin. Redistribution may occur via the phloem. Most North American grapevines (and correspondingly most rootstock varieties) are sensitive to limeinduced chlorosis. Thus, choice of rootstock cultivars, such as Fercal, 41B, or 140 Ruggeri, is important when grafted vines are grown on calcareous soils. Alternative treatments include spraying the foliage with iron chelate (Feeethylenediamine-N,N0 -bis(2-hydroxyphenyl)acetic acid), adding ferrous phosphate to the soil (Dı´az et al., 2009), planting a cover crop of Festuca ovina (Bavaresco et al., 2010) or Festuca rubra, or, if the soil is not too

Fertilization

calcareous or alkaline, adjusting the pH with sulfur. It is not too surprising that V. vinifera roots, with millennia of adapting to calcareous soils, is notably lime tolerant (Cambrolle´ et al., 2015). Boron Boron is required for nucleic acid synthesis, in the maintenance of cell-membrane integrity, and in calcium use. It can be translocated in both xylem and phloem. Although required only in trace amounts, the range separating deficiency and toxicity is narrow. Deficiency symptoms develop as blotchy yellow chlorotic regions on terminal leaves. Chlorosis soon spreads to the leaf margin. Apical regions of the shoot may develop slightly swollen dark-green bulges. Early dieback may result in the development of stunted lateral shoots. Boron deficiency also induces poor fruit set (due to retarded germ-tube elongation) and the development of many shot berries. Deficiency symptoms tend to develop on sandy soils in high-rainfall areas, on soils irrigated with water low in boron, or on strongly acidic soils. Application of borax to counteract deficiency can lead to toxicity if not evenly distributed. Toxicity symptoms begin with the development of brown to black specks on the tips of leaf serrations. The necrotic regions spread and become continuous. The inhibition of growth may result in leaves wrinkling and puckering along the margin. Copper Copper acts primarily as a cofactor in respiratory oxidative reactions and the synthesis of proteins, carbohydrates, and chlorophyll. It is variously translocated in the phloem or the xylem. Copper-deficiency symptoms have been only rarely observed in grapevines, possibly due to the historical use of copper-based fungicides (notably Bordeaux mixture). When deficiency occurs, the leaves are dwarfed and pale green, shoots develop short internodes, cane bark has a rough texture, and root development is poor. Toxicity produces leaf chlorosis similar to that of lime-induced chlorosis. Toxicity most commonly occurs in soils where copper has accumulated following prolonged use of copper-containing fungicides (Scholl and Enkelmann, 1984). Copper buildup in the soil has also been implicated in some vineyard “replant diseases.” Molybdenum Molybdenum is required for nitrate reduction, as well as the synthesis of proteins and chlorophyll. Only rarely has molybdenum been found to be deficient in vineyard soils. However, on acidic soils, and with cultivars such as Merlot, deficiency can be associated with poor fruit set and millerandage (“hen and chickens”). This can be treated effectively with foliar sprays (Williams et al.,

229

2005; Longbottom et al., 2010). When molybdenum deficiency is severe, necrosis develops and spreads rapidly from the leaf margins inward. The demarcation zone between healthy and necrotic tissue is pronounced, and the unaffected areas appear normal. Affected leaves often remain attached to the shoot and fall with difficulty. Chlorine Chlorine is involved in both osmotic and ionic balance, as well as the splitting of water during photosynthesis. Although required in fairly large amounts, chlorine is not known to be limiting in vineyard soils. More commonly, it is associated with toxicity under saline conditions. It produces a progressive, well-defined necrosis that moves from the edges of the leaf toward the midvein and petiole. Because physiological disruption occurs at levels well below those causing visible toxicity, the use of chlorine-tolerant rootstocks on saline soils is advisable, for example, Salt Creek and Dog Ridge (Table 4.7).

Organic fertilizers The addition of manure to vineyards was once standard practice. With mechanization, specialization, and concentration in most agricultural industries, ready access to ample supplies of animal manure became increasingly compromised. At the same time, production costs of inorganic fertilizers fell, and manure changed from being an asset to an environmental disposal problem. In addition, commercial fertilizers were of definable and known composition, could be chosen for immediate or delayed application, were readily obtained when desired, and easier to store, handle, and use. Nonetheless, several trends have encouraged some growers to return to organic fertilizers. Increased energy (and therefore fertilizer) costs, concerns about groundwater pollution, and the belief that inorganic fertilizers encourage the destruction of the soil’s humus content have all encouraged a reduction in commercial fertilizer use. However, the issue is far more complex than believed or commonly presented (Kirchmann and Ryan, 2004). For example, manure can leach as much or more nitrate per kilogram of nitrogen as ammonium nitrate (Fig. 4.48), and can aggravate phosphorus loss (Wortmann and Shapiro, 2008). Nonetheless, enhancement of the soil’s aggregate structure may reduce these effects. Nitrogen availability is typically the major factor limiting the decomposition (mineralization) of organic material. As such, its presence markedly affects relative mineralization vs. humifaction. Because most manures have a relatively high carbon-to-nitrogen ratio, the initial

230 240 Leaching of total N (kgha–1)

FIGURE 4.48 Sum of total nitrogen leached over 3 years in a lysimeter study fertilized with 100 kg N/ ha in the form of ammonium nitrate and differently treated poultry manures during the first year. ON, control, no additions. FromKirchmann, H., Johns A.E.J., Bergstro¨m, L.F., 2002. Possibilities for reducing nitrate leaching from agricultural land. Ambio 31, 404 e408., reproduced by permission.

4. Vineyard practice

200 160 120 80 40 0 ON

Fresh manure

Anaerobic manure

Aerobic manure

estimated to contain up to about 90% of the nitrogen lost from vineyards at harvest. As with liquid manure, winery wastes must be added with caution, if a wet slurry. Otherwise, nutrient leaching can pollute groundwater. It is recommended to compost the material prior to addition (Bertran et al., 2004), with pH adjustment toward neutrality. The direct use of winery or distillery waste is not recommended, especially on acid soilsdit could lead to manganese buildup to toxic levels (Boubals, 1984). Equivalent problems with winery wastewaters have not been noticed (Hirzel et al., 2017). Pomace can also be a source of viral contamination, potentially increasing the incidence of nematode-transmitted viral infections.

Farmyard manure (FYM)

75 Organic C content (t ha–1)

effect of adding manure can be a reduction in nitrogen availability. This results because microbes are more effective at nitrogen uptake than vine rootsdthe nitrogen being bound up in their biomass. In addition, fresh manure may contain sufficient urea or uric acid to be toxic, “burning” roots before conversion to ammonia. Tillage, associated with manure incorporation, increases oxygen availability, facilitating rapid microbial nitrogen utilization. The “back to nature” viticulture movement has been prompted by a growing market for organic wines. In concert, many governments have enacted laws designed to limit nitrate and phosphate water pollution. This has spurred interest in means by which vineyard production can be maintained with limited or no application of inorganic fertilizers. As of this writing, this has yet to be demonstrated as a long-term viable option, nor have optimal conditions been found. In addition, assessing any consistent benefit of conventional vs. organic viticultural practice is fraught with difficulties (see Lester and Saftner, 2011). Animal manure, if repeatedly added to soil, can markedly enhance its organic content (Fig. 4.49). If well matured (composted), it is also a good source of slowly released nutrients and humic materials (Haynes and Naidu, 1998). Furthermore, manure enhances the activity of nitrogen-fixing bacteria, such as Azotobacter. In contrast, their activity is repressed by the application of nitrogen fertilizer or liquid animal waste. Additional benefits of aged manure include improved soil aeration (Baumberger, 1988) and a shift in soil pH toward neutrality (Fardossi et al., 1990). However, fresh manure, high in liquid components, must be applied sparingly. Even during warm weather, microbial activity may be insufficient to rapidly bind nutrients, resulting in nutrient loss and groundwater contamination. Alternative organic fertilizers include compost, green manures, chopped vine prunings, residue from destemming, and winery waste (pomace and dregs). The last is

Ammonium nitrate

50 FYM discontinued 1971 NPK fertilizer No fertilizers

25

0 1850

1900

1950

2000

Time (years)

FIGURE 4.49 Changes in soil organic carbon content on a Rothamsted continuous barley experiment. Treatments consisted of no fertilizer, NPK fertilizer applied annually (48 kg N/ha), farmyard manure (FYM) applied annually at 35 t/ha, and FYM applied from 1952 to 1971 and none since. From Johnston A.E., 1986. Soil organic matter, effects on soils and crops. Soil Use Manag. 2, 97e105, reproduced by permission.

Fertilization

Humic colloids, produced during humifaction, can play a vital role in maintaining soil fertility. They not only sorb nutrients, but favor the development of soil aggregates, improving air and water permeability (Wortmann and Shapiro, 2008). As humus decomposes, its sorbed and bound nutrients are slowly released to the soil solution. Although microbial activity clearly promotes the development of a favorable soil structure (Weil and Kroontje, 1979), there is no clear evidence of improved nutrient availability (Kirchmann et al., 2004). Humic substances consist primarily of refractory compounds, derived principally from microbial changes to the structure of lignins, plant phenolics, and complex polysaccharides. These colloidal particles combine with clay colloids to form the soil’s aggregate structure (Fig. 4.50). Cations, such as Ca2þ and Fe3þ, help stabilize these aggregates, further encouraging formation of a desirable crumb structure. Humus also provides much of the surface on which soil microbes grow. They, in turn, function as the main food source for soil invertebrates. Of these, the most obvious are earthworms, but these are far outnumbered by much smaller insect larvae, collembola, mites, nematodes, etc. Due to their size, earthworms are important in enhancing soil porosity and thereby facilitating aeration and water penetration, as well as dispersing nutrients throughout the soil’s upper layers. Smaller invertebrates, such as collembola and insect larvae, act as the principal herbivores, chewing dead plant and fungal remains, exposing their remnants to further microbial action. Although generally beneficial, excess and poorly timed application of organic fertilizers is deleterious. Under such conditions, they can disrupt vine growth and fruit development (Morlat, 2008; Morlat and Symoneaux, 2008). Manure and compost are most easily incorporated at the inception of vineyard establishment. This is especially advantageous if the organic content of the soil is low. Alternatively, organic amendments may be disced in periodically. Alternatively, earthworm activity can incorporate such material within a short period, if added in small amounts such as a top dressing. This is best done in early spring or autumn. Although addition slowly releases nutrients over an extended period, its primary benefit is improved soil structure and its nutrient retentive properties. Additional information on composts and other means of enhancing soil organic content can be found in Cass and McGrath (2005) and McGourty and Reganold (2005). Despite the benefits of composts and manures, there are several disadvantages. Synchronization of nutrient release to grapevine need is difficult. Mineralization and nutrient release is affected not only by a wide variety of soil and atmospheric conditions (rainfall, temperature, pH, moisture content, and texture), but also by the amendment’s chemical composition (Dahlin et al., 2005).

231

Inadequate release can lead to nutrient deficiencies, whereas untimely liberation can result in loss by leaching (Kirchmann and Thorvaldsson, 2000). Leaching is particular likely during cool, wet periods. In contrast, application of inorganic fertilizers can usually be better timed to vine need and root uptake, resulting in more efficient and effective nutrient use (Fig. 4.51). If the primary intention of adding organic amendments is as a fertilizer, application should be timed for nutrient liberation to coincide with feeder root production, often peaking at or just after flowering and fruit set (Comas et al., 2005). These roots often have a short life span, absorbing nutrients primarily during the first few days following emergence (Volder et al., 2005). Farm manure Although manure has advantages, it possesses several inconveniences, beyond local unavailability. Variability in composition (Table 4.12) is a major concern, complicating assessing appropriate application rates. Important factors influencing the nutritional composition of manures are its source, the nature and amount of straw present, and handling and storage before application. Additional potential issues may include toxicity (e.g., copper supplementation in pig feed), zinc deficiency (poultry manure), nitrogen deficiency (resulting from decomposition of large amounts of incorporated straw), and nitrogen loss (ammonia volatilization during urine breakdown). Where economically feasible, some heavy-metal contaminants can be immobilized with the addition of a nanocomposite of sodium carbonate, biosilica, and attapulgite (Wang et al., 2017). Because animals use only a portion of the organic and nutrient content of their feed, much of the mineral nutritive value remains in the manure. About half of the nitrogen, most of the phosphates, and nearly 40% of the potassium found in the original feed remain in the feces. Fresh manure contains both readily and slowly available nitrogen. The readily available form (mostly urea and ammonia) is rapidly lost within a few weeks, via volatilization, if not bound to soil colloids or taken up by microbes. The slowly available, organically bound nitrogen is more stable (Klausner, 1995). Most of this is decomposed within 1 year, whereas the more resistant may take several years to mineralize. Thus, repeat application over many years is essential for development of relatively uniform nutrient availability. Brady (1974) estimated that roughly 1000 kg (w1 ton) of manure supplies about 2.5 kg nitrogen, 0.5 kg phosphorus, and 2.5 kg potassium. Most plant remains are either hemicellulose, lignins, or lignineprotein complexes. Additional organic material consists of the cellular remains of bacteria, primarily peptidoglycans (a complex polymer of two alternating amino sugars). They can constitute up to 50% of the manure’s dry mass.

232 FIGURE 4.50 Model of soil aggregate organization with major binding agents indicated. From Tisdall, J.M., Oades, J.M., 1982. Organic matter and water stable aggregates in soil. J. Soil Sci. 32, 141e163, reproduced by permission.

4. Vineyard practice

Major binding agent Solid 2000 μm

Roots and hyphae (medium-term organic)

Pore

Root 200 μm

Plant and fungal debris encrusted with inorganics (persistent organic)

Hypha Aggregates or particles

Hypha 20 μm

Bacterium Packets of clay particles

Microbial and fungal debris encrusted with inorganics (persistent organic) 2 μm

Microbial debris (humic materials)

Clay particles

Amorphous aluminosilicates, oxides and organic polymers sorbed on clay surfaces and electrostatic bonding, flocculation (permanent inorganic) 0.2 μm

Clay plates

Cement

One of the rarely noted disadvantages associated with manure is the extensive release of methane by ruminant animals (Moss et al., 2000). Microbial digestion of plant material in the rumen generates copious amounts of methane, a gas about 20 times more active as a “greenhouse” gas than carbon dioxide. In addition, if the manure is liquid, or becomes so during maturation, much of the nitrogen is lost to the atmosphere or washed into the surrounding soil, potentially contaminating groundwater and local streams, ponds, and lakes. However, when well matured, application to vineyards beneficially enhances the soil’s organic

content, its texture, and the development of a vibrant microbial and invertebrate biome, and puts to good use what otherwise could be a polluting farm waste. Green manures Green manures are another long-established addition for improving soil structure and nutrient content. The procedure involves growing a crop followed by plowing it under while still green. If plowing occurs while the crop is still succulent, its nitrogen content is typically adequate to promote rapid decomposition, without limiting short-term vine nitrogen availability.

233

Fertilization

60 N-uptake

Fertilizer-derived N (%)

50

Leaching

40

30

20

10

0 Ammonium nitrate

Poultry manure - slurry

Red clover manure

FIGURE 4.51 Nitrogen in harvested crops and leaching during periods of 2 (red clover manure) and 3 years (NH4NO3 and poultry manure), as percentage of N applied with the respective N source during the initial year. From Dahlin, S., Kirchmann, H., Katterer, T., Gunnarsson, S., Bergstrom, L., 2005. Possibilities for improving nitrogen use from organic materials in agricultural cropping systems. Ambio. 34, 288e295, reproduced by permission. TABLE 4.12

Average values for moisture and nutrient content of farm animal manures.

Moisture Source Portion Percentage content

Nitrogen (%)

Horse

Phosphorus (%)

Potassium (%)

Manure 80

75

0.55 (0.50e0.60) 0.33 (0.25e0.35) 0.40 (0.30e0.50)

Urine

20

90

1.35 (1.20e1.50) Trace

1.25 (1.00e1.50)

Mixture d

78

0.7

0.55

Manure 70

85

0.40 (0.30e0.45) 0.20 (0.15e0.25) 0.10 (0.05e0.15)

Urine

30

92

1.00 (0.80e1.20) Trace

1.35 (1.30e1.40)

Mixture d

86

0.6

0.45

Manure 60

80

0.55 (0.50e0.60) 0.50 (0.45e0.60) 0.40 (0.35e0.50)

Urine

40

97

0.40 (0.30e0.50) 0.10 (0.07e0.15) 0.45 (0.20e0.70)

Mixture d

87

0.5

Manure 67

60

0.75 (0.70e0.80) 0.50 (0.45e0.60) 0.45 (0.30e0.60)

Urine

33

85

1.35 (1.30e1.40) 0.05 (0.02e0.08) 2.10 (2.00e2.25)

Mixture d

68

0.95

Poultry Mixture d

55

1.00 (0.55e1.40) 0.80 (0.35e1.00) 0.40 (0.25e0.50)

Cattle

Swine

Sheep

0.25

0.15

0.35

0.35

0.4

1

Ranges in parentheses. Data from van Slyke, L.L., 1932. Fertilizers and Crop Production. Orange Judd, New York.

Green manures commonly are planted in midsummer to early fall, to limit or avoid vines experiencing water and nutrient stress. This is especially important under nonirrigated arid conditions, or on poor soils. However, some competition may be desired to limit vine vigor under conditions of high rainfall, or on fertile soils (Caspari et al., 1996). Green manures, grown as a groundcover, can be useful in limiting water

loss, fertilizer leaching, and soil erosion on slopes. For example, ground covers can dramatically restrict water runoff during downpoursd15% vs. 80% (Rod, 1977). Although loss is less marked when measured over the whole season, ground covers can reduce water runoff from 19% to 1.5% on slopes of more than 40
(Rod, 1977). In addition, ground covers can facilitate weed control, bind nutrients otherwise lost by leaching, keep

234

4. Vineyard practice

down dust (valuable in mite control), and reduce erosion (Battany and Grismer, 2000). If green manure crops are left until spring, they can trap snow and limit frost penetration. Leguminous and cereal crops have usually been used as green manures. Legumes have the advantage of forming symbiotic associations with Rhizobium. These root noduleeforming bacteria fix nitrogen (Fig. 4.52), potentially replacing that removed annually with each harvest. The amount of nitrogen incorporated depends on the rhizobial strain, the host legume, the soil moisture conditions, and the nitrate content of the soil. Their use is an eco-friendly way of supplying nitrogen to the soil. However, if used for this purpose, the soil should not receive nitrogen fertilization. Adding nitrate reduces the activity of rhizobial nitrogen fixation. Production levels can vary from 10 to more than 200 kg/ha/year (Keller, 1997). High seed costs, the need for Rhizobium inoculation, and higher water demands can limit legume feasibility. Occasionally, these disadvantages may be reduced with self-seeding legumes, such as burr clover. In contrast, cereal crops require less water, the seed is less expensive, and the plants loosen compacted soil. An effective compromise is to use a mix of both (Winkler et al., 1974). This has the additional advantage of providing a greater range of habitats for beneficial insects (potential biological control agents). Nevertheless, the species chosen should not provide habitats favoring rodent, pest, or disease infestations. Positioning nesting or resting sites for owls and hawks in, or adjacent to,

Yield of corn (kg grain/hectare)

6000

Rye Vetch

5000

4000

No cover crop

3000

2000

1000 0

45

90

180

Nitrogen added (kg/hectare)

FIGURE 4.52 Effects of legume (vetch) and nonlegume (rye) cover crops on the 7-year-average yield of corn receiving three levels of nitrogen fertilizer on a sandy loam soil. Cover crops were grown each year. Data from Adams, W.E., Morris, H.D., Dawson, R.N., 1970. Effects of cropping systems and nitrogen levels on corn (Zea mays) yields in the southern Piedmont region. Agron. J. 62, 655e659.

vineyards can help control rodent pests, and has the additional advantage of minimizing bird damage at harvest time (Saxton et al., 2009; Saxton and Kean, 2010). The effect of ground cover choice on the grape flavor has rarely been considered. Work by Feng et al. (2011) is beginning to shed light on this issue. Its potential to reduce crop size and influence fruit composition may also become part of a technique used to minimize the negative effects of climate warming. Conversely, herbicidal management of weed growth under vines may be preferable, where enhanced yield is to grower benefit. As always, there are countervailing influences to every procedure. Compost Except for green manures, most organic fertilizers are best first composted. It is less variable in chemical composition, and its nutrient content is released slowly. Compost can also have additional benefits, such as acting as a mulch (reducing water evaporation and soil erosion), assisting in weed control, and reducing pesticide and metabolite leaching (Ferna´ndez-Bayo et al., 2015). Cocomposting winery solid and liquid waste converts both products into a material safely added to vineyard soils. Solid wastes from wine production constitutes about 20%e25% of the grape mass (Sessa et al., 2013)destimated to consist of about 62% pomace, 14% lees, 12% stalks, and 12% dewatered sludge (Cotoras et al., 2014). Because of its variable composition, experimentation is required to establish a formula for efficient and effective composting. Sewage sludge is occasionally used as a liquid additive, but it must be first checked for heavy-metal contamination (Perret and Weissenbach, 1991). In addition, too much liquid complicates composting. Simple composting involves mounding the waste into windrows, frequently 1.5 m high, 2 m wide, and 4 m or more in length. These have sufficient size to insulate the interior, permitting its temperature to rise to 55
Ce70
C. The heat, generated by microbial activity, selects for thermophilic bacteria and fungi that quickly degrade most readily decomposable organic material. The combination of heat and microbial decomposition also tends to pasteurize (but not sterilize) the waste, reducing most pathogens and weeds to undetectable levels (Noble et al., 2009). Its effect on grape virus inactivation appears not to have been investigated. Data with other viruses suggest that exposure to temperatures near 70
C for 3 weeks is effective in their inactivation. Thus, systems that do not expose all the material to adequate temperatures are probably best avoided if the compost is to be applied to vineyards. Because composting is most efficient when microaerobic, the mounds are turned periodically. Frequency

235

Organic viticulture

depends on the rapidity with which the internal temperature rises. Temperatures above 65
Ce70
C inhibit the action of the most desirable thermophilic microbes. Turning not only avoids overheating and incorporates material from the exterior, but also provides adequate oxygenation for the required microbial activity. This also limits off-odor production from anaerobic activity. To further assist oxygen penetration, bulking agents such as wood chips, corn cobs, corn stalks, or straw are often incorporated. They provide aeration channels, as well as drainage passageways for liquid escape, but excessive addition can act as a nitrogen drain, retarding decomposition of the main ingredients. Wood chips have the advantage that they do not decompose readily and can be separated for reuse. In so doing, they can act as a natural inoculum of desirable microbes for the next compost pile. Commercial inocula are available, but are seldom necessary if some finished compost is incorporated into new piles. Moisture levels ideally should be between 50% and 60%. Higher levels tend to shift metabolism toward anaerobiosis and malodor production. In addition, higher moisture levels can promote fermentation of ethanol to acetic acid, reducing the pH. At below 40% moisture content, decomposition is retarded. Alternative composting techniques include windrows with aeration supplied by pumping air through perforated tubes under static silo-like vertical systems, agitated beds, plugeflow systems, and rotating drums (Arvanitoyannis et al., 2006). Choice depends primarily on space availability, need for rapid conversion, and economy. Where the first two are not of primary concern, windrows are usually the preferred choice. Where the substrate consists primarily, or solely, of spent pomace, the pH must be raised to near neutral (6.5e7). This is required for the optimal activity of thermophilic microbes involved in decomposition. Raising the pH usually involves the incorporation of lime into the mix. It is estimated that composted pomace can return up to 30%e50% of the nutrients removed at harvest. Rates of application tend to vary from 2.5 to 10 kg/hectare. If the soil is saline or sodic, it may be necessary to add a source of calcium before application. Sodium levels may be higher in the pomace than desired, especially if winery wastewater has been incorporated. This often comes from sodiumbased cleansing agents used in the winery. Potassium contents may also be undesirably high. If the liquid component comes from municipal sources, additional sources of potential soil contamination can be a concern. Once bacterial mineralization brings the carbon/nitrogen ratio close to 20:1, microbial activity in the compost slows, and the temperature in the pile falls.

Further composting shifts to fungal action. This is particularly important in the humifaction of much of the remaining organic material. It partially involves the condensation of lignin-derived phenolics with ammonia. The humus so derived is refractory and mineralizes only slowly in soil. Thus, it makes an excellent soil amendment. Subsequent maturation involves degradation or volatilization of potential phytotoxins, such as ethylene oxide, ammonia, and lowmolecular-weight fatty acids (notably acetic, propionic, and butyric acids). After some 12e24 weeks (depending on the temperature, moisture, and nutrient content, as well as turning frequency), the compost can be safely used as an organic amendment or mulch. Composting appears to be accelerated by the addition of biochar, reducing both nitrogen and carbon loss (Sa´nchez-Garcı´a et al., 2015). Biochar Biochar is the lightweight, porous charcoal produced by the anaerobic pyrolytic degradation of organic material. Its use as a viticultural soil amendment is comparatively new, but it has a long history of agricultural use. Its application to soil has several potential advantages (Elad et al., 2011). Its surface area/volume ratio is particularly high, permitting it to effectively retain water and sequester inorganic nutrients in a biologically available form. Biochar also tends to adjust soil pH toward neutral, can absorb soil-derived toxins and contaminants, may encourage mycorrhizal and other desirable rhizospheree root associations, and favors maintenance of a beneficial soil microflora. Biochar addition has also been noted to enhance vineyard productivity without a diminution of grape quality (Genesio et al., 2015). In this regard, this property is enhanced by its combination with compost (Mackie et al., 2014; Wu et al., 2016). There is also evidence for biochar enhancing resistance to several soil pathogens. Because of its production method, biochar introduces no pests or pathogens. Biochar also tends to retain its desirable properties for extended periods, due to its particulate inorganic nature. Finally, its production from vineyard and/or winery waste, where the economics are appropriate, could be a source of biofuel for winery operations (Zhang et al., 2017).

Organic viticulture Organic viticulture’s goal is ostensibly to enhance vineyard sustainability. However, in practical terms, organic viticulture has largely focused on avoiding the use synthetic pesticides and inorganic fertilizers. As noted above, organic fertilizers can be as effective as inorganic fertilizers, and are more environmentally friendly (having lower energy production demands).

236

4. Vineyard practice

Their ability to retain nutrients, releasing them slowly, is desirable (if this coincides with vine need). It also facilitates a friable soil structure, improves water retention and percolation, and may support a more vibrant microbiome. However, that organic viticulture is inherently environmentally superior is largely unsubstantiated. Increases in soil fauna and flora diversity are not always observed (Lotter et al., 1999; Ma¨der et al., 2002), and pesticides are still used. If a truly “natural” viticulture was superior, then diseases should not be controlledd disease being part of the natural environment. Although man-made pesticides are eschewed, inorganic (e.g., sulfur- and copper-based compounds), as well as organic toxicants (e.g., oils and soaps), are used. Any substance that is a disease/pest control agent must, by its very nature, be inhibitory or lethal to the intended target. Such pesticides have typically been assumed to be environmentally safe, and their residuals harmless in wine production and to the environment. However, prolonged use of copper-based fungicides results in copper accumulation in the soildto a point where it can disrupt both its fauna and its flora. In addition, the grape yeast flora may be less complex than that of grapes from conventional viticulture (Milanovic et al., 2013). Organic pesticides, such as Stylet Oil, reduce grapevine photosynthesis and grape soluble solids (Finger et al., 2002). Pesticides used in organic viticulture can disrupt natural disease control agents, as can some conventional pesticides. For example, sulfur sprays and dusts increased the mortality of Anagrus spp., a biocontrol agent of leafhoppers (Martinson et al., 2001), as well as suppressing populations of predatory (Phytoseiulus) and mycophagous (Orthotydeus lambi) mites. The latter can reduce the incidence of powdery mildew (English-Loeb et al., 1999). Extensive use of natural insecticides, such as those with pyrethroids (derived from Chrysanthemum), was once considered environmentally safe. They are now known to disrupt economically important insect populations (e.g., bees) and accumulate to toxic levels in stream sediments (Weston et al., 2004). Use of another natural pesticide, nicotine, is no longer permitted, and its derivatives, neonicotinoids, are now being banned. In addition, various oils used in organic pest control can affect the taste and odor of grapes and wine (Redl and Bauer, 1990). These comments should not be construed as an attack on “natural” controls. No rational person advocates using anything more toxic or disruptive than necessary. Applying any pesticide, of whatever nature, should be done as sparingly and wisely as possible. What is required in any comparison of methods is objectivity, based on verifiable data, obtained by sound experimentationdnot apocryphal views pontificated with missionary zeal. To this end, natural pesticides should require the same exhaustive assessment for

efficacy, safety, and residue accumulation as synthetic agents. Lack of data should not be allowed to generate a false sense of security. Although health concerns about the risks of pesticide use have induced several governments to impose or contemplate pesticide deregistration, little thought has gone into the potential health risks of increased mycotoxin contamination of foods and beverages. As of this writing, though, the levels of mycotoxins (e.g., ochratoxin A [OTA]) have not been shown to be higher in organic than in conventionally produced wines (Chiodini et al., 2006; Gentile et al., 2016). Furthermore, in an extensive study of natural vs. synthetic pesticides, the percentage of potentially carcinogenic residues found in food was the same (about half) (Gold et al., 1992; Ames and Gold, 1997). Because of their low concentration, none was considered to constitute a significant cancer risk. Producers espousing organic viticulture are as desirous of producing high-quality wines as their conventional counterparts. However, few studies have attempted to compare their sensory qualities. In those that have been conducted, little difference was detected (Henick-Kling, 1995; Hill, 1988).

Disease, pest, and weed management Grapevines can be attacked by a wide diversity of biologic agents. There is insufficient space in this book to deal with all these maladies. Thus, only a few of the more important and/or representative examples of grapevine disorders are given. Detailed discussions of grapevine maladies for specific countries can be found in specialized works such as Bettiga (2013) and Wilcox et al. (2015) (North America), Galet (1991) and Larcher et al. (1985) (Europe), and Coombe and Dry (2006) and Nicholas et al. (1994) (Australia). It may appear to the grape grower that grapevines are abnormally afflicted by diseases and pests, but both are comparatively rare in nature. Viticulture (a form of monoculture) does exacerbate the situation, though. Grapevines inherently contain a wide range of endogenous and induced defenses. Thus, no matter how prevalent and damaging some diseases and pests can be, they are usually those to which V. vinifera was not exposed during its evolution (e.g., phylloxera and downy and powdery mildews). In contrast, most Vitis spp. in North America evolved in their presence and developed, to varying degrees, resistance to these causal agents. Regrettably, the major pests and disease agents of Vitis vinifera were all inadvertently introduced from North America in the 1800s. Their impact is still shown in European statistics. Between 1992 and 2003, viticulture employed just under 70% of all fungicide used in

Disease, pest, and weed management

Europe, but occupied only 3%e4% of agricultural land use (Anonymous, 2007). Most of this comes from the use of inorganic sulfur- and copper-based pesticides (also used in organic viticulture). Use of sulfur on grapevines in the United States is estimated at 14 million kg/ year. Although astounding, it partially relates to amounts used at between 3 and 5 kg/ha/treatment. In the past, typical use was in the 10e20 kg/ha range (Morton and Staub, 2008). This change is partially due to the adoption of synthetic organic pesticides. They began to replace inorganic fungicides in the 1940s. They were usually effective in lower amounts (1.5e3 kg/ha). In contrast, most modern fungicides (e.g., thiazoles) require only about 100 g/ha to be effective. Even newer, systemic acquired resistance effectors (e.g., acibenzolar-Smethyl) are effective at w30 g/ha. Correspondingly, the total amount of fungicides (by weight) used has been on the decline since the mid-1940s (Gianessi and Reigner, 2006). Further reductions in the amounts used relate to alternative measures of disease and pest control, more effective means of application, and more information on when application is needed (e.g., integrated pest management [IPM]). Regrettably, more effective and efficient use of any control measures demands much more understanding than in the pastdwhen spraying was done on a scheduled, prophylactic basis. For IPM to be implemented industry wide, both pest control advisers and grower acknowledgment of their expertise/assistance are required (Hillis et al., 2016). Although the use of pesticides will be required into the foreseeable future, their application is expensivedin terms of not only initial purchase, application costs, and inadvertent potential vine toxicity, but also disruption of the soil and grape flora and fauna, environmental contamination, and human health concerns. In terms of expenditure, it is estimated that, depending on the season, avoiding the need for fungicide application (just for powdery mildew) could save growers up to $48 million per year (California)dabout 20% of production costs (Fuller et al., 2014). Disease development generally occurs when there is a disjunction between plant defenses and biotic/abiotic stresses. Part of this appears to relate to the relative rate at which the host (or pathogen) releases cytoplasmic vesicles (exosomes) containing small RNAs. Once taken up by the host (or pathogen), interfering RNA silences host immunity (or pathogen virulence) genes (Cai et al., 2018). Of plant defenses, the first involves constitutive protective coatings of the leaf, flower, and fruit (e.g., cuticular waxes) or the suberized surfaces of woody structures. Induced defenses initially involve cell-membrane pattern-recognition receptors. These respond to distinctive, highly conserved, microbial constituents. These

237

constituents are often termed PRIs (plant resistance inducers) or PAMPs (pathogen-associated molecular patterns). Their effectors can be fungal chitins or ergosterol, or bacterial flagellins or peptidoglycans. In addition, plants may possess a series of complex and interactive intracellular mechanisms that respond to specific pathogen effector compounds. In the tit-for-tat world of infection, effective pathogens may evolve a means of inactivating host immune responses. Correspondingly, the host may develop recognition mechanisms for these effectors (called effector-triggered immunity), inducing one or more additional defense mechanisms. An extreme example is the rapid death (apoptosis) of infected and adjacent cells, indirectly also causing the death of the pathogen. Such a hypersensitive reaction is often induced by dominant resistance (R) genes. In addition, immune responses may involve slower chemical and anatomical changes by invaded and adjacent cells and tissues. Plants occasionally respond similarly to physicochemical irritants (e.g., pollutants). Host defenses can involve the release of oxygen radicals (that kill the pathogen as well as tissue cells in the immediate vicinity of the causal agent), the production and release of antimicrobial compounds (e.g., resveratrol and ε-viniferin) or enzymes (e.g., chitinase and glucanases that digest fungal cell walls), activation of the secretion of compounds (e.g., callose that walls off the pathogen), and/or production of hormonal substances (e.g., ethylene or jasmonic, salicylic, and abscisic acids) that activate systemic defense responses throughout the plant. The last appears to function by the release of glutamate from stressed cells, activating a rapid, progressive, long-distance, calcium-based plant defense signaling reaction (Toyota et al., 2018). Some of these actions appear to have lingering effects, by inducing semipermanent epigenetic changes (gene methylation and/or histone modification) (Luna et al., 2012). Plants may also possess RNA-silencing capacities against several pathogens. Typically, such responses effectively prevent the vast majority of potential invaders from inciting an ongoing infection. Pathogens are, thereby, defined as those invaders that either avoid activating or inactivate host responses sufficiently rapidly and/ or redirect the host’s physiology toward acceptance (typically the property of viruses). Although rapid activation of defenses is generally beneficial, undue/ unnecessary activation of systemic host defenses can be an energy drain, reducing a vine’s capacity to grow and fully ripen its fruit. Thus, it is against effective invaders, and to limit unwarranted vine responses, that vineyardists often do battle. Pathogens are generally classified as obligate parasites or necrotrophs. Both can be equally destructive,

238

4. Vineyard practice

but the former must be more subtle in their pathology. As they live only on living tissue, they must not be so destructive as to kill host cells (at least before the pathogen reproduces). In contrast, necrotrophs kill host cells to obtain their nutriment. They generally produce a wide range of enzymes and metabolites that kill host cells ahead of penetration. One of the fundamental questions still unanswered in plant pathology is why most plants remain resistant (or tolerant) to the bewildering array of potential diseasecausing agents with which they are beset. For example, of the multiple species of powdery mildews, only one affects grapevines. The site of effective attack can also be surprisingly limited (e.g., phylloxera). Were the source(s) of the typical resistance of hosts to the vast majority of potential pathogens better understood, it might be easier to design genetic modifications donating the “gold standard” of resistancedlong-lasting immunity (or at least, tolerance). Similar to changes affecting irrigation and fertilization, wide-ranging changes in pest, pathogen, and weed management are in progress. At one end of the spectrum are schemes relying totally on organic and biodynamic concepts (Jenkins, 1991; Reeve et al., 2005). Although the latter are currently in vogue, the principles of sustainable viticulture, based on IPM, have a solid scientific footing (Broome and Warner, 2008). Those of biodynamic viticulture are too often based on quasi-philosophical concepts using longdisgraced notions, the equivalent to the e´lan vital of 1800s biology. “Natural” pesticides, such as phosphorous acid (Foli-R-Fos 400), canola oil (Synertrol Horti Oil) (Magarey et al., 1993), and potassium silicate (Reynolds et al., 1996) can occasionally be as effective as standard pesticides. However, this is not a consistent finding, with higher concentrations often required (Schilder et al., 2002). In addition, there is no guarantee that “organic” pesticides are necessarily more environmentally friendly or safe for human consumption than so-called “synthetic” control agents (Bahlai et al., 2010). As already noted, to be effective, they must be inhibitory/toxic. Only compounds or adjustments that modify the ecological niche (to the detriment of the pest), genetic resistance, or the canopy microclimate are likely to have minimal or limited detrimental impact on vineyard sustainability. IPM is one of the main concepts generating better and more cost-effective pest and disease control. The term “management” reflects a paradigm shiftdthat of limiting damage to an economically acceptable level (its economic threshold). This is more rational and feasible than attempting what is often impossibledabsolute control. IPM combines the expertise of specialists in diverse but cognate fields, notably plant pathology, economic entomology, plant

nutrition, weed and soil science, microclimatology, statistics, and computer science, amplified with data from drones (unmanned aerial vehicles) (Romboli et al., 2017). Coordinated programs usually reduce pesticide use significantly (Broome and Warner, 2008), while achieving improved effectiveness. IPM often includes factors such as environmental modification and assessment, biological control, better synchronization and rotation of pesticide use, and the avoidance or limitation of undesirable interactions. Timing pesticide application to coincide with vulnerable periods in a pathogen’s life cycle usually reduces the number of applications, while increasing effectiveness. It also promotes optimal pesticide selection and application (e.g., restricting selective, systemic, curative agent use to situations when disease incidence is high). This approach is less disruptive to indigenous biological control agents and has become more feasible with advances in disease forecasting. When combined with enhanced pest/disease monitoring, miniaturization of solar-powered weather stations (Plate 4.10) (Hill and Kassemeyer, 1997), remotesensing devices (Beghi et al., 2017; Gatti et al., 2017), geographic information system technology, and smartphone apps (e.g., pmassessment.com.au), epidemiological models have become more accurate in predicting local disease severity situations. Risk assessment (the cost/benefit ratio) is being increasingly proposed to regulate if, when, and to what degree pesticide application is warranted. For example, spider mites are often considered a serious

PLATE 4.10 Remote weather station providing vineyard meteorological data. Photo courtesy Jackson, R.S.

Disease, pest, and weed management

grapevine pest. However, little significant effect on yield and quality may accrue from heavy infestation (e.g., Panonychus ulmi) (Candolfi et al., 1993). Vine capacity may be less affected than appearance might suggest, due to compensation by the vine for foliage damage (Hunter and Visser, 1988; CandolfiVasconcelos and Koblet, 1990; Fournioux, 1997). Compensation is most evident early in the season. Biological control measures may also reduce, if not eliminate, the need for control agents. In this regard, ground covers can provide alternate hosts for biological control agents (as long as they do not harbor serious vine pests and pathogens). Even better, ideally in the long term, is the incorporation (pyramiding) of several resistance genes into cultivars (e.g., Regent, Rondo, Prior, Flore´al, Vidoc, and Voltis) (Schneider et al., 2014). Regrettably, these may be rejected due to agronomic issues (Daniela et al., 2013). Nonetheless, the greatest impediment to the adoption of diseaseresistant cultivars is consumer reticence to try wines bearing unfamiliar names. If true that millennials consume a higher proportion of organic wines, they may generate the needed consumer base for wines from resistant cultivars. For most consumers, though, newer breeding techniques, such as CRISPR (clustered regularly interspaced short palindromic repeats), may permit the incorporation of effective disease resistance into existing cultivars, without modifying their crucial varietal flavors. Although the economic and ecologic benefits of IPM are obvious, its successful implementation is far from simple. A major factor is its considerable developmental costs. It takes years to develop an effective program, requiring the dedication of specialists in many fields. Without their various skills, forecasting consequences with the requisite accuracy needed would be impossible. For example, the most efficient fungicides against a particular plant pathogen may be toxic to parasites of an equivalently significant pest. As well, reduction in pesticide use may lead to outbreaks of secondary pests. Adoption of an IPM program, largely dependent on biological control agents for the western grape leafhopper in California, coincided with an infestation of variegated leafhoppers. Even seemingly unrelated changes in viticultural practice can affect IPM efficiency. For example, herbicide use to reduce loss of soil organic matter, and eliminate sites for overwintering pests, can increase the incidence of fungal diseases. Impeding the adoption of IPM is difficulty in predicting its economic benefits. In most instances, the financial losses ascribable to specific pest and disease agents are guesstimates at best. In some instances, reduced yield may permit the optimal ripening of the remaining fruit. Pesticide application is too often driven more by fear than needdthe “ounce of prevention” concept. As a

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result, full implementation of IPM strategies tends to be regional, and directed toward the most economically significant disease and pest agents. Although not perfect, IPM offers a more integrated and rational approach to disease and pest management than any other process. It can also deliver both economic savings and environmental benefits. Although IPM is preferable, investigation of individual components can be of considerable use. Research on pesticide combinations can avoid potential mutual interference, as well as reducing application frequency (Marois et al., 1987). Improvements in nozzle and sprayer design now provide better and more uniform spread, reducing runoff and drift, while achieving better pest/pathogen contact. Ensuring that a greater percentage of the target receives a toxic dose can delay the development of resistant strains. The most efficient nozzles available are those that give a small negative charge to the droplets when released (electrostatic sprayers). Plant surfaces, being positively charged, attract droplets, facilitating their uniform deposition, and limit removal by rain. Restricted variation in droplet size (optimum between 25 and 100 mm) and reduced water volume minimize runoff and drift (from the vaporization of very small droplets). Improved uniform coverage also has the advantage of reducing the development of pesticide resistance. Nonuniform spread facilitates resistance evolution by selectively eliminating susceptible and moderately tolerant strains, leaving only the most insensitive to propagate and multiply. Greater application efficiency may also be favored by night spraying, when conditions are calm (avoiding wasteful drift) and droplet evaporation is reduced. In addition, assessment of effective leafarea coverage can be used to fine-tune dosage application (Siegfried et al., 2007). Another advantage of IPM is that it emphasizes control diversity, rather than reliance on any one agent or method (Basler et al., 1991). Experience has indicated that dependence on any single control measure is likely to be ultimately unsuccessful. The same is equally probable with biological or genetic control schemes, especially if used to the exclusion of other control procedures. Because most pathogenic agents multiply and evolve exceedingly rapidly, they are disappointingly adept at developing resistance to single selective pressures.

Pathogen control Chemical methods Faced with increasing pesticide resistance, the difficulty and expense of finding new control agents, and increasingly regulated use, limiting the development of resistance has become imperative (Staub, 1991).

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Relatively nonselective contact agents are best used when prophylactic protection against a wide range of pathogenic fungi and bacteria is requireddtheir broad-spectrum action minimizes the likelihood of developing resistance. Conversely, their broadspectrum action is also one of their disadvantagesdthey are equally likely to be toxic to biological control agents. Nonetheless, occasional use, as needed, can reserve selective and curative action of systemic agents for critical situations, when their precise action is most needed. By being incorporated into the plant, and frequently translocated beyond the site of application, systemic agents can inactivate pathogens and pests within host tissues, something contact pesticides cannot do. Systemic agents also tend to be less toxic to other organisms. For example, use of the systemic fungicide penconazole against downy mildew had only minimal effect on the phylloplane flora (Perazzolli et al., 2014). As such, they are less likely to disrupt biocontrol agents. In addition, systemic agents can often be applied at lower dosages. Dosage rates for contact agents tend to be in the range of 1e3 kg/ha, whereas systemic application rates are usually 0.4e0.5 kg/ha. Disease forecasting, the prediction of disease outbreaks based on meteorological data, is particularly useful in timing pesticide application. Where developed and pertinent, disease forecasting can reduce application rates by half (e.g., downy mildew control) (Menesatti et al., 2013). Regrettably, disease forecasting models are expensive to develop and often of localized applicability. In the absence of an appropriate model, scheduling may be based on the phenologic (growth) stage of the vine and the pathogen’s life cycle. The precision possible with phenological models (Fig. 4.2) is well adapted to the spray timing. Early, precise, local indicators of disease development are a useful adjunct to PV. For example, one of the early host responses to Plasmopara viticola infection is the production of stilbene phytoalexins. Detection of its violet-blue leaf autofluorescence could be a predictor of the impending disease severity (Latouche et al., 2015). Development of resistance to broad-spectrum, contact pesticides tends to be slow or nonexistent. Single mutations are unlikely to provide adequate protection against the extensive, nonspecific disruption of membrane structure and enzyme function induced by these pesticides. When the availability of many nonselective fungicides began in the late 1940s, the destructive potential of several marginally important pests was forgotten. Their potential destructiveness reappeared only when the use of selective pesticides began to replace nonselective pesticides (Mur and Branas, 1991). For example, the increased incidence of the omnivorous leafroller in California can be partially attributed to the reduced use of nonselective pesticides

against grape leafhoppers (Flaherty et al., 1982). This has, in the case of organic viticulture, encouraged the use of nonselective pesticides, such as oils (e.g., Stylet Oil), plant poisons (e.g., pyrethroids), or bacterial toxins (e.g., Bt toxin). Regrettably, organic-certified pesticides may be as lethal to biocontrol agents as other pesticides (Biondi et al., 2012). In contrast, selective, systemic agents tend to have highly specific toxic actions. These usually involve precise molecular sites on, for example, a particular mitochondrial enzyme or specific membrane component. Regrettably, this specificity facilitates their neutralization by pathogen mutation. Initially, resistant populations tend to be less viable than their sensitive counterparts, due to a partial impairment of important host functions involved in resistance. However, after prolonged selective pressure, subsequent mutations may restore full biological fitness, such as overexpression of the genes of an alternative metabolic pathway. Other examples of competitively neutral resistance factors may involve mutations regulating ABC (ATPbinding cassette) and MFS (major facilitators superfamily) transport proteins (Del Sorbo, 2000). These have the capacity to translocate fungicides out of the cell before they can produce significant metabolic damage. At this point, terminating application of the pesticide is likely to have minimal effect on the population of resistant strains. Without any debilitating effect on growth, resistant strains can be as ecologically competitive as pesticide-sensitive strains. Both single- and multiple-fungicide resistance is known in Botrytis cinerea (Fig. 4.53), and may exhibit no significant fitness penalties (Ferna´ndez-Ortun˜o et al., 2015). Thus, for long-term effectiveness, selective/systemic chemicals are best limited to use only in situations in which their precise in-tissue toxicity is required (Delp, 1980; Northover, 1987). Rotational use is another means by which the “life span” of systemic pesticides may be extended. By alternating among pesticides that have differing modes of action, the advantage of resistance genes against any one control agent may be delayed, if not completely avoided. Furthermore, absence of sustained pesticide pressure minimizes the likelihood of enhanced ecologic fitness being selected for among resistant strains. Unfortunately, success in retarding the accumulation of resistance using rotation is not guaranteed (Baroffio et al., 2003). Thus, minimal use of selective fungicides, and a greater reliance on biologic, genetic, or environmental management practices, is required. An alternative approach involves a combination of nonselective and selective pesticides. The broadspectrum activity of nonselective agents reduces the pest or pathogen population, thereby reducing the

Disease, pest, and weed management

selective advantage of resistant genes to a selective agent. This approach has been accredited with delaying the emergence of resistance genes in some pathogen populations (Delp, 1980). Surveying resistance in local populations is an important component in assessing the continuing efficacy and value of any pesticide.

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A variant tactic is the joint application of two or more selective pesticides. Theoretically, this should reduce resistance development (but has not be confirmed empirically). For example, if the probability of insensitivity to any one agent were equal to 1  108, the combined likelihood of any organism surviving (possessing or developing simultaneous resistance) both agents

FIGURE 4.53 Evolution of resistance to fungicides in Botrytis cinerea in French vineyards. (A) Mean frequency of a given resistant phenotype in all plots in which this resistance was found. (B) Mean frequency of plots displaying a given resistant phenotype with respect to all tested populations. Phenotypes are abbreviated as follows: specific resistance to dicarboximides (ImiR1), antimicrotubules (BenR1 and BenR2), anilinopyrimidines (AniR1), hydroxyanilides (HydR3), carboxamides or succinate dehydrogenase inhibitors (CarR), and multidrug resistance (MDR1, MDR2, and MDR3). From Walker, A.S., Micoud, A., Re´muson, F., Grosman, J., Gredt, M., Leroux, P., 2013 French vineyards provide information that opens ways for effective resistance management of Botrytis cinerea (grey mould). Pest Manag. Sci. 69, 667e678, reproduced by permission of John Wiley and Sons.

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would be (1  108) (1  108) ¼ 1  1016. If three pesticides were applied together, the likelihood of a pest developing resistance to all three would fall to 1  1024 (effectively nil). Although feasible, the cost of such application argues against it, and its value has as yet to be confirmed empirically. In both practices just noted, it is important that each agent possesses unrelated toxic actions, typically associated with being in different chemical groups (Table 4.13). If the chemicals have similar modes of toxicity, the selective pressures they produce are likely to be equivalent. In this situation, resistance to any member of a group may donate resistance to other members of the groupda phenomenon termed crossresistance. Retarding the development of pesticide resistance can be favored by improved application. This frequently involves the use of improved sprayers (Furness, 2002), combined with more accurate assessment of spray coverage (Furness, 2009). The development of site-specific robotic sprayers should promote appropriate application only where needed (Berenstein and Edan, 2017). This has already been developed for weed control (targeted herbicide application combined with mechanical cutting). Sites where pesticide coverage is inadequate (below the toxic threshold) provide conditions favoring resistance development (selectively favoring the tolerant strains). The incorporation of adjuvants (stickers and/or spreaders) into spray mixtures can also improve the likelihood of agent effectiveness (MacGregor et al., 2003). Spreaders facilitate dispersion over plant surfaces (by reducing surface tension that can disrupt distribution), whereas stickers minimize wash off. Regrettably, stickers also limit redistribution of the agent over leaf and fruit surfaces as they enlarge. Adjuvants may also disrupt one of the preexisting plant defenses against invasiondthe waxy cuticular coating of the epidermis. This is one of the potential problems with all soap/oil mixtures used in pest management. Alternatively, encapsulation of pesticides can facilitate attachment, extend release, and decrease any potential phytotoxicity (Nuruzzaman et al., 2016). This is of particular value with microbially derived pesticides that have had problems of efficacy related to photolability, volatility, or other forms of rapid inactivation. An example is the micelle formulation of geranylorcinol used against B. cinerea (Taborga et al., 2015). Other examples of pesticide formulation, involving nanoscale encapsulation, are noted in Oliveira et al. (2018). One of the inherent problems with any control agent sprayed or dusted is dilution, whether by rain, degradation by light, microbial action, or expansion growth of the host surfaces. Thus, the effective concentration becomes lower, reducing the effective dose. This could

permit the easier, progressive selection of resistant strains. Finally, application of other control measures (sanitation, environmental modification, etc.) can further delay the development of pesticide resistance. Because mutation is largely a random event, and therefore partially dependent on population size, the smaller the pest population, the more slowly resistant mutations are likely to occur and be propagated. None of these techniques will necessarily prevent the eventual development of resistance, but they can significantly delay its occurrence. The delay will depend on factors such as the widespread use of protective measures, the number of growth cycles per year, existing genetic diversity in the pest or pathogen population, the population size, and the facility with which the pest or pathogen is dispersed. Traditionally, control agents have been applied as either liquid sprays or dusts. Both have potential problems, such as loss due to drift and wash off, dilution by rain, contamination of groundwater, and damage to other organisms. Direct injection of systemically translocated compounds into the trunk would counter many of these factors. The application of metalaxyl against downy mildew by this method is as effective as spraying (Du¨ker and Kubiak, 2009). Its disadvantage is the complexity of injecting individual vines. Recently, a new and novel class of control agents has entered the market. They are termed effectors, and they activate systemic acquired resistance. They induce metabolic pathways equivalent to those initiated by various stress factors. In the case of disease, application of effectors preadapts the vine before invasion begins. Examples are b-aminobutyric acid, which enhances vine resistance to downy mildew (Cohen, 2002; Harm et al., 2011); benzothiadiazole (BTH) and acibenzolar-Smethyl (salicylic acid analogs), which are effective against powdery mildew (Campbell and Latorre, 2004), bunch rot (Iriti et al., 2004), and downy mildew (Harm et al., 2011); and jasmonic acid, useful in limiting the damage caused by Pacific spider mites (Tetranychus pacificus) and phylloxera (Omer et al., 2000). Some fungicides also have part of their effectiveness due to activating plant defenses (Belle´e et al., 2018). Methyl jasmonic acid is a naturally synthesized, intracellular regulator (effector) mediating several diverse defense responses in plants, as does salicylic acid, but via another metabolic pathway. Methyl jasmonate has also been used to enhance the concentration of resveratrol and related stilbene phytoalexins (Vezzulli et al., 2007). The combined action of methyl jasmonate and benzothiadiazole can also increase the phenolic content and color of grapes, as well as enhancing the concentrations of esters, higher alcohols, and terpenes found in wines (Ruiz-Garcı´a et al., 2014). For maximal benefit, they are

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TABLE 4.13

Examples of fungicides used in controlling grape diseases, arranged according to chemical family.

Chemical group

Activity groupa

Common and trade names

Anilinopyrimidine

I

Pyrimethanil (Scala); other examples, cyprodinil (Vangard, Chorus)

Benzimidazole

A

Carbendazim (Delsene); other examples, benomyl (Benlate), triophanate methyl (Topsin-M)

Benzothiadiazole

*

Acibenzolar-S-methyl (Actigard)

Dicarboximide

B

Iprodione (Rovral); other examples, vinclozolin (Ronilan), chlozoline (Serinal), procymidone (Sumisclex)

Dithiocarbamate

Y

Maneb (Maneb); other examples, zineb (Zineb), ferbam (Carbamate), maneb + Zn (Mancozeb, Dithane)

DMI-pyrimidine

C

Fenarimol (Rubigan)

DMI-triazole

C

Myclobutanil (Rally, Nova); other examples, difenoconazole (Score), hexaconazole (Anvil), tebuconazole (Elite), triadimefon (Bayleton)

Hydroxyanilide

J

Fenhexamid (Elevate, Teldor)

Phenylamide

D

Mefenoxam (Metalaxyl, Ridomil); other examples, furalaxyl (Fongarid)

Phosphonate

Y

Fosetyl-aluminum (Aliette); other example, phosphorous acid

Phenoxy quinoline

M

Quinoxyfen (Legend)

Phthalimide

Y

Captan (Captan, Merpan); other examples, chlorothalonil (Bravo)

Strobilurin

K

Trifloxystrobin (Flint); other examples, azoxystrobin (Abound, Amistar), kresoxim-methyl (Stroby), pyraclostrobin (Cabrio)

DMI, demethylation-inhibiting. a Grouping of fungicides by their similar mode of action; *Activators of inherent plant defenses, instead of a fungicidal group.

Structure (principal example)

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best combined with a control agent against the main pathogen or pest. Biological control Synthetic and organic pesticides are likely to remain the main arsenal of agents against pests and diseases into the foreseeable future. Nonetheless, increasing emphasis on biological control is environmentally beneficial but can also indirectly act in delaying development of resistance to pesticides (Englert and Maixner, 1988). For efficacy, it is often necessary to maintain a broad diversity of plants in the vicinity (Thomson and Hoffmann, 2009). Control agent populations often cycle considerably throughout the year (Bernard et al., 2006). To assist in maintaining sufficient numbers of biological control agents, crops of flowering plants (Scarratt et al., 2007) are often grown between rows to supply alternative food sources. Nonetheless, this may not be sufficient for meaningful control (English-Loeb et al., 2003). Thus, a supply of alternate hosts may be encouraged in the vicinity, both as supplemental food sources and as sites for overwintering. In addition, the release of commercially reared competitive/parasitic species may be required to reach effective control populations. By establishing themselves on the host, competitors may restrict the establishment of potential pest species (e.g., exclusion of the Pacific spider mite by the Willamette spider mite) (Karban et al., 1997). Various fungi, yeasts, and bacteria can also displace fungal pathogens from invasion sites. Although insects are susceptible to many bacterial, fungal, and viral pathogens, few have shown promise in becoming effective biocontrol agents. A few exceptions include the granulosis virus, against the western grape leaf skeletonizer (Harrisina brillians) (Stern and Federici, 1990), and Beauveria bassiana (GHA strain), against western flower thrips. Another example is Bt toxin, produced by Bacillus thuringiensis. The toxin is active against several lepidopteran pests, such as the omnivorous leafroller (Platynota stultana) and the grape leaffolder (Desmia funeralis). Bt toxin produces “holes” in the intestinal lining of the insect, permitting intestinal bacteria access to the hemolymph (“blood”). This can induce septicemia (Broderick et al., 2006). In some regions, where Bt toxin has been extensively used, signs of resistance have appeared. This may be countered by the use of a subgroup of Bt toxins, in the Cry1Ca complex. They are toxic to lepidopteran insects currently resistant to standard Bt toxin (Avisar et al., 2009). An unsuspected benefit of its widespread use in crops such as corn and cotton has been area-wide suppression of pests in varieties and crops not possessing the Bt toxin (Naranjo, 2011). Regrettably, the development of resistance, and some expression difficulties in

transgenetic varieties (De Rocher et al., 1998), may make the expense of engineering cultivars to produce Bt toxins of short-term value. The same fate might also befall genetic engineering of grapevines possessing arthropod resistance with the Galanthus nivalis agglutinin (GNA) gene, with the insecticidal protein from Pseudomonas chlororaphis, or with fungal resistance supplied by endochitinase genes from Trichoderma harzianum (Lorito et al., 2001). Even natural biocontrol agents, such as the parasitic wasp (Microctonus hyperodae), are not insensitive to inactivation by natural selection (Pennisi, 2017). Pheromones are another agent in the arsenal of biocontrol agents available for insect management. Pheromones are species-specific, airborne hormones produced by insects to locate mates. Thus, they can selectively attract insects to pesticide traps, or induce mating disorientation when applied throughout a vineyard. Other attractants that could selectively attract pests to traps are grape volatiles. These may be used by fertile females to locate oviposition sites (Tasin et al., 2005). Alternative techniques for effective mating disruption include the release of large numbers of artificially reared, but sterile, individuals during the mating season, or the use of vibrational signals used by insects to attract mates (Polajnar et al., 2015, Plate 4.11). One regrettable consequence of reliance on mating disruption has been the resurgence of minor pests serendipitously controlled by other control agents (Gallardo et al., 2016). Although the initial cost of grafting to resistant rootstocks is considerable, its beneficial effects can be long term. In some cases, grafting may be the only effective means of limiting damage. This strategy was first used

PLATE 4.11 The transducer used to generate a select vibrational signal to wires (or posts) to disrupt the mating of the leafhopper, Scaphoideus titanus. Photo courtesy of Valerio Mazzoni.

Disease, pest, and weed management

in the late 1800s to control the phylloxera infestation in Europe. Resistant or tolerant rootstocks are also one the principal means by which the damage of several nematodes, viruses, and soil-based toxicities is limited. Little investigated, however, is the role of grapevine rootstocks in influencing resistance to leaf and fruit pathogens (Erb et al., 2009). The biological control of fungal pathogens is less well developed. This partially reflects the growth of fungal pathogens within plants, away from exposure to parasites or predators. Nevertheless, inoculation of leaf surfaces with epiphytic microbes may prevent the germination and subsequent penetration of fungal pathogens. The phylloplane flora may inhibit pathogens by competing for organic nutrients required for germination, activating systemic host defenses, inciting direct mycoparasitism, or provoking antibiosis. A commercially successful example involves Trichoderma harzianum (Trichodex). It and related agents are especially effective against stem-invading fungi causing Eutypa, esca, and Petri diseases (Hunt, 1999), as well as enhancing resistance to Plasmopara viticola (Perazzolli et al., 2012). Alone, though, it is inadequate to control Botrytis cinerea, and requires rotation with conventional fungicides, or combination with Rhodotorula glutinis and Bacillus megaterium (Greygold). Interestingly, combined with iprodione, the efficacy of both control agents is enhanced. Another promising antifungal bacterium is Lysobacter capsici AZ78, acting against downy mildew. Its efficacy is improved when combined with copper (Puopolo et al., 2014). For powdery mildew, where the majority of the fungus remains on the host’s surface, use of the mycoparasite Ampelomyces quisqualis (AQ10) has occasionally proven effective (Falk et al., 1995). Mycoviruses may be effective against fungal pathogens, but the only one currently used is an RNA virus (BCRV1) again Botrytis cinerea (Yu et al., 2015). Another new and fascinating approach involves endophytesdmicrobes that grow and multiply asymptomatically within plant tissues. They appear to be symbiotic, improving plant growth (e.g., producing IAA), activating plant defenses, producing antibiotics (Barrow et al., 2008; Shoresh et al., 2010), or supplying nutrients (e.g., enhancing potassium and iron solubilization) (Rodriguez and Redman, 2008; West et al., 2009). The most wellstudied examples are vesicularearbuscular mycorrhizal fungi. Others under investigation include bacterial symbionts, although some may be latent pathogens. Often endophytes occur in sufficiently low numbers to avoid visual observation, requiring culturing or detection by their distinctive DNA or fatty acid contents. When observed microscopically within plant tissues, endophytes are typically found in the xylem (as with most endophytic bacteria), or the root cortex (as with Trichoderma). Their presence and distribution often vary

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considerably throughout the season, some occurring systemically, whereas others are limited to the presumed point of entry. The geographic diversity in these organisms is marked, but their effects seem similar. An unexpected observation has been that those found in domesticated grapevines are more likely to be more beneficial than those detected in wild vines (Campisano et al., 2015). Several endophytes can also desensitize the host to a variety of environmental stresses, such as salinity, heavy metals, drought, or heat. This ability has led some researchers to advocate their use in lieu of more expensive and controversial genetic breeding procedures (Barrow et al., 2008; Rodriguez et al., 2008). This could be expanded to include adjustment of the rhizoflora in and around roots (Pineda et al., 2010), and the phylloplane flora of aerial plant surfaces. Even strains of Pseudomonas carrying the biocontrol genes ph1D and hchAB have been correlated with improved photosynthesis and grape aroma (Svercel et al., 2010). These strains were isolated from vineyards under long-term viticulture. This could be an example of where long-term monoculture can have beneficial, rather than the more usual undesirable, effects (e.g., reduced biodiversity and diminished soil productivity). Other examples of potentially useful endophytic bacteria include strains of Pseudomonas syringae and P. aeruginosa that produce syringomycin E and rhamnolipids. Their combined action is effective against a range of fungal pathogens (Takemoto et al., 2010). Endophytic strains of Pseudomonas fluorescens and Bacillus subtilis have been shown to reduce the size of crown gall in infected plants (Eastwell et al., 2006). Most bacterial endophytes appear to be neutral to beneficial, with the exception of latent strains of Rhizobium (Agrobacterium) vitis. Most are fastidious, xyleminhabiting strains of Pseudomonas and Enterobacter, but can also include Azotobacter, Azospirillum, Acetobacter, and Burkholderia species. They tend to be distinct from those found in the rhizosphere (Bell et al., 1995), but some, such as Burkholderia, can colonize both the rhizosphere and internal tissues (Compant et al., 2005). Bacterial entrance occurs primarily via pruning wounds (West et al., 2009). At least some fungal endophytes may be latent or nondamaging strains of pathogens, for example, Phaeomoniella chlamydospora and Phaeoacremonium spp. (Halleen et al., 2003). Other fungal endophytes, though, parasitize pathogens, for example, Acremonium byssoides and Alternaria alternata on Plasmopara viticola (Burruano et al., 2008; Musetti et al., 2006). Mycorrhizae have often been associated with enhanced resistance to nematodes, such as Xiphinema index (Hao et al., 2012), and to other pathogens (Jung et al., 2012), and even signaling adjacent plants of

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potential attack (Babikova et al., 2013). Mycorrhizal associations can activate defense mechanisms (e.g., jasmonic and salicylic acid-dependent genes), both locally and systemically (Cameron et al., 2013). One of the more challenging aspects of using biocontrol can be the special requirements for storing and application of the agent. Because spores and biological toxins often have relatively short half-lives, they need to be kept under cool, dry conditions. In addition, they are often quickly inactivated on exposure to solar UV radiation. Thus, if applied to aerial parts of the vine, they are best sprayed in the evening to extend their effective period. These limitations have retarded grower acceptance (Hofstein et al., 1996). Progress with microencapsulation may extend the effective life span of control agents. Combination with other control agents may also be beneficial, as shown with copper and Trichoderma viride (Vincekovic et al., 2016). One of the more intriguing examples of biological control involves cross-protection. This is a phenomenon in which virally infected cells are immune to subsequent infection by a related or more virulent strain. Transforming vines with viral coat or dispersal genes can potentially have the same effect (Beachy et al., 1990; Krastanova et al., 1995). Another component in many biological control schemes is augmentation of the soil’s organic content, by applying manure, compost, or a mulch (Hoitink et al., 2002). It has its greatest value when dealing with root pests and pathogens. The effect is thought to arise by suppressing the growth and survival of the pathogenic agent, by enhancing the competitive/degradative activity of the soil’s microflora and fauna. This applies especially to the region just around the rhizosphere. Thus, some of the benefits may also accrue from improved vine nutrition. This could involve better access to soil nutrients (directly or via mycorrhizal association) and augmentation of host defenses. Similar in concept is the application of a solution of organic nutrients (e.g., liquid manure). Augmentation of the phylloplane flora may both directly and indirectly suppress the germination or growth of leaf and fruit pathogens (Sackenheim et al., 1994). Conversely, they may also complicate disease control, by degrading fungicides (Salunkhe et al., 2015). Biological control has generally been assumed to be safer to humans and the environment than chemical control. Risk assessment concerning nontarget organisms has usually supported this view, but improved methods of use may limit the chance of exceptions (Barratt et al., 2010). For example, application of fungal biocontrol agents (e.g., Trichoderma and Gliocladium) may provoke allergic respiratory problems and a range of cellular toxic effects in humans (see Brimmer and Borland, 2003). In other instances, some agents have shown

action against beneficial mycorrhizal associations, disrupted plant metabolism, or induced energy expenditure in activating unnecessary systemic plant defenses (creating a metabolic drain). Environmental modification Modifying the microclimate around plants has long been known to potentially minimize disease and pest incidence. By improving the light and air exposure around grapevines, canopy management can increase the toughness and thickness of the epidermis and its cuticular covering. An open canopy structure also facilitates the rapid drying of fruit and foliage surfaces. This reduces the time available for fungal penetration, and may limit or inhibit spore production. Furthermore, an exposed canopy enables more efficient application of pesticides (synthetic or organic). Berry exposure can be enhanced by applying gibberellic acid. It promotes cluster stem elongation and separation of the fruit (Plate 3.9). Reduced berry compactness also favors production and retention of a typical epicuticular wax coating (Fig. 4.54). However, the effect of gibberellic acid on grape composition is complex, and appears to be cultivar specific (Teszla´k et al., 2005). For example, it can affect grape coloration and phenolic and mineral content. In addition, it can increase the formation of o-aminoacetophenone (Christoph et al., 1998; Pour Nikfardjam et al., 2005b)da compound frequently associated with the development of an “untypical aged flavor” in wine. By suppressing the action of IAA oxidase, gibberellic acid can increase auxin content. This appears to favor o-aminoacetophenone synthesis (Pour Nikfardjam et al., 2005a). Less compact clusters can also be achieved with minimal pruning and early leaf removal around the inflorescence. The improved air exposure, and its drying effects, probably explains the reduced incidence of Botrytis bunch rot and infestation with Lobesia botrana. Prebloom application of the growth regulator prohexadionee calcium to the flower cluster is another method of improving fruit exposure. Although the growth regulator reduces yield, berry size, and weight, as well as anthocyanin content, flavanol and flavonol concentrations are enhanced. The wines may also show a more aromatic (fruity/floral) aspect and astringent/bitter attributes (Avizcuri-Inac et al., 2013). Fruit exposure may also be enhanced by basal leaf removal. The technique has been so successful in reducing dependence on fungicidal sprays that several wineries have written the practice into their contracts with growers (Stapleton et al., 1990). Basal leaf removal also eliminates most first-generation nymphs of the grape leafhopper (Erythroneura elegantula) (Stapleton et al., 1990). This can improve subsequent biological control by Anagrus epos, a parasitic wasp. It provides more

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(A)

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(B)

FIGURE 4.54 Scanning electron micrograph of the epicuticular wax of mature berries. (A) Typical platelet structure of noncontact areas. (B) Shallow depression (D) and pore (P) typical of contact area between adjacent berries. From Marois, J.J., Nelson, J.K., Morrison, J.C., Lile, L.S., Bledsoe, A.M., 1986. The influence of berry contact within grape clusters on the development of Botrytis cinerea and epicuticular wax. Am. J. Enol. Vitic. 37, 293e295, reproduced by permission.

time for wasp populations to increase and spread to grapevines from overwintering sites on wild blackberries and other plants. Balanced plant nutrition generally favors disease and pest resistance, by promoting optimal development of anatomical and physiological defenses. Nutrient excess or deficiency can have the inverse effect. For example, high nitrogen levels suppress the synthesis of a major group of grape antifungal compounds, the phytoalexins (Bavaresco and Eibach, 1987). They also favor cell elongation, associated with weaker walls, as well as promoting vigorous growth, dense foliage, and the development of high innercanopy humidity. With adequate irrigation, the consequences of nematode root damage are often mitigated. Adequate irrigation also avoids the serious consequences of water deficit. Conversely, excessive irrigation can favor disease development by promoting luxurious canopy development and increasing berry cluster compactness. This is particularly important for cultivars that inherently produce compact clusters, notably Zinfandel and Chenin blanc. Weed control usually reduces disease incidence. This may result from the removal of alternate hosts, on which pests and disease-causing agents may survive and propagate. For example, dandelions and plantain are often carriers of tomato and tobacco ringspot viruses, and Bermuda grass is a reservoir for sharpshooter leafhoppersdthe primary vectors of Pierce’s disease. Conversely, cover crops are generally viewed as beneficial, supporting populations of biocontrol agents (Vogelweith and Thie´ry, 2017). Thus, cover crop selection needs to be chosen carefully, relative to

pest and disease issues, as well as other local viticultural and economic constraints. Soil tillage can occasionally be beneficial in disease control. For example, burial of the survival stages of Botrytis cinerea (sclerotia and infected tissue) promotes their degradation. In addition, the emergence of grape root borers (Vitacea polistiformis) is restricted by burial of their pupae (All et al., 1985). Although environmental modification can limit the severity of some pathogens, it can enhance other problems. For example, soil acidification, used in the control of Texas root rot (Phymatotrichum omnivorum), has increased the incidence of phosphorus deficiency in Arizona vineyards (Dutt et al., 1986). The elimination of weeds may inadvertently limit the effectiveness of some forms of pest biological control (removing survival sites for pest predators and parasites). The alternative host of one pest may be the reservoir of predators of another. A novel and new approach to disease control involves thermal treatment of the vines (with tractor-mounted heaters). It apparently can be effective in controlling powdery mildew. Genetic control Improved disease resistance is one of the major goals of grapevine breeding. It was first seriously investigated as an alternative to grafting in phylloxera control. Subsequently, rootstock breeding efforts have focused on developing rootstocks possessing improved lime, drought, salt, virus, and nematode resistance. Breeding rootstocks is easier because disruption of the genetic traits that regulate grape flavor is not involved. In addition, a new name for the rootstock has none of the

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marketing disadvantage it has for the scion. Nonetheless, work on developing new scions with significantly improved pest and disease resistance has continued (e.g., Bronner by Norbert Becker, Germany; Artaban, Flore´al, Vidoc, and Voltis by the INRA, France; Divico and Divona by Agroscope, Switzerland). These appear to possess durable resistance to several damaging grapevine fungal diseases. Regrettably, enhanced resistance may aggravate haze development (due to higher concentrations of pathogenesis-related [PR] proteins in the fruit), resulting in reduced aging potential (due to the lower retention of tannins), similar to French-American hybrid wines. However, for organic and biodynamic wines, such varieties have a distinct advantage. By reducing disease control costs, prices for organic wines may approach those of conventionally produced wines, increasing their market acceptance. Also, those preferring organic wines may be more interested in the environmental benefits of their production than in the varietal origin or aging potential. Although sources of disease resistance may still exist in remnant populations of wild vinifera and ancient cultivars (Toffolatti et al., 2016; Hoffmann et al., 2008), most potentially useful resistance genes occur in other Vitis species. For example, Vitis rupestris and V. riparia were the primary sources of powder and downy mildew resistance (French-American hybrids) and V. rupestris is a source of resistance to black rot. Other sources of useful resistance exist in V. amurensis and V. rotundifolia. Modern genetic approaches have the best chance of incorporating durable multidisease resistance, without modifying enologically critical varietal traits (see Grapevine improvement, Chapter 3). As noted earlier, plants possess broad, nonspecific defense mechanisms against pest/pathogen attack (in addition to preexisting structural and chemical defenses). Systemic acquired resistance often involves the production of phytoalexins (a range of stilbene phenolics) as well as terpenoids, glycosteroids, and alkaloids with antimicrobial properties. In addition, plants may synthesize PR proteins (e.g., chitinases and thaumatinlike proteins). As part of the induced cascade of events, a salicylic acidedependent signaling pathway is activated (Jung et al., 2009). Pathogens that overcome the primary defenses may prompt a second line of defense. This depends on specialized receptor proteins. These activate a cascade of reactions involving hydrogen peroxide and nitric oxide. When rapid and intense, localized tissue death (apoptosis) results in what is termed a hypersensitive response. If effective, it kills the invading pathogen (Tameling and Takken, 2008)dincluding infected and adjacent cells. Traditionally, most research has focused on R genes. They act in a dominant manner, donating immunity to

pathogens producing a particular effector. This generates strong selective pressure on the pathogen to mutate, negating the resistance. Thus, breeder and pathogen were often interlocked in a cat-and-mouse game of the breeder adding new R-gene alleles and the pathogen mutating to neutralize their effect. Examples of R-type genes (derived from Vitis rotundifolia) include RPV1 (resistance to Plasmopara viticola 1) and RUN1 (resistance to Uncinula necator 1). Another example, derived from V. vinifera, is REN1 (resistance to U. necator 1). Up to seven different R genes active against powdery mildew have been discovered in different North American and Asian Vitis spp. (Qui et al., 2015). Of R-type genes, REN4, from Vitis romanetti is particularly interesting, due to its unique mode of action. It both is non-race specific and acts prior to haustorial production, suppressing spore germination (Ramming et al., 2011). An alternative, and potentially longer-term, approach to genetic-based resistance may involve components of non-host resistance. These are called S (susceptibility) genes, due to at least one of their influences being pathogen/host compatibility (van Schie and Takken, 2014). Loss of gene function may confer resilient resistance. Such genes may be widely conserved across the plant kingdom. For example, there are up to 17 VvMLO (Vitis vinifera Mildew Locus O) genes known in grapevines (Winterhagen et al., 2008). Inactivation (knockdown) of three VvMOL genes produces a marked reduction in susceptibility to Uncinula necator (Pessina et al., 2016). The gene inactivation potential provided by CRISPR, without otherwise modifying the cultivar’s genome, is particularly interesting. By avoiding the insertion of foreign DNA, involved in current forms of genetic engineering, existing concerns about genetically modified organisms (GMOs) should be avoided. In addition, as the cultivar possesses only a modified (mutated) form of a single, already existing gene, its original name need not change. That would avoid a major disadvantage of other forms of genetic modificationdcultivar name change. In most instances, complete resistance (immunity) is preferred. However, even slowing the rate of disease spread (tolerance) may provide adequate protection in most years, especially where several cycles of pathogen/pest reproduction are required for expression of severe damage. Regrettably, tolerance to one pest can occasionally favor infection by another (e.g., tolerance to Xiphinema index increases the likelihood of grapevine fanleaf virus [GFLV] transmission). In addition, tolerance to a systemic pathogen (by masking presence) could favor the likelihood of its spread by grafting or other forms of mechanical transmission. The increasing availability of molecular markers, such as simple sequence repeats and single-nucleotide

249

Eradication and sanitation In most situations, the eradication of established pathogens is impossible, due to their survival on alternative hosts (e.g., weeds and native plants), or the presence of long-lived dormant stages. Eradication has greater potential for success with newly introduced, exotic pathogens. In this situation, it is normal practice to destroy all the vines in affected areas. Clearly, this induces extreme hardship for the owners involved, and requires at least financial compensation from the government. Consequently, studies are in

100

Native Vitis Hybrid

75

V. vinifera

50

25

0 V. ae s V. tiva ci lis n V. ere rip a a Ba ria c C o h D a n M e C n c e oir ar h l l éc a u o r ha n l F ac oc C ab h C fra ab n Sa c u M Pi e v no rlo tn t oi r

polymorphisms, has begun to ease mapping of the locations of resistance genes (Fischer et al., 2004; Di Gaspero et al., 2007). This potentially facilitates gene amplification or inactivation, or isolation and subsequent insertion via transduction. For example, loci associated with powdery mildew resistance are positioned on chromosomes 15 and 18, whereas resistance factors for downy mildew have been identified on chromosome 18. This information is useful in a process termed marker-assisted breeding (Eibach et al., 2007). The known location of similar genes in other plants can aid the identification and localization of their equivalents in grapevines. This opens the possibility of translocating useful genes without crossing broad generic boundariesda frequent complaint leveled against most GMOs. The difference is termed cisgenic vs. transgenic engineering (Schouten et al., 2006). The VVTL-1 gene from Chardonnay has been isolated, modified to become constitutive, and inserted into Thompson Seedless (Dhekney et al., 2011). It expresses significantly enhanced disease resistance to several serious fungal grapevine pathogens. Simply inserting the protein-coding component of an S- or R-resistance or R-resistance gene is unlikely to be adequate for activity. It requires the simultaneous insertion of appropriate promoter, terminator, and controller regions for proper function. Thankfully, most resistance loci possess several related genes/alleles, as well as their control regions (see Dry et al., 2010), which may enhance durable resistance. Finally, a caveat for all such research is the possibility that improving a particular property may unexpectedly negatively affect another. An example is the enhanced disease resistance of French-American hybrids. Their wines tend to be low in phenolic content, limiting aging potential. Most grape (as well as added) tannins are lost during fermentation as they bond and precipitate with PR proteins (Fig. 4.55). Although limited aging potential can be viewed as negative, it improves early drinkability by limiting bitterness and astringency. Because of long-term efficacy of most forms of genetic (or chemical) defenses is never certain, it is advisable to incorporate a range of control measures into any management system.

% Added Tannin Recovered in Pellet

Disease, pest, and weed management

FIGURE 4.55 Tannin precipitated from varietal wines after an addition of 150 mg/L seed tannins. Error bars represent standard deviations for winemaking replicates (n ¼ 3). Reprinted with permission from Springer, L.F., Chen, L.A., Stahlecker, A.C., Cousins, P., Sacks, G.L., 2016 Relationship of soluble grape-derived proteins to condensed tannin extractability during red wine fermentation. J. Agric. Food Chem. 64, 8191e8199, Copyright 2016. American Chemical Society.

progress to assess the feasibility of reducing the impact of eradication. For above-ground pathogens, removing the vine down to the crown is being investigated. The production of new shoots from long dormant buds permits the more rapid reestablishment of the vineyard. The upper parts of the vines are either buried, with a mulch layer added to encourage rapid decomposition of infected material (Sosnowski et al., 2009), or burned. Seed propagation is frequently used to eliminate (eradicate) host-specific systemic pathogens from annual crops. However, this is inapplicable with grapevinesd disrupting the combination of genetic traits that gives each cultivar its unique attributes (see Chapter 2). Thus, except where disease-free individuals can be found, the elimination of systemic pathogens involves thermotherapy, meristem culture, or a combination of both. Thermotherapy can vary from placing young rooted shoots at 35
Ce38
C for 2e3 months to dormant canes being given higher temperature treatment, but for shorter periods. The treatment is effective against several viruses, such as the grapevine fanleaf, tomato ringspot, and fleck viruses, as well as the leafroll agent (typically associated with closteroviruses). Hot-water immersion is also appropriate and preferable for some pathogensdthe specifics depending on the pathogen and its location (on or within the vine). Hot-water treatment can be effective in eliminating pathogens such as the bacteria Xylella fastidiosa, Xanthomonas ampelina, and Rhizobium (Agrobacterium) vitis; the phytoplasmas associated with grapevine yellows diseases (e.g., flavescence dore´e, bois noir); the fungus Phytophthora cinnamomi and those causing Petri disease; and root nematodes,

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such as Xiphinema index and Meloidogyne spp. Procedures and recommended precautions for hot-water treatment are provided in Hamilton (1997), Waite et al. (2001), and Waite (2005). Because hot-water treatment stresses vine tissues, it is recommended that treated cuttings be protected from adverse weather conditions, usually in a greenhouse or equivalent, for several months prior to planting out in field nurseries. Following treatment, the cutting may be dipped in a combination of biocontrol agents to reduce the incidence of reinfection (Graham, 2007). Treating vines infected by several systemic pathogens may require a combination of procedures. Thermotherapy may be combined with meristem culture to enhance elimination of systemic pathogens. However, meristem culture can give growers apprehension. The treatment can modify the morphological and physiological traits of propagated vines. Expression of juvenile traits, such as spiral phyllotaxy, reduction in tendril production, more jagged and pubescent leaves, stem coloration, and reduced fertility are fairly typical. The origin of these juvenile traits probably involves tissue dedifferentiation. This occurs when embryonic tissues are regenerated via micropropagation (Hackett, 1987). Nonetheless, these traits usually disappear as vines mature and/or are cane propagated (Grenan, 1994; Deloire et al., 1995). A novel temperature-based treatment is cryotherapy (Bettoni et al., 2016). It involves a brief exposure of shoot tips to liquid nitrogen. It has the potential to eradicate a range of pathogens, notably systemic viruses and bacteria. It has shown promise in eliminating GVA (grapevine virus A) (Wang et al., 2003) and three variants of GLRaV (Pathirana et al., 2015). For systemic pathogens not eliminated by temperature treatments, but that do not invade meristematic tissues, elimination is often possible by meristem culturing. Propagation of small shoot-tip fragments has been used to eliminate viruses such as GFLV and GLRaV-1; the virus-like agents of stem pitting, corky bark, and leafroll; the viroid of yellow speckle; and the bacterium Rhizobium (Agrobacterium) vitis. Once freed of systemic pathogens, vines usually remain disease-free, if grafted to disease-free rootstock and planted in pathogen-free environments. Although most serious grapevine viruses are not insect transmitted, they may be nematode transmitted. Thus, use of resistant rootstock is advisable where the soil is infested with root-feeding nematodes. Alternatives are leaving the land fallow for up to 10 years, or fumigating. Sanitation and hygiene may not eliminate disease or pest problems, but they may reduce their severity by destroying resting stages or removing survival sites.

Quarantine Most, if not all, wine-producing countries possess laws regulating the importation of grapevines. Some of the best examples illustrating the need for such regulations are those involving grapevine pathogens. Two of the major grapevine diseases in Europe (downy and powdery mildew) were imported unknowingly from North America in the 19th century. The phylloxera root louse was also accidentally introduced, probably on rooted cuttings. Several viral and virus-like agents are now globally widespread, presumably via the importation of asymptomatic, but infected, rootstocks or scions. Thankfully, some other potentially devastating diseases are as yet not global. For example, Pierce’s disease is still largely confined to southeastern North America and Central America. However, it is causing severe problems in parts of California, due to spread by the glassy-winged sharpshooter, and has been noted in some parts of Europe. Although phylloxera is present in most wine-producing regions, it has not spread throughout all parts of the countries in which it occurs. Thus, limiting grapevine movement within regions can still have a significant impact on the spread of those pathogens with limited natural means of dispersal. Disinfection of footwear and machinery is another component of limiting spread, both within and between vineyards. Because it is difficult to detect the presence of some pests and disease-causing agents, only dormant canes are permitted entrance into most jurisdictions. This especially impedes the introduction of root and foliar pathogens. Nonetheless, fungal spores, insect eggs, and other minute dispersal agents may go undetected. Consequently, imported canes are typically quarantined for several years, until they are determined to be free, or freed, of known pathogens. Most pests, as well as fungal and bacterial pathogens, express their presence during the quarantine period. For latent viruses and viroids, detection usually requires grafting or mechanical transmission to indicator plants, a process termed indexing. Less time-consuming analytic techniques, such as ELISA (enzyme-linked immunosorbent assay) (Clark and Adams, 1977), cDNA (Koenig et al., 1988), or polymerase chain reaction (PCR) probes (Constable et al., 2012), can both facilitate and speed detection. Nonetheless, because no technique is 100% effective, repeat sampling is advisable. For example, it may take up to a year after infection for some viruses to become reliably detectable. Even with these caveats, modern assessment techniques permit the release of imported stock earlier than previously. Unfortunately, quarantining is both expensive and an irritant to the importer. A possible solution is the

Disease, pest, and weed management

development of encapsulated somatic embryos (Das et al., 2006). Being produced under sterile conditions, and from disease-free tissue, they could be shipped directly to propagation facilities in the host country. Theoretically, this should completely avoid the potential for pathogen introduction along with cuttings.

Consequences of pathogenesis for fruit quality The negative influence of pests and diseases is obvious in symptoms such as blighting, distortion, shriveling, decay, and tissue destruction. More subtle effects can involve reduced vine vigor and berry size, as well as delayed/inhibited ripening. Sequelae such as reduced root growth, poor grafting success, decreased photosynthesis, and increased incidence of bird damage on weak vines (Schroth et al., 1988) are easily missed or misinterpreted. In some instances, detection of infection is impeded by minimal symptoms, or the absence of uninfected individuals for comparison. This was initially the case with several viral and viroid diseases. Most pest and disease research is concerned with understanding the pathogenic state and how its effects can be minimized. However, in making practical decisions on disease control, it is important to know the effects of disease on vine health and yield, and grape and winemaking quality, and the potential secondary negative influences provoked by particular control measures. All pests and disease agents disrupt vine physiology to some degree and, therefore, potentially influence fruit yield and quality. For example, the sensory quality of wine was detectably reduced from vineyards possessing as little as 5% esca-affected vines (Lorrain et al., 2012). However, agents that attack berries clearly have the greatest impact on fruit quality. These include three of the major grapevine scourgesdBotrytis cinerea, Plasmopara viticola, and Uncinula necator. Grapevine viruses and viroids, being systemic, can both directly and indirectly affect berry characteristics. For example, leafroll-associated viruses retard berry ripening and suppress
Brix and fruit coloration (red cultivars). Wine quality is also reduced, with fruit aromas diminished and earthy flavors enhanced (Alabi et al., 2016). Surprisingly, only about 60% of tasters were able to distinguish between wines made from infected and those made from healthy vines. Insect pests can cause fruit discoloration and malformation, as well as creating lesions favoring invasion by pathogens and saprophytes as well as secondary pests. Of fruit-infecting fungi, the effects of Botrytis cinerea have been the most extensively studied. Under special environmental conditions, infection produces a “noble” rot, yielding superb wines (see Chapter 9). Typically, however, the fungus produces a bunch (“ignoble”) rot. Subsequent invasion by acetic acid bacteria probably explains the high levels of fixed and volatile acidity in the fruit.

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Under moist conditions, secondary invaders, such as Penicillium and Aspergillus, contribute additional offflavors, such as geosmin, 2-methylisoborneol, 1-octen3-one, and 1-octen-3-ol (La Guerche et al., 2006). These saprophytes can also produce mycotoxins. Examples are isofumigaclavine, festuclavine, and roquefortine, produced by Penicillium spp. (Moller et al., 1997); OTA, primarily derived from Aspergillus carbonarius (O’Brien and Dietrich, 2005); fumonisins, synthesized by Aspergillus niger (Mogensen et al., 2010); and trichothecenes, generated by Trichothecium roseum (Schwenk et al., 1989). In sufficient amounts, they can be mammalian cytotoxins and carcinogens. The presence of secondary saprophytes, such as Mucor species, can disrupt the activity of lactic acid bacteria during malolactic fermentation (San Roma´o and Silva Alema´o, 1986). B. cinerea produces several phytotoxins, but none known to affect humans (Krogh and Carlton, 1982). Their presence may be the source of the disrupted yeast growth noted by Blakeman (1980). In addition to increased fixed and volatile acidity, fungi associated with bunch rots reduce nitrogen and sugar contents. This can create difficulties during fermentation. Large accumulations of b-glucans, synthesized by B. cinerea, can create clarification problems. More than a minimal level of bunch rot generally excludes their use in making red wines, if for no other reason than the oxidation of anthocyanins and other grape phenolics by fungal laccases. Cladosporium bunch rot also reduces the quality (color and flavor) of wines produced from affected red grapes (Bricen˜o et al., 2009). Botrytis infection can also increase problems associated with protein haze (Girbau et al., 2004). This appears to result from the enhanced production of thaumatin-like (PR) proteins by infected tissues. The effects of sour rot on wine quality have been studied by Barata et al. (2008). Sour rot is the result of microbial invasion of the fruit by a diverse set of agents, notably following feeding by insects and birds. Other than the origin of a sweet, honey-like note, generated by marked increases in ethyl phenylacetate and phenylacetic acid, and reduction in fatty acid and their esters, it is uncertain what specific feature(s) triggers consumer rejection. Wines made from grapes infected by powdery mildew tend to be of higher pH and phenol contents than otherwise. There may also be a bitterish aspect (Ough and Berg, 1979), and other flavor modifications (Fig. 4.56). A reduced anthocyanin content appears to be due to physiologic disruption during grape development (Amati et al., 1996). These consequences are augmented with increased skin contact. This may involve the conversion of several ketones to 3-octanone and (Z)-5-octen-3-one (Darriet et al., 2002). As little as 1%e5% infection can occasionally be detected

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Estery LSD = 0.31 Fruit flavour persistence ns

5.0 1–5% infection

10–30% infection

Pineapple LSD = 0.34

4.0 Overall fruit flavour ns

Citrus LSD = 0.29

3.0

2.0 Melon ns (P = 0.06)

Bitterness ns 1.0 0% infection

0.0 Cooked apricot LSD = 0.34

Astringency LSD = 0.30

>30% infection Acidity LSD = 0.27

Earthy LSD = 0.31

Fungal LSD = 0.30

Viscous/oily LSD = 0.30 Sweetness LSD = 0.22

Cooked tomato LSD = 0.36

FIGURE 4.56 Effects of the level of powdery mildew (0%, 1%e5%, 10%e30%, >30% infection) on the sensory attributes for Chardonnay wine. LSD, least significant difference (P ¼ .05); ns, not significant. From Stummer, B.E., Francis, I.L., Zanker, T., Lattey, K.A., Scott, E.S., 2005. Effects of powdery mildew on the sensory properties and composition of Chardonnay juice and wine when grape sugar ripeness in standardised. Aust. J. Grape Wine Res. 11, 66e76, reproduced with permission.

sensorially (Stummer et al., 2005). Variability in flavor consequences may relate to the degree to which diseased grapes are colonized by secondary saprophytes. Browning in white wines is common, but can be partially offset by the bleaching action of sulfur dioxide. Infected white grapes also possess higher concentrations of hazeproducing proteins. Fermentation may also take up to twice as long (Ewart et al., 1993). Wines made from fruit infected with downy mildew possess atypical flavors. For example, those made from 20% infected Merlot and Cabernet Sauvignon fruit showed intense green, herbaceous notes, combined with fig and cooked fruit flavors. The former were generated by (Z)-1,5-octadien-3-one and 3-isobutyl-2methoxypyrazine, whereas the latter by 3-methyl-2,4nonanedione, g-nonalactone, and g-decalactone (Pons et al., 2018). With flavescence dore´e, the production of a dense, bitter pulp makes commercial wine production from affected fruit virtually impossible. Of virus and virus-like infections, leafroll has been the most investigated relative to grape and winemaking quality. Potassium transport is disrupted and berry titratable acidity decreased. This typically generates

wines of higher pH and poorer color. Sugar accumulation in the berries is usually decreased, due to suppressed transport from the leaves. Ripening is often delayed. GLRaV-1 and rugose wood viruses shift the relative proportions of phenolic accumulation from seeds and skins, but do not appear to affect grape anthocyanin or total phenolic contents (Tomazic et al., 2003). In contrast, joint infection with both GFLV and grapevine fleck virus (GFkV) reduces the level of the more stable (oxidation-resistant) anthocyanins in Nebbiolo grapes (Santini et al., 2011). The physiological disorder BSN (desse`chement de la rafle, Stiella¨hme), causes grape shriveling and fruit fall around and after ve´raison. Wines produced from grapevines so affected often lack balance, are high in acidity, and are low in ethanol, several higher alcohols, and esters (Ureta et al., 1982). Specific effects of disease development on wine aroma have seldom been reported. Exceptions are the reduced varietal character of grapes infected by B. cinerea (La Guerche et al., 2005), or modification by the presence of the ajinashika virus (Yamakawa and Moriya, 1983). Although not a direct consequence of pathogenesis, the application of protective chemicals may indirectly

Disease, pest, and weed management

affect wine quality. For example, the copper in Bordeaux mixture can compromise the quality of Sauvignon blanc wines and related cultivars. It can reduce the concentration of important varietal aroma compounds, such as 4mercapto-4-methylpentan-2-one. This effect apparently can be reduced by prolonged maceration (Hatzidimitriou et al., 1996), or avoiding the use of Bordeaux mixture (Darriet et al., 2001). Fungal and pest control agents, as well as herbicides, may also have phytotoxic effects. These can vary from direct visible damage, as caused by sulfur at high temperatures, to more subtle changes, such as reduced sugar accumulation in berries (Hatzidimitriou et al., 1996) and disruption of photosynthesis (Saladin et al., 2003). Even more indirect influences can suppress and modify the soil flora and fauna. For example, long-term use of Bordeaux mixture has resulted in the substantial accumulation of copper in vineyardsdbeing in the range of 130e1280 mg/kg in European vineyards (Wightwick et al., 2010). This partially results from copper’s binding with organic matter in the soil, but mainly from the formation of copper and iron oxyhydroxidesdthese bind tightly to the soil’s clay fraction. The highest concentrations tend to be found in the upper layers of the soil. An indirect effect is suppression of the soil’s microbial activity, and an indirect augmentation of its organic content (Parat et al., 2002).

Fungal diseases With few exceptions, fungal pathogens grow as long, thin, branching, microscopic filaments (individually called hyphae, and collectively, mycelia). Most fungi produce cell-wall ingrowths (septa) along the hyphae. The ingrowths are usually incomplete and leave a central opening through which nutrients, cytoplasm, and organelles may pass. Thus, fungi possess the potential to adjust the number and proportion of nuclei and various organelles within the organism as they grow. This gives fungi a degree of genetic flexibility unknown in other organisms. The filamentous growth habit also provides them with the ability to physically puncture plant-cell walls. This property, combined with their degradative powers (by secreting hydrolytic enzymes) and prodigious spore production, helps explain why fungi are the predominant disease-causing agents of plants. Most parasitic fungi reproduce primarily by forming asexually generated spores (conidia). They may or may not be produced in an enclosing structure. They may also produce spores generated by meiosis, usually in the spring or summer. These are named relative to their taxonomic grouping (ascospores, Ascomycota; basidiospores, Basidiomycota; and oospores, Oomycota). Fungi that primarily reproduce without a sexual mode are variously termed hyphomycetes or fungi imperfecti.

253

Botrytis bunch rot Several hyphomycetes can induce bunch rot, either alone or together. However, the principal agent is Botrytis cinerea. The fungus appears to exist as two coinhabiting, but distinct, subspecies (Fournier et al., 2005), differing in host range, conidial size, and vegetative compatibility. Within each group, differences occur in features such as fungicide resistance, virulent aggressiveness, and presence of transposable elements. Regardless, both cryptic species infect grapevines, showing no differences in propensity for bunch vs. noble rot. They may occur sympatrically. As a necrotroph, B. cinerea induces necrosis in advance of tissue penetration, primarily infecting damaged, mature, or senescent tissues. In addition, unlike most other Botrytis species (Staats et al., 2005), B. cinerea is a nonspecialized pathogen, infecting an incredibly wide range of species and plant tissues (including gymnosperms). Consequently, spores infecting grapes may arise from a range of host species, within and around vineyards. Nonetheless, most earlyseason infections appear to arise from spores produced on overwintered mycelia within the vineyard (Jacometti et al., 2007) (Fig. 4.57). Spores may also arise from black, multicellular, resting structures (sclerotia) (Fig. 4.58A). In both instances, these are usually conidia (asexual spores). Occasionally, ascospores may be produced from the sclerotia in multicellular fructifications (apothecia) (Fig. 4.58B). Pathogenesis results from the combination of a wide range of toxins and enzymes. The first to be studied extensively were pectinases. By degrading the pectins that hold plant cells together they have a macerating action. Their action is probably aided by the production of oxalic acid, which lowers the pH of intercellular fluids. Pectinases may also initiate disruption of the cell membrane. This action, combined with the release of phytotoxic chemicals, such as secobotrytriendiol (Dura´n-Patro´n et al., 2000), botcinolide, botrydial, and necrosis- and ethyleneinducing proteins (NEP1 and NEP2) (Staats et al., 2007), as well as laccase, induces tissue necrosis. The potent polyphenol oxidase activity of laccase can also generate a wide range of toxic quinones. Initial penetration of plant tissues is undoubtedly aided by its release of cutinases and lipases. Initial infections usually develop on aborted and senescing flower parts, notably where they attach to the receptacle (Viret et al., 2004). When the remnants of flowers are trapped within growing fruit clusters, they are well positioned to initiate fruit infections later in the season. Although removing infected flower remains can delay the onset of later fruit infections, it is currently not a practical control measure. Another source of fruit

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4. Vineyard practice

FIGURE 4.57 Disease cycle of Botrytis bunch rot. From Flaherty, D.L., Jensen, F.L., Kasimatis, A.N., Kido, H., and Moller, W.J., 1982 Grape Pest Management. Publication No. 4105. Cooperative Extension, University of California, Oakland, CA, reproduced by permission.

(A)

(B)

FIGURE 4.58 Production of Botrytis cinerea conidia on (A) conidiophores and (B) ascospores from asci, arising from sclerotia. Photo courtesy, Jackson, R.S.

Disease, pest, and weed management

infection originates from latent infections that occurred in the spring. These form when hyphae invade the vessels of young green berries (Pezet and Pont, 1986). The fungus subsequently becomes inactive, until the fruit is nearly ripe. At this point, cell walls soften, acidity levels fall, sugar contents rise, flavonoid phenolics are more polymerized, and the concentration of some antifungal compounds has declined (Keller et al., 2003). Such latent infections may be the primary source of infection, notably those during dry autumn conditions. Thus, the initial focus of control is best directed at limiting early infections, with spraying coinciding with flowering, 80% cap fall, and bunch closure. An equation involving temperature and wetness duration, correlated with flower phenology, has been proposed to coordinate spraying to achieve maximum efficiency (Ciliberti et al., 2015). Under protracted rainy or humid conditions bunch rot can rapidly develop de novo. The disease is particularly destructive late in the season, when resistance mechanisms decrease in ripening fruit. Such infections may be quickly followed by secondary invaders, notably Acetobacter and saprophytic fungi (e.g., Penicillium, Aspergillus, Cladosporium, and Rhizopus spp.). Metabolism of tartaric acid, and degradation of resveratrol and other phenolic phytoalexins, by B. cinerea facilitates the growth of secondary invaders. These can often be more damaging to fruit quality than the initial Botrytis infection. For example, Penicillium expansum is the likely source of the earthy smelling compound geosmin in infected grapes (La Guerche et al., 2005), whereas Acetobacter is well known to be a source of acetic and gluconic acids. Several insects, such as the European grape berry moth, the light brown apple moth, and fruit flies, can aggravate disease incidence. They can be agents for both transporting and infecting fruit with conidia (Mondy et al., 1998b). Infections of leaves, shoots, and other vine parts by Botrytis occur, but these are primarily important as overwintering sites. In addition to physiological changes that increase susceptibility to attack are histological modifications (Kretschmer et al., 2007). Microfissures may develop around stomata (Fig. 4.59A) and micropores (cracks) in the cuticle (Fig. 4.59B). Both provide sites facilitating fungal entrance and the release of plant nutrients that favor spore germination. Weathering of the cuticular waxy plates on plant surfaces also favors infection by aiding spore adherence. The loss of wax is most noticeable where berries press and rub against one another (see Fig. 4.54B). The cutin content can decrease by more than 60% by ve´raison from its preanthesis level (Comme´nil et al., 1997). Rapid berry enlargement, especially during heavy rains, can induce skin splitting and the release of juice, further

255

favoring infection. Degradation of procyanidins late in the season has also been correlated with sensitivity to Botrytis (Pezet et al., 2003). Additional factors affecting fruit susceptibility include skin characteristics and cluster compactness. Reducing fruit compactness (e.g., leaf removal around the cluster shortly after bloom), as well as application of growth regulators (e.g., prohexadioneecalcium), can occasionally be as effective as fungicides in reducing infection (Hed et al., 2009; Molitor et al., 2011). Open clusters not only facilitate air infiltration (surface drying) and improved cuticular development, but also give growers the option for longer maturation times. b-Aminobutyric acid can enhance both features as well as being an effector of systemic acquired resistance (Kocsis et al., 2018). In contrast, heavy rains, protracted periods of high humidity, and shallow vine rooting increase susceptibility. Shallow rooting exposes the vine to waterlogging, which can favor rapid water uptake and berry splitting before the xylem becomes plugged near the end of ripening. Subsequently, rainy spells can cause berry splitting via osmotically driven water uptake through the skin (Lang and Thorpe, 1989). In addition, protracted moist periods provide conditions that favor spore production, germination, and disease spread. After germination, spores produce one or more germ tubes that grow out through the spore wall. Fruit penetration occurs shortly thereafter, often through microfissures in the epidermis. Subsequent ramification initially progresses more or less parallel to the berry surface and through the hypodermal tissues. Infection may fail to spread to the mesocarp under dry conditions (Glidewell et al., 1997)da situation that occurs during noble rot. Depending on the temperature and humidity, copious numbers of spores may be produced within days. Conidia are borne on elongated, branched filaments. These erupt either directly through the epidermis or via stoma. The white to gray color of the young spores gives rise to the common name for most Botrytis diseasesdgray mold. On maturity, the spores turn brown. They can be so densely packed as to give infected tissues a feltlike appearance. Early in infection, white grapes may take on a purplish coloration (Plate 9.1). All infected fruit eventually turns reddish brown, presumably due to the phenol-oxidizing action of laccase. Nonetheless, the production of H2O2 (under the aegis of glucose oxidase) can equally oxidize grape phenolics, enhancing the generation of brownish pigments. Effective management often requires both sprays and environmental adjustment. Those fungicides that remain localized to the surface act protectively, whereas systemic forms can act within plant tissues. Thus, systemic agents possess both protective and curative

256

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(A)

(B)

FIGURE 4.59 (A) Drawing of Botrytis cinerea penetrating a berry peristomatal microfissure from a scanning electron micrograph. (B) Scanning

electron micrograph of a section through a cuticular micropore. (A) From Bessis, M.R., 1972. E´tude en microscopie electronique a` balayage des rapports entre l’hoˆte et le parasite dans le cas de la pourriture grise. C. R. Acad. Sci. Paris, Se´r. D 274, 2991e2994; (B) From Blaich, R., Stein, U., Wind, R., 1984. Perforation in der Cuticula von Weinbeeren als morphologischer Faktor der Botrytisresistenz. Vitis 23, 242e256, reproduced by permission.)

properties. With the development of fungicide resistance (dicarboximides), or deregulation (benzimidazoles), there has been a shift to chemicals such as iprodione (Rovral), cyprodinil (Vangard), fludioxonil (Switch), pyrimethanil (Scala), fenhexamid (Elevate, Teldor), tolylfluanid (Euparen Multi), and purified paraffinic oil (Stylet Oil). Their application, especially at early flowering, late flowering, and pre-bunch closure, may occasionally achieve 80%e90% control. Later application is usual required only when conditions become especially conducive to infection. Nonetheless, the discovery of strains resistant to all botryticides suggests that the efficacy of site-specific fungicides in gray mold control may be limited (Rupp et al., 2017). Neither sulfur- nor copper-based fungicides are effective against B. cinerea. Fungicide application is enhanced by leaf removal around the clusters. BTH, a newer class of disease control agents, possesses the novel property of inducing systemic acquired resistance (Iriti et al., 2004). It has been found to activate phenol synthesis, including anthocyanin and resveratrol, the latter of which enhances disease resistance (Iriti et al., 2004). Regrettably, BTH has been noted to undesirably affect the aroma profile of some wines (Vitalini et al., 2014). Acibenzolar-S-methyl and chitosan are other control agents activating systemic acquired resistance. Registration of fungicides use is regulated by government agencies, as are dosage rates and residual levels in wine. The latter typically sets limits on the last spraying date prior to harvest. Because importing countries may set even more stringent maximum permitted residual

levels, growers and wineries may need to adjust application dates relative to the countries to which their wines are exported. Plowing under infested plant remains, as well as applying a green manure, helps to reduce vineyard survival of the fungus. By itself, though, it is ineffective as a control measure. The use of less vigorous rootstocks, divided canopy systems, and basal leaf removal can separately, or in combination, help generate a more open canopy and speed drying of vine surfaces. The application of gibberellic acid can also favor drying by opening tight fruit clusters. This is probably applicable only for table and raisin grapes, as gibberellic acid increases the incidence of untypical aged flavor in wine. Because Botrytis cinerea is destructive to multiple plants, and rapidly develops fungicide resistance, biological control is under investigation in many parts of the world. It varies from the application of compost and manure extracts to adding suppressive mycoviral, bacterial (Burkholderia phytofirmans), yeast (Pichia membranifaciens), and filamentous fungal (Pythium radiosum) agents. Rhamnolipids from P. aeruginosa, noted earlier, may also be effective, not only as direct inhibitors, but also as activators of plant defense mechanisms (Varnier et al., 2009). The most commercially successful biocontrol agent for Botrytis infections appears to be Trichdex, a formulation of Trichoderma harzianum spores (O’Neill et al., 1996). Because the action of biocontrol agents may be suppressed by fungicides, joint application is counterindicated. This feature should not affect the application of an extract made from grape canes (a source of

Disease, pest, and weed management

stilbenes). Not only does the extract suppress fungal growth, but it also activates expression of several grapevine resistance genes (De Bona et al., 2019). Jacometti et al. (2010) provide a review of control practices, stressing alternative measures. Although the most frequent cause of bunch rot is Botrytis cinerea, similar damage can be induced by other fungi. These often include species of Penicillium and Aspergillus, and in more subtropical climates, Colletotrichum spp. (ripe rot) and Greeneria uvicola (bitter rot). These may attack alone or in combination with secondary invaders. Bunch rot can also be induced by pathogens that typically affect vegetative organs, notably Phomopsis viticola and Guignardia bidwellii (Steel et al., 2013). Powdery mildew (oidium) The fungus that induces powdery mildew is a member of a large group of generally host-specific, obligate, parasitic fungi. They cause related diseases on a wide range of flowering plants. The species that attacks grapevines, Uncinula (Erysyphe) necator, is specialized to members of the Vitaceae (Halleen and Holz, 2001) (Fig. 4.60). Even low levels of infection can negatively affect grape quality. In addition, U. necator can augment

257

susceptibility to other diseases, pests, and spoilage organisms (Gadoury et al., 2007, 2012). It is often the most destructive pathogen afflicting V. vinifera. After germination, spores of U. necator produce mycelium that grows over the epidermis. Periodically, specialized projections penetrate downward into epidermal cells. These generate short extensions within the cell (haustoria), establishing intimate contact with the cytoplasm. Nutrients extracted by the haustoria permit continued growth and sporulation of the surface mycelium. The underlying palisade cells express the greatest physiological disruption, soon becoming necrotic. This probably results from redirection of nutrients from these and adjacent cells to infected epithelial cells, resulting in their starvation. Most of the fungus remains external to the vine. U. necator is an endemic pathogen of indigenous grapevines in the eastern and central parts of North America, where long exposure has resulted in native species having developed a degree of tolerance. In contrast, Vitis vinifera evolved in isolation of the pathogen. Its young tissues possess little inherent resistance to the pathogen. When U. necator was accidentally introduced into Europe about 1840, presumably on imported

FIGURE 4.60 Disease cycle of powdery mildew. Drawing by R. Sticht (Kohlage), from Pearson, R.C., Goheen, A.C. (Eds.), 1988. Compendium of Grape Diseases APS Press, St. Paul, MI, reproduced by permission.

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vines from the United States, it initiated the first of a series of devastating diseases to strike European vineyards. Genome studies indicate that there were probably two separate introductions (Brewer and Milgroom, 2010). The same investigation suggests that movement of the pathogen to California, and subsequently Australia, was associated with importation of cuttings from Europe. It has generally been thought that U. necator evolved in eastern North America. However, the discovery of several resistance genes in Asian species (e.g., Vitis romanetti (REN4) in China and a V. vinifera cultivar (REN1) from Central Asia), makes this view somewhat less tenable. Unlike many pathogens, U. necator is not highly dependent on specific climatic conditions for sporulation or infection. The fungus infects over a wide range of temperatures (10
Ce32
C) and is little affected by low humidity. It can attack any green vine tissue. Thus, vineyard conditions that favor higher tissue temperatures, such as basal leaf removal, open canopy training systems, avoidance of excessive vine vigor, and PRD, may limit disease severity. These factors can act synergistically (Austin et al., 2011). In temperate climates, overwintering can occur as infections on inner bud scales. Subsequent sporulation can initiate infection upon budbreak. If shoots are sufficiently infected early in the season, they become severely stunted, forming what are called “flag shoots.” Fungal survival also involves resting structures (cleistothecia). Lodged in bark crevices, when washed from diseased tissue, they are ideally positioned to initiate new infections (Gubler and Ypema, 1996). Overwintering cleistothecia swell and rupture after rains, ejecting ascospores. These can wash, or be blown, onto young tissues and initiate early infection following budbreak (Pearson and Gadoury, 1987). In North America, asynchrony of cleistothecial spore discharge with cultivar budbreak can occasionally reduce spring spore load, limiting the seriousness of early infection (Moyer et al., 2014) and increasing the efficacy of fungicide application. In regions where cleistothecia participate in the infection cycle, it may be essential to initiate control measures earlier than when infection develops only from overwintered hyphae. Application of lime sulfur or flowable sulfur, prior to budbreak, is often effective in limiting early cleistothecial-based infections (Gadoury et al., 1994). Early infections can result in leaf and fruit distortion, by killing surface tissues before they expand and reach maturity. Severe infection leads to leaf and fruit drop, as well as death of shoot tips. Rapid spore (conidial) production generates the white powdery appearance (mildew) typical of infected tissue. Conidiophores and the chains of spores arise perpendicularly from the

prostrate surface mycelia covering host tissue. The spores initiate further rounds of infection during the season. Fungal growth and sporulation are optimal between about 20
C and 30
C. Above 32
C, fungal metabolism essentially stops. Later in the season, cleistothecial production can give infected tissues a red- to black-speckled appearance. Fruit is most sensitive to infection shortly after initiation, with many cultivars rapidly developing immunity thereafter (Gadoury et al., 2003). This appears to involve hyphal inability to effectively penetrate the mature epidermis. Immunity may be complete within as little as 4 weeks (Ficke et al., 2003). This may also be due to enhanced synthesis of stress and PR proteins, such as osmotin and traumatin-like proteins (Monteiro et al., 2003). Although growing tissue remains susceptible, older tissues become increasingly resistant, with mature leaves seldom being infected. Despite this, fruit may succumb to a diffuse, nonsporing form of infection visible only microscopically. It predisposes the fruit to insect and other pathogen attack, and the wine quality is degraded by abnormally high concentrations of volatile acidity and ethyl acetate (Gadoury et al., 2007). Early control not only is important for fruit protection, but also minimizes foliage damage and its consequences for the current and subsequent years’ growth. Although the rachis remains susceptible for a prolonged period, this does not appear to affect fruit development or augment fruit infection. Control of berry infection is most effective when applied at and shortly after flowering and fruit set, but may need to be extended under conditions favorable to the diffuse infection noted above. Where early leaf infection has historically been serious, control should commence earlier, and may need to be extended up to ve´raison. Disease management is based primarily on sanitation and fungicidal sprays, principally sulfur. Removal of overwintering fungal tissue on leaves, stems, and fruit limits early disease onset. Delaying early onset can often postpone severe disease development until after harvest. Developing an open canopy also reduces disease incidence, as well as improving uniform spray application. Wettable or sulfur dusts are the principal control agents. Although not significantly incorporated into plant tissues, the predominant location of the fungus on plant surfaces permits sulfur to act as both an effective preventative and a curative agent. Unfortunately, sulfur’s action is temperature dependent, being much less effective below 20
C, and becoming increasingly phytotoxic above 30
C. In addition, sulfur’s use may promote increased spider mite populations, by suppressing predators such as the mycophagous mite Orthotydeus lambi. Sulfur application is recommended to cease several weeks before harvest, to minimize potential H2S production during fermentation (Kwasniewski et al., 2014). This likelihood

Disease, pest, and weed management

can be further minimized by short skin-contact times and prefermentative clarification. Alternative control agents include demethylationinhibiting fungicides (e.g., Bayleton, Rally, and Rubigan), or strobilurin fungicides (e.g., Abound, Flint, and Sovran). They can be more effective and less phytotoxic, but are more costly. The potential for rapid resistance to these highly specific agents requires that they be used sparingly, and never more than twice in sequence. Agents such as silicon, bicarbonates (e.g., baking soda), oils (e.g., canola oil), cinnamic aldehyde, and phosphate fertilizer may also be effective. Chitosan (a deacetylated derivative of chitin) activates chitin/chitosan receptors. These can induce a series of systemic acquired resistance factors, providing effective control even under high disease pressure conditions (Iriti et al., 2011). Most of these agents are available in commercial form. Ampelomyces quisqualis (AQ10), as well as other mycoparasitic fungi, have been used as biological control agents. Regrettably, they tend to be limited by the environmental conditions they require. Natural infection by A. quisqualis develops too late in the season to be an effective control agent. The existence of several sources of gene-based resistance factors offers the hope of an eventual durable form being incorporated into vinifera cultivars. For example, race specificity of individual R alleles (e.g., RUN1, RUN2, and REN2) might be offset by pyramiding these factors (Feechan et al., 2015). As noted, early prevention is critical to avoiding rapid spread and severe disease development. In addition, yield loss is often directly related to the timing of the initial infection. The development and widespread use of disease-risk models (Broome et al., 1995) have improved the timing and effectiveness of fungicide application, while reducing the amount required. Downy mildew (oidium) Downy mildew is the second of three inadvertently introduced pathogens that quickly spread, devastating European vineyards in the mid- to late 1800s (the others being powdery mildew and phylloxera). All were indigenous to North America, but alien to Europe. Despite the name similarity to powdery mildew, and some superficial resemblance, the causal agent, Plasmopara viticola, is genetically unrelated. It is now classified, with similar fungal-like protists, in the phylum Oomycota, separate from the true fungi (Eumycota). Although going under a single species designation, Pl. viticola is considered to consist of five cryptic species. Of the three that infect cultivated grapevines in North America, one is restricted to interspecies hybrids, one attacks V. vinifera and V. labrusca, and the last occurs in the southeastern parts of North America

259

(Rouxel et al., 2014). Pl. viticola in Europe appears to consists of two geographic and genetically distinct populations, localized in either western or eastern regions. At the spore dispersal stage, the sporangia germinate to produce several flagellated zoospores (Fig. 4.61). Zoospores possess a short motile stage, during which they swim in a thin film of water on the host tissue toward the stoma. Here, they adhere to the epithelium, lose their flagella, and begin to penetrate the host. Shortly after successfully establishing itself in the host, the fungus may initiate sporangial production. This typically occurs at night. The sporangia remain viable for only a few hours after sunrise. This may explain the highly diverse and localized dispersion of Pl. viticola genotypes (Gobbin et al., 2005). Because infection is highly dependent on rainfall or dew, high humidity, and a restricted temperature range, downy mildew tends to produce less widespread damage than powdery mildew. Similar to powdery mildew, downy mildew is an obligate parasite, produces haustoria, and attacks all green parts of the vine. However, Plasmopara viticola hyphae do not remain exterior to the plant. In contrast, they ramify extensively throughout host tissues. Under moist conditions, sporulation develops rapidly. On leaves, spore-bearing hyphae (sporangiophores) erupt preferentially through stomata on the lower surface. Leaf invasion is the primary source for spores inducing fruit infections. Infected shoot tips become white with spore production and show a distinct Sshaped distortion. The shoot subsequently turns brown and dies. Grapes are most vulnerable when young, but all parts of the fruit cluster remain susceptible until maturity. As with leaf infection, severe development of the disease can result in premature fruit abscission. Fruit infection has the greatest direct effect on grape quality and yield. Nonetheless, severe leaf infection can indirectly affect yield in the subsequent year, due to reduced carbohydrate generation. During the summer, the fungus produces a resting stage within infected tissues, termed an oospore. Oospores may remain dormant for several years. When conditions favorable to germination occur, they produce a sporangium, within which spores (sporangiospores) develop. Typically, this occurs in the spring, but can occur throughout the season (Kennelly et al., 2007). In mild climates, both oospores and dormant mycelia (in infected leaves) may initiate spring infections, though in most locations infections are induced by spores derived from oospores. Bordeaux mixture and several nonsystemic fungicides provide protection against infection, but are not curative. For this, systemic fungicides such as fosetyl

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FIGURE 4.61 Disease cycle of downy mildew. From Agrios, G.N., 1997. Plant Pathology, fourth ed. Academic Press, New York, NY, reproduced by permission.

aluminum and phenylamides (e.g., Metalaxyl) are required. The uptake of these fungicides by plant tissues reduces their dilution or removal by rain. Depending on local conditions, early and effective control often limits the need for subsequent or multiple fungicide applications (Jermini et al., 2010). As of this writing, there are no biological control measures that are effective, although several fungi have been shown to be toxic to Pl. viticola. These include Alternaria alternata, Epicoccum nigrum, Acremonium byssoides, and Fusarium proliferatum. In addition, application of chitosan oligomers (a deacetylated derivative of chitin) activates resistance by enhancing phytoalexin, chitinase, and b-1,3glucanase production in plant tissues (Aziz et al., 2006). Callose synthesis upon invasion may also be involved as a defense mechanism in resistant vines. The production of an open canopy by basal leaf removal, or a more open training system, has only a

minimal effect on disease incidence. Consequently, chemicals remain the only effective treatment for this pathogen under conditions favorable for disease development. Black rot of grapes Unlike the diseases noted thus far, black rot is of economic significance only in eastern North America and selected regions in Europe, South America, and Asia. Most indigenous Vitis spp. show considerable resistance to attack, having evolved in the presence of the pathogen for millions of years. In contrast, cultivars of V. vinifera are very susceptible. Depending on the occurrence of favorable (humid) weather conditions, and the initial inoculum, the disease can cause crop losses of up to 80%. Three subspecies of the causal fungus, Guignardia bidwellii (Phyllosticta ampelicida), are recognized. One affects only species of the Vitis subgenus; a second infects both

Disease, pest, and weed management

subgenera (Vitis and Muscadinia), while the third attacks only species of the related genus Parthenocissus. Infection can occur on new growth at any time during the growing season (Fig. 4.62), but does not develop on mature leaves or fruit following ve´raison. On leaves, lesions develop as small creamy circular spots. As these enlarge (up to 10 mm), they darken to a tan color, and finally turn reddish brown. The spots are surrounded by a band of dark-brown tissue. Characteristic small, black, roundish raised structures (spore-bearing pycnidia) develop in older parts of the lesion. Production of conidia requires high humidity for several hours, a condition that most frequently occurs at night. Black, elongated lesions usually develop on petioles and the fruit stalk. These may encircle the structure, killing any tissue distal to the lesion. On young shoots, similar but larger black cankers develop. These produce pycnidia during the growing season. Lesions develop surprisingly quickly on the fruit, converting whole berries into blue-black, shriveled “mummies” within a few days. Many cycles of localized infection may occur during a single season. During the fall and winter months, structures superficially resembling pycnidia develop in

261

mummies on the ground. These fruiting bodies, called pseudothecia, develop as a result of sexual reproduction. In the spring, the ascospores mature and are ejected into the air following even light rainfalls. They initiate a new round of infection. On wild vines, the fungus probably survives most effectively on fruit mummies. However, in commercial vineyards, most mummies are collected and destroyed. Thus, survival is most probably associated with infected portions of canes not removed during pruning. Spore production from overwintered tissue can continue well into midsummer. Control used to be based on the use of protective contact fungicides, such as maneb and ferbam. However, current preference is to use systemic fungicides, such as tridimefon (Bayleton) and myclobutanil (Eagle 40). They have the added benefit of being potentially curative. Strobilurin fungicides are less curative, but may be valuable due to their simultaneous action against other pathogens, notably powdery and downy mildews (Hoffman and Wilcox, 2002). Sanitation (destruction of mummified fruit and infected cane wood) is particularly important in reducing the spring inoculum load. This can significantly delay and reduce the economic

FIGURE 4.62 Disease cycle of black rot of grapes caused by bidwellii. From Agrios, G.N., 1997. Plant Pathology, fourth ed. Academic Press, New York, NY, reproduced by permission.

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4. Vineyard practice

significance of infection. In climates where the incidence of black rot is frequent, and especially where mechanical pruning is practiced, inspection and destruction of infected cane wood should be practiced. For a discussion of integrated control measures see Hoffman et al. (2004). Eutypa dieback and other related syndromes Eutypa dieback is probably the most serious manifestation of trunk disease/decline syndromes. Eutypa dieback induces a slow, but insidious, attack on the woody components of the vine. Symptoms include progressive death of spurs, cordons, and eventually the whole vine, associated with the expanding zone of wood necrosis and decay. Accompanying leaf symptoms may include deformation, stunting, chlorosis, tearing, and splotchy areas of necrosis, but usually only commence after pathogen establishment in the trunk is well developed. The disease often kills the vine within 10 years if unchecked. The effect on vineyard yield over time, in susceptible and more resistant cultivars, is compared in Fig. 4.63. The major causal agent, Eutypa lata (E. armeniacae), preferentially invades wounds of the perennial shoot 35

× +

(A) 30 25

+

20

+ +

15

+ × ×

Yield (t/ha)

×

×

+ ×

×

× +

+ + × ×

5 + ×

+ +

+

10

0

+ ×

+

+ + + × × + × + × + ×

+

× y = 1.37 + 3.65x – 0.151x2

+

×

r2 = .62

(B)

30 25 ×

20 15

×

× +

× + + ×

× × + +

+ + ×

10

× +

× +

×

× +

+

× +

+ +

+

+ +

+ + +

+ +

+

y = 32.9(1 – exp(–0.35x)) – 11.6

5

r2 = .89 0

2

4

6

8 10 12 14 16 18 20 22 24 26 28 Vineyard Age (Yr)

FIGURE 4.63 Relationship between vineyard age and yield for (A) six Chenin blanc vineyards and (B) eight Barbera vineyards in Merced County, California. Symbols represent different vineyards. The data illustrate the relative disease susceptibility and resistance of the Chenin blanc and Barbera to Eutypa dieback with time. Data from 1964 to 1990. From Munkvold, G.P., Duthie, J.A., Marois, J.J., 1994. Reductions in yield and vegetative growth of grapevines due to Eutypa dieback. Phytopathology 84, 186e192, reproduced by permission.

system (Fig. 4.64). Its most serious manifestations derive from xylem infections, principally originating from wounds produced during graft conversion or training system conversion. Wounds produced during annual pruning can also produce infection sites. Nonetheless, these infections tend to be less significant, as cane wood is less susceptible than the permanent older wood. In addition, much of the cane wood is removed during pruning, before the fungus can advance into more permanent parts of the vine. However, cane wood infection may be more significant where minimal pruning is employed. Where vines are spur pruned, a double pruning technique has been suggested by Weber et al. (2007). In the procedure, nonselective trimming of the canes to 30e45 cm occurs in late fall, followed by a more traditional pruning to two-node spurs in late winter. At this time, the likelihood of infection is much reduced. Fungal growth in the xylem is slow, with minimal invasion of the cambium and phloem. As invasion progresses, an enlarging, elongated, lens-shaped canker forms. It often remains undetected for years, due to the overlying bark. Disease symptoms often appear months or years after infection, and are typically detected when leaf and shoot symptoms develop. These are usually localized to shoots directly associated with and above the lesion. Symptoms are most apparent in the spring, when stunted shoots are most evident, compared with rapidly elongating healthy shoots. The young leaves on affected shoots are usually upturned, small, distorted, and chlorotic, and possess a tattered margin. Shoot internodes are markedly dwarfed. Fruit formation on infected wood is limited and usually dehisces before maturity. These effects are caused by several phenolic mycotoxins produced by the causal agent. Eventually, the portion of the vine associated with the lesion dies. This feature gives rise to the expression “dead arm.” The canker is usually observed only by removing the outer bark. Well-established cankers contain rows of flask-shaped perithecia (pseudothecia), structures in which ascospores are produced. Cutting tangentially along the wood, and through the canker, exposes the perithecia as round objects containing a jelly-like material (translucent when wet; white and sheetlike when dry), or matte black when empty after spore discharge. The infected wood typically possesses a V-shaped appearance and has a lightgrayish to dark-brown color. Spores in perithecia generally mature in late winter or early spring. Subsequent release follows periods of sufficient rainfall (>1 mm), often being most marked in very early spring. Sprinkler irrigation may be an important source of water for spore discharge in dry regions.

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Dead infected wood

Dieback-infected grapevine

Fungus fruits (perithecia) produced in old dead wood Canker

Spores produced in spring, summer, autumn Spores germinate in wood cells

Fresh pruning wound Spores discharged in wet weather

FIGURE 4.64 Disease cycle of Eutypa dieback. From Flaherty, D.L., Jensen, F.L., Kasimatis, A.N., Kido, H., and Moller, W.J. 1982 Grape Pest Management. Publication No. 4105. Cooperative Extension, University of California, Oakland, CA, reproduced by permission.

Eutypa dieback is particularly difficult to control. Except in areas where grapevines are the major woody plant, sanitation alone is relatively ineffective in reducing disease incidence. This results from E. lata infecting many indigenous woody species common to grape-growing regions. Spores are also effectively wind dispersed over more than 100 km. Thus, a local source of infection is not necessary for disease development. Finally, normal (surface) fungicidal spraying is ineffective in preventing spore germination in exposed xylem. Management techniques include wood removal to about 5e10 cm below apparent xylem staining. This is required as the fungus may occur considerably in advance of wood staining. The wound so created is usually treated with a creamy suspension of Flusilazole, Carbendazim, or 20% boric acid. Several organic fungicides also appear to be effective (Cobos et al., 2015). The suspension must soak into the exposed xylem to provide adequate protection. Destruction (preferably burning) of the infected wood is recommended as diseased tissue can remain a significant source of spore production after removal. In addition, any pruning wounds created on 2-year or older wood should be treated. Application of a protectant should be performed as soon as possible after pruning. Adjustment of tractor-driven sprayers can be both effective and more economical than hand application

(Ayres et al., 2016). Conversely, a suspension of Trichoderma harzianum (or T. atroviride) may be applied (Mutawila et al., 2016). Alternative biocontrol agents include Bacillus subtilis and Erwinia herbicola. Where possible, pruning should occur when the xylem is active and spore production low. The formation of lignin and suberin at the wound site markedly reduces disease incidence (Munkvold and Marois, 1990). Thus, very early-autumn or late-spring pruning minimizes the likelihood of infection. The discovery that the feeding of arthropods (e.g., ants and millipedes) on pruning wound sap can effectively transmit trunk disease organisms (Moyo et al., 2014) may require integration of their control during pruning. Another set of syndromes, resembling those shown by Eutypa dieback, but without foliar symptoms, can be induced by several genera in the Botryosphaeriaceae ´ rbez-Torres and Gubler, 2009; Pitt et al., 2010). These (U include Botryosphaeria, Diplodia, Dothiorella, Lasiodiplodia, and Neofusicoccum. Their respective importance can vary significantly from country to country, from region to region, and with the season. All produce wedge-shaped cankers in the trunk associated with cordon dieback, necrotic canes, stunting, shoot and bud necrosis or whitening, graft failure, and fruit rot. Some of these fungi have also been implicated in young vine decline, such as Petri disease (see next section).

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Esca, black measles, Petri, and black foot diseases These terms refer to various expressions of a disease complex associated with brown wood streaking and vine decline. They appear to be principally caused by Phaeomoniella chlamydospora. Esca and black measles are respectively European and North American terms for its expression in mature vines (Mugnai et al., 1999). Petri disease, slow decline, and black goo are various names given to its expression in young, grafted vines. Drought stress appears to enhance symptom expression. Its expression can remain largely or completely undetected for years. Certain expressions of grapevine “replant problems” may be another manifestation of this disease complex, although assorted bacteria and nematodes have also been implicated in replant problems. Confusion about these maladies arises from their frequent association with a range of fungi, and their presence in the absence of disease symptoms (Bruez et al., 2016). Fungi, not infrequently isolated from diseased and dying vines, or showing aggravating symptoms, include several hyphomycetes, notably Phaeoacremonium minimum (syn. aleophilum) and associated species. Other fungi involved may include Phomopsis spp., occasionally Fusarium oxysporum, and several white rot fungi (e.g., Fomitiporia punctata and F. mediterreanea), as well as Armillaria and Phellinus. The last two are thought to be particularly important in esca expression in Europe. The syndrome complex has probably been present for centuries, but only comparatively recently has its presence been recognized. One of the factors awakening people to its presence was the extensive replanting necessitated by an outbreak of phylloxera in California. This occurred as a new strain of phylloxera attacked the predominantly used rootstock, AR#1. The replanting provided conditions where the slow vine decline was accentuated by the numbers being observed. Once identified, other trunk syndromes were recognized as being alternative expressions of possibly the same disease. Another possible cause for the apparent increase in its expression, in this case in mature vines, was abandoning the use of sodium arsenate to paint pruning wounds. Depending on when pruning is done, wounds may remain susceptible to infection by P. chlamydospora for weeks to months. Esca expression can be intermittent, and may reveal itself in either chronic or acute form. Symptoms of chronic disease are characterized by a progressive foliage deterioration, whereas the acute phase (apoplexy) results in sudden wilting and vine death (Andreini et al., 2014). In the chronic form, leaf symptoms can begin at any time. They start in basal leaves and move apically, appearing as yellow to red patches.

These develop necrotic centers as the patches coalesce (“tiger stripes”). They eventually become irregular brown zones of necrosis between the veins and the margins of the leaf, leading eventually to leaf dehiscence. Symptoms may be associated with the production of fungal toxins (e.g., naphthalenones, such as scytalone and isosclerone). Fruit symptoms vary with the region and cultivar. In France and northern Italy, berries are often visually asymptomatic, but do not fill or mature properly. Budbreak may also be delayed, depending on the rootstock. In southern Europe and California, affected berries may develop brown/violet patches (the black measles syndrome). Foliar and fruit symptoms may occur simultaneously or separately. Vines showing chronic symptoms one year may appear healthy and symptomless in subsequent years. Disease symptoms are probably the result of the release of several phenolic toxins, but distinct from those associated with dead arm. Symptoms of infection in the wood appear as pale brown discolored sections in the xylem, surrounded by a dark ring, especially in the trunk or cordon. This lesion may occur alone, or in combination with lesions produced by white rot fungi or Eutypa dieback (see Creaser et al., 2002). Frequently, infection occurs in the absence of leaf or fruit symptoms. Expression of the disease in young vines (Petri disease) most frequently results in, or from, partial graft failure associated with infection by P. chlamydospora. However, infection by itself does not necessarily induce disease development (Edwards and Pascoe, 2003). Stress conditions, such as water deficit or overcropping before the vine is established, appear to favor disease expression. The vine may show a slow decline within the next few years. If decline occurs later, it would normally be considered an example of esca. On investigation, the graft site usually shows lesions (streaks or dots). These may exude a thick black ooze (black goo). Microscopically, xylem vessels may show tyloses that have grown in from infected parenchyma cells. The exudate contains phenolic substances synthesized by the necrosing parenchyma cells. External symptoms include poor budbreak, stunted shoot growth, mild foliar chlorosis associated with necrotic edges, wilting, and dieback. Toxins produced inhibit the establishment of effective xylem connections, severely restricting water and nutrient transport under hot, dry conditions. This probably explains why extensive watering and nutrient application (compost) may diminish symptoms or revive diseased plants. Although most prevalent and severe in grafted vines, own-rooted vines can also develop the disease. Although the origin of infection in young vines is still insufficiently established, the prevailing view is that it originates principally from symptomless

Disease, pest, and weed management

infected vines from which the rootstock or scion wood was obtained. This is consistent with the use of hotwater treatment to eradicate the pathogen (Fourie and Halleen, 2004). Treating the wood prior to grafting with several fungicides has also been found to be beneficial (Fourie and Halleen, 2006). Subsequent vine establishment in pathogen-free potting soil, prior to planting in the vineyard, might prevent early field infection (when vines seem to be the most susceptible). The same may apply to black foot disease (below). In contrast, esca may develop from infection via pruning wounds, or from asymptomatically infected scion stock. Another disease that develops under similar conditions is Cylindrocarpon and Campylocarpon black foot disease. It can be another source of grapevine decline in new plantations. In this case, though, the fungus causes a root and butt rot. In cold climates, the practice of removing vines from their supports and burying them for winter protection may also produce lesions facilitating fungal entrance (Petit et al., 2011). Subterranean symptoms include few feeder roots, low root biomass, and necrotic root lesions. Vegetation is chlorotic and stunted. No effective specific control exists, although interesting results with endomycorrhizal infection have been promising (Petit and Gubler, 2005). Inoculation of roots with Glomus intraradices reduced both the number of root lesions and the disease severity, while increasing root dry weight. Ochratoxin-producing aspergilli Although technically not disease causing, growth of strains of Aspergillus carbonarius and A. nigra is of concern due to their production of OTA. It is one of the most dangerous mycotoxins, being associated with immunosuppressive, teratogenic, as well as nephro- and neurotoxic effects. Red wine has been estimated to be the source of about 10% of total OTA intake in the European Union (Miraglia and Brera, 2002). Consequently, limiting its presence in grapes is of major importance. Aspergilli can readily establish themselves on and in grapes through surface wounds, as secondary invaders. Their control, other than indirectly via limiting insect damage and other fungal diseases, is with biocontrol agents. These can include yeasts, such as Metschnikowia pulcherrima and Kluyveromyces thermotolerans, or the yeastlike basidiomycete Aureobasidium pullulans (Zhang et al., 2016).

Bacterial diseases Bacteria are an ancient group of microorganisms, existing predominantly as colonies of more or less independent cells. Most possess a rigid wall, providing them with their standard spherical to rod shapes. However,

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one group, the phytoplasmas, possess no rigid cell wall and are amorphous, making them almost impossible to identify under a regular light microscope. Bacteria are typically restricted to entering plants either via natural openings (e.g., stoma and lenticels), or through wounds produced by grafting or insect and nematode feeding. Nevertheless, some root pathogens produce enzymes that can degrade plant cell walls, permitting direct penetration of young roots. Overwintering occurs as dormant cells in soil, or in plant parts or vectors. Grapevines may also act as hosts for fastidious, endophytic bacteria (Bell et al., 1995), as well as rhizosphere bacteria. Some are growth promoting, such as Burkholderia (Compant et al., 2005), as well as facilitating disease resistance (Miotto-Vilanova et al., 2016). Crown gall Crown gall is a disease affecting many woody plants. It is induced by species of Rhizobium (Agrobacterium) (Young et al., 2001). The species most commonly infecting grapevines is Rhizobium vitis (Palacio-Bielsa et al., 2009). Exterior to the vine, R. vitis is typically isolated only from the rhizosphere around young roots. The pathogen can survive for at least 2 years, possibly longer, in dead and dying roots (Burr et al., 1995). R. vitis can invade young vine roots (Fig. 4.65), possibly through its production of polygalacturonidases. These enzymes may also assist the bacterium in gaining access to the xylem. Alternatively, infection may occur via wounds produced by nematode feeding (e.g., Meloidogyne hapla) (Su¨le et al., 1995). Other potential penetration sites may involve wounds produced as a result of grafting, soil cultivation, or vine burial (as winter protection). Upon gaining access to the xylem, where the bacterium primarily establishes itself, it can be passively transported throughout the plant. Such movement occurs primarily in early spring. Subsequently, the bacterial population may drop precipitously in the shoot system, rising again in the fall (Bauer et al., 1994). Thus, although the bacterium can grow systemically throughout the vine, its distribution is far from uniform, and is most frequently localized to the root system. Nonetheless, it has been detected in dormant buds and leaves (Orel et al., 2017). The strains of Rhizobium vitis that inhabit wild vines in North America and Europe (Burr et al., 1998) appear to be nontumorigenic. In contrast, most strains in commercial vineyards are tumorigenic (gall forming). This divergence may have originated from the accidental dispersion of asymptomatic, tumorigenic strains on rootstocks, associated with their global distribution in phylloxera control. Despite R. vitis primarily inhabiting the root xylem, and occasionally inciting localized root necrosis, its serious pathogenicity occurs at the trunk base. In this

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Transformed cells divide rapidly T-DNA is integrated into plant chromosomes, plant cell is transformed

T-DNA leaves bacterium and enters wounded plant cell

Bacteria multiply and spread intercellularly

Cell hyperplasia and hypertrophy lead to gall formation

Galls on stem and root of heavily infected plant

Bacteria entering stem or root through wound Crown gall bacteria overwintering in soil Plant cell

Bacterium becomes attached to wounded plant cell

Older gall with several new centers of activity

Plant infected Healthy with crown plant gall

Ti-plasmid Chromosome

Bacteria from gall surface move into soil

FIGURE 4.65 General disease cycle of crown gall. From Agrios, G.N., 1997. Plant Pathology, fourth ed. Academic Press, New York, NY, reproduced by permission.

location, R. vitis can provoke uncoordinated cell division in the vascular tissue, inducing gall formation. Individual galls are commonly self-limiting, and subsequently rot and separate from the stem. New galls may originate next to old galls. In young vines, galls may encircle the trunk, killing the vine. In mature vines, the consequences of gall formation depend on their number, size, and distribution. Heavy galling significantly disrupts vascular flow, reducing vine vigor and fruit yield (Schroth et al., 1988). Galls are also invasion sites for other pathogens, notably Pseudomanas syringae f. syringae and Armillaria mellea. Gall formation appears to require cellular microlesions in parenchyma cells, such as those produced by frost damage. Correspondingly, gall initiation typically occurs during the spring in cold climates. Galls develop most commonly near the transition from root to shoot (crown), hence the name “crown gall.” Graft conversion (topping) can also be a significant gall activator, resulting in graft failure. Wounding indirectly stimulates gall induction by activating parenchyma cell multiplication. This favors bacterial attachment and the transfer of a tumorigenic (Ti) plasmid from the bacterium into dedifferentiating parenchyma cells. Upon plasmid transfer and activation, the infected cell transforms into a tumorous cell.

This is associated with the overproduction of auxins and cytokinins, provoking an uncoordinated multiplication of callus-like tissue. It is this tissue that generates the gall. The plasmid also codes for the production of a unique group of amino compounds: the opines nopaline, octapine, and vitopine. Opines serve as specific nutrients for bacterial growth. No fully effective control of crown gall exists. The best option, where possible, is to plant disease-free stock in virgin soil. Where the soil is already infested, the best options are to either graft to a crown galle resistant rootstock or initiate some form of biological control (Filo et al., 2013). Resistant rootstocks such as NAZ4, NAZ6, Gloire de Montpellier, C3309, or 101-14 Mgt do not prevent infection, but reduce gall severity. Severity may also be reduced by prior colonization with suppressive strains of Rhizobium vitis (e.g., ARK1, F2/5) or the rhizosphere bacterium Rahnella aquatilis HX2 (Chen et al., 2007). Their effectiveness depends on the strain(s) of Rhizobium vitis in the soil. Inoculation of disease-free rootstocks with Pseudomonas aureofaciens and P. fluorescens also appears to markedly reduce infection and disease severity (Khmel et al., 1998). Where crown gall is serious, as in northeastern North America, multiple trunking is often practiced as a precaution. It is unlikely that all trunks will succumb

Disease, pest, and weed management

simultaneously, leaving one or more trunks to sustain and reestablish the vine. The disease is typically more serious on V. vinifera cultivars than on V. labrusca and French-American hybrids. Eradicating the bacterium from scions and rootstocks used in propagation can often, but not consistently, be achieved with hot-water treatment (Burr et al., 1996). Thus, micropropagation from apical shoot tissue is used where elimination is obligated, as with international movement of cuttings. When propagating disease-free clones, the use of pasteurized soil and greenhouse equipment is essential to prevent the spread of R. vitis during bench grafting. Pierce’s disease Pierce’s disease is induced by another xyleminhabiting bacterium, Xylella fastidiosa spp. fastidiosa (Hopkins and Purcell, 2002). Considerable genetic diversity exists within the species (Hendson et al., 2001), but most pathotypes are ill adapted to multiplying and causing disease in grapevines. Other strains of the pathogen cause similar diseases in many tree crops and can exist as an asymptomatic endosymbiont in a wide range of annual crops, vines, and weeds (Wistrom and Purcell, 2005). These can act as reservoirs for the pathogen, from which transmission to susceptible hosts may occur via xylem-feeding insects (Redak et al., 2004). Because of host specificity, and differential cultivar sensitivity, transmission does not necessarily result in disease. The primary insect transmitters are sharpshooter leafhoppers (Cicadellidae) and related spittle bugs (Cercopidae). Geographical distribution of the disease is limited by the presence of suitable vectors and the bacterium. Except for several isolated observations on grapes in Europe (Boubals, 1989; Berisha et al., 1998), and its recent appearance in parts of California and Taiwan, Pierce’s disease is primarily isolated to its endemic habitats in the southeastern United States, Mexico, and Central America. The tropical to subtropical localization of Xylella-induced diseases suggests that winter temperatures are a significant factor limiting pathogen spread. In areas where the pathogen is endemic, indigenous species of Vitis are resistant or relatively tolerant to infection. Disease severity is often a reflection of the relative incidence of the pathogen (Ruel and Walker, 2006). Although its occurrence can severely restrict the commercial cultivation of V. vinifera, there is considerable variability in cultivar susceptibility. Of popular varieties, Chardonnay and Pinot noir are particularly sensitive, Cabernet Sauvignon and Sauvignon blanc are less so, and Riesling and Zinfandel show moderate resistance. Until recently, Pierce’s disease was of limited significance in Californiadpresumably due to the absence of an effective vector. This situation has changed since

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the appearance of the glassy-winged sharpshooter (Homalodisca coagulata). It is an indigenous insect within the historical range of the disease in the southeastern United States and northeastern Mexico. It is capable of flourishing on the nutritionally meager solution found in the xylem. In sensitive vines, transmission often results in rapid bacterial colonization and movement throughout the plant. Subsequently, the bacteria adhere to the interior surfaces of xylem vessels. This is accompanied by the accumulation of xanthan gums in vessel lumina. Later, tyloses grow into affected vessels, causing further blockage (Stevenson et al., 2004). These sequelae disrupt water flow and place the vine under potential water deficit. Symptoms are exacerbated in hot or arid climates, but are not in themselves produced by water stress (Thorne et al., 2006). Details of the mechanisms of pathogenesis can be found in Chatterjee et al. (2008). Leaf symptoms (scorch) develop as a progressive inward browning and desiccation of the blade. In advance of the necrosing region, concentric areas of discoloration commonly develop. They are yellow in white cultivars and red-purple in red cultivars. The blade may eventually drop, leaving the petiole still attached to the shoot (Stevenson et al., 2005). This produces a symptom called “matchstick.” Late in the season, “green islands” may remain on canes, surrounded by brown mature bark. These islands are associated with regions where peridermal differentiation is absent. In severely affected vines, budbreak is delayed, and shoot growth is slow and stunted. The first four to six leaves are dwarfed, and the main veins are bordered by dark-green bands. Subsequent leaves generally are more typical in size and appearance. Infected V. vinifera cultivars may survive for 1e5 years, depending on the age of the vine when infected, the variety, and the local conditions. Young vines are particularly susceptible and frequently succumb within 2 years. Until the late 1990s, with the spread of the glassy-winged sharpshooter, infection in southern California developed in mid- to late season. Frequently, the bacterium did not survive the winter, and recovery was common (Hill and Purcell, 1995). This is no longer the situation with the new, more effective vector. In warm climates, where the pathogen and vector are common, the only effective control is growing resistant or tolerant cultivars. Where the disease is established, but localized, vineyard plantings should ideally avoid areas where reservoirs of the pathogen and vector are common, notably river banks populated with vines and shrubs, such as blackberry and elderberry. Because transfer from indigenous plants is less efficient, replanting riparian environments with native plants can reduce

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the likelihood of transmission. Transfer is predominantly from symptomless carriers, rather than from vine to vine. Thus, planting a 7-m-wide conifer or hardwood belt around vineyards in susceptible sites has been investigated as a transmission buffer. Insecticidal control of vectors has generally been unsuccessful in halting disease spread, but can be effective in dramatically reducing local vector populations. For example, imidacloprid and thiamethoxam are particularly active against sharpshooters. Thankfully, these insecticides appear to have little effect on sharpshooter egg parasitoids. In California, release of egg-parasitoid wasps (Gonatocerus spp.) has markedly reduced the population of glassy-winged sharpshooters. Other biological control agents of potential interest include mycoparasites, such as Hirsutella spp. and Beauveria bassiana, as well as various natural predators. In addition, competitive exclusion, associated with infection by asymptomatic strains of X. fastidiosa, may provide resistance to subsequent exposure to pathogenic strains. Particular rootstock/scion combinations can reduce progression and limit disease severity (Wallis et al., 2013). Thus, although not strictly a control measure, it may be a useful adjunct in disease management. Yellows diseases Several grapevine yellows syndromes have been identified, all induced by one or more types of phytoplasmas (cell-wall-less bacteria) (Lee et al., 2000). Because of their amorphous shape and small size, they are almost impossible to differentiate microscopically from cell fragments. Disease symptoms are also often poorly diagnostic. Thus, unambiguous identification requires the use of molecular techniques, such as PCR. Although associated with infection by members of the provisional genus Candidatus (Anonymous, 2004), they are usually classified by their 16S ribosomal RNA fragments (Tran-Nguyen et al., 2008). For example, flavescence dore´e is induced by a member of the elm yellows disease group (16SrV); grapevine yellows in the eastern United States and northern Italy by a member of the western X disease group (16SrIII); Vergilbungskrankheit (Germany), bois noir (France), and southern European (Mediterranean) grapevine yellows by members of the stolbur subgroup of aster yellows (16SrIG); and Australian grapevine yellows by an Australian subgroup of aster yellows (16SrIJ). Each group appears to be transmitted by different plant- or leafhoppers: Vergilbungskrankheit and bois noir by Hyalesthes obsoletus; Australian grapevine yellows, probably by Orosius argentatus; and flavescence dore´e and eastern American forms by Scaphoideus titanus (littoralis). The vectors are not specialized grapevine pests, feeding and multiplying on a wide range of host plants. Typically,

alternative so-called “dead-end” hosts (such as grapevines) often express more severe symptoms than those on which the pathogen and standard host have developed a form of coexistence. In grapevines, phytoplasmas multiply exclusively in the phloem, where they may disrupt nutrient flow and cause growth substance disturbances. During feeding, the vector acquires the bacterium in its salivary glands. After reproducing in the insect, the bacterium accumulates in the salivary glands. This favors transmission via the fluid ejected by the proboscis just prior to feeding. Some forms of the disease are also transmissible, at a low frequency, by grafting. Because the vector, S. titanus, is endemic to eastern North America, it was thought that the phytoplasma inducing flavescence dore´e may have been introduced into Europe, along with S. titanus, in the late 1940s (Maixner et al., 1993). However, this now appears unlikely. The strains causing yellows diseases in France and eastern North America are genetically distinct. Grapevine yellows diseases are frequently characterized by several symptoms. Newly infected vines show delayed bud burst and shoot growth in the spring. Internodes are shortened and cane development may show a zigzag pattern. Leaf blades may become partially necrotic and roll downward, more or less overlapping one another, and become brittle. The foliage often turns yellow in white cultivars, red in red cultivars. Alternatively, angular colored spots may develop on leaf blades. In sensitive cultivars, the most distinctive symptom is a drooping posture that develops in the summer, due to poor lignification of the vascular tissues. In addition, shoots do not turn brown as they mature, or develop only patchy brown areas. Shoot tips may die back during the growing season and develop black pustules. Affected shoots usually die during winter. The fruit tends to shrivel and develop a dense, fibrous, bitter pulp. Symptoms typically begin to develop in the year following infection, termed the crisis year. Symptoms may be more pronounced with joint phytoplasma/virus infections. Symptoms may disappear in subsequent years, associated with induction of responses typical of systemic acquired resistance (Landi and Romanazzi, 2011). Two distinct expressions of flavescence dore´e occur in Europe. In the Nieluccio type, the disease becomes progressively more severe each year until the vine dies. In the Baco 22A type, the vine recovers after symptoms develop in the crisis year. If they are reinfected within a few years of a previous infection, recovered vines show only a localized, rather than a systemic, reaction. There is also considerable variation in cultivar sensitivity to the various forms of grapevine yellows. For example, Pinot noir is particularly susceptible to flavescence dore´e, but is little affected by bois noir. Other forms of yellows diseases may or may not show remission. In

Disease, pest, and weed management

European regions, where flavescence dore´e and bois noir occur sympatrically, symptoms of flavescence dore´e appear earlier in the season than those of bois noir (Angelini et al., 2006). A characteristic feature of bois noir, that gives it its name, is the frequent blackening of poorly ripened wood in the fall. No effective disease control is known for regions where both vector and pathogen are established. The only effective option is growing varieties that show recovery (Baco 22A expression), or that are relatively insensitive to infection. Insecticide use delays but does not stop pathogen spread. Avoidance of infected stock is required during propagation as the pathogen may be spread by grafting. Nonetheless, this recommendation is difficult to apply effectively, with graft spread being most likely during the infection year, when the source material is symptomless. Recovered vines apparently are noninfectious, and can be safely used in vine propagation. Dormant scion wood can also be cured of infection as well as vector eggs by hot-water treatment (Caudwell et al., 1997).

Viruses, virus-like, and viroid diseases Viruses and related pathogens are submicroscopic, noncellular, infectious agents, dependent on the host cell for self-replication. Those that attack grapevines possess only RNA as their genetic material. Differentiation between viruses and viroids is based primarily on the presence or absence, respectively, of a protein coat enveloping the nucleic acid. Viroids also possess a much smaller RNA genome than the majority of plant viruses. Virus-like diseases are those possessing transmission characteristics similar to those of viruses, but for which no consistent association with a pathogenic agent has yet been established. Infection is usually systemic, affecting all tissues, with the occasional exception of the apical meristem, pollen, and seed. Where apical meristem infection is absent, viral elimination is possible via micropropagation (excision of meristematic tissue and embryogenesis in tissue culture). This is particularly important where traditional vegetative propagation techniques have facilitated the perpetuation of systemic infections. Because of the systemic spread associated with grafting, the widespread adoption of grafting probably explains the global dispersion of most grapevine viruses and viroids. Although grapevine viruses and viroid infections are graft transmissible, some are also spread by nematode, aphid, mealybug, and fungal vectors (Walter, 1991). Some can also be mechanically transmitted via pruning equipment; thus the importance of frequent surface sterilization of pruning equipment. In addition to micropropagation, cultivars may be cured of some viral infections by heat treating dormant

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stem cuttings or young vines. Regrettably, the treatment appears ineffective in eliminating viroids (Duran-Vila et al., 1988). The production and propagation of virus- and viroidfree nursery stock are ongoing projects in most wineproducing countries. This is based on the belief that cured clones grow better and generate better-quality grapes than infected vines (Komar et al., 2007). Although generally true, clones free of all known systemic pathogens do not consistently outperform their infected counterparts (Woodham et al., 1984). Nonetheless, eliminating all systemic infections is desirable, if only because symptomless carriers can be a source of agents inducing debilitating diseases or limit grafting success in susceptible cultivars. Because disease-free vines remain pathogen-free while micropropagated in culture vessels, their import and propagation in the host country may reduce, if not eliminate, the need for quarantining and indexing imported vine cuttings. Detection of viral and viroid infection has historically been based on indexing. This involves inoculation of an “indicator” plant that produces distinctive disease symptoms after infection. Identification with serological techniques, especially with ELISA, cDNA, or PCR probes, can confirm indexing results, and may eventually replace the long and expensive indexing process. This is necessary as vineyards may be unknowingly infected by several viruses. In one example, eight viruses and two viroids were discovered (Jo et al., 2015). In addition, modern sequencing procedures are identifying an increasing number of grapevine viruses. Of the 65 known, only about half appear to be of economic significance. The major viruses infecting grapevines fall into one of three main groups: nepoviruses, closteroviruses, and vitiviruses (formerly classified under trichoviruses). Nepoviruses are polyhedral single-stranded RNA (ssRNA) viruses, with a genome divided unequally into two linear segments. They include the fanleaf, tomato ringspot, tobacco ringspot, and peach rosette mosaic viruses. All are nematode transmitted and cause several forms of grapevine decline. Closteroviruses are flexuous, filamentous, ssRNA viruses, with a single linear genome. A few are known to be occasionally transmissible by mealybugs or scale insects. They probably cause most instances of leafroll. Vitiviruses are also flexuous, filamentous, ssRNA viruses. The main examples are GVA and GVB. They are most frequently associated with instances of stem pitting (Kober stem grooving) and corky bark, respectively. Both viruses can be transmitted by mealybugs. Examples of other viral groups causing or associated with grapevine diseases are trichoviruses (grapevine berry inner necrosis virus), luteoviruses (grapevine ajinashika virus), grapevine fleck virus (possibly belonging to the tymovirus group), a foveavirus inducing rupestris stem pitting, a

270

4. Vineyard practice

capillovirus-like virus occasionally associated with rugose wood, as well as two recently identified viral diseases: grapevine red blotch disease (caused by a geminiviruseGRBaV) (Sudarshana et al., 2015) and grapevine vein clearing (a caulimoviruseGVCV) (Beach et al., 2017). Most, if not all, grapevine viruses are graft transmissible. The exact causal nature of many presumably viral and viroid diseases in grapevines remains unclear. Investigation is confounded by many cultivars being symptomless carriers, the slow development of symptoms, the apparent inconsistent association with a recognizable pathogen(s), and the complex etiology of symptom expression. In some instances, several viroids or viruses, or both, may be required for expression, or may modify disease expression. For example, veinbanding disease develops only when grapevines are jointly infected with both GFLV and GYSVd1 (Szychowski et al., 1995). Fanleaf degeneration Fanleaf degeneration possesses the longest known historical record of any grapevine virus. Its symptoms are identifiable in herbarium specimens more than 200 years old. Because of its long history in Europe, and the absence of infection in free-living North American grapevines, GFLV is assumed to be of European or Near Eastern origin. Its current distribution is believed to be due to grafting and the dispersion of European cultivars worldwide. Natural spread in vineyards is slow because of the limited movement in soil of its major nematode vectors, Xiphinema index and X. italiae (about 1.5 m/year). Because grapevine roots typically do not form natural grafts, vine-to-vine transfer is unlikely. Although transferable to other plants, the virus is limited to grapevines under field conditions. This may result from the limited host range of its vectors. The impact of infection varies widely, depending on cultivar tolerance and environmental conditions. Tolerant cultivars are little affected by infection, whereas susceptible varieties show progressive decline. Nonetheless, fanleaf degeneration is generally viewed as the most damaging grapevine viral disease. Yield losses can reach up to 80%, with any fruit that does mature being of poor quality. Infected vines have a shortened life span, increased sensitivity to environmental stresses, and reduced grafting and rooting potential. Three distinctive syndromes have been recognized, based on particular strainecultivar combinations. These are malformation, yellow mosaic, and vein-banding. Fan-shaped leaf malformation is the most distinctive foliage expression. Chlorotic speckling and a leathery texture commonly accompany leaf distortion. Shoots may be misshapen, showing fasciation (stem flattening), a zigzag pattern at the nodes, atypically variable

internode lengths, double nodes, and other aberrations. Fruit set is poor and bunches are of reduced size. The yellow mosaic (chromatic) syndrome develops as a strikingly bright-yellow mottling of the leaves, tendrils, shoots, and inflorescences in the early spring. Discoloration can vary from isolated chlorotic spots to uniform yellowing. The third expression, vein-banding, develops as a speckled yellowing on mature leaves, bordering the main veins in mid- to late summer. In both discoloration patterns, leaf shape is normal, but fruit set poor, with many shot berries (hen and chickens appearance). Fanleaf degeneration also shows a characteristic intracellular development of trabeculae. These appear as strands of cell-wall material spanning the lumen of xylem vessels. Where both vector and virus are established, planting vines grafted on rootstock resistant to the virus is usually required (vector tolerance still permits infection). Fumigation of the soil with nematicides can, to varying degrees, reduce but not eradicate nematode vector presence. Allowing the land to lie fallow can be useful in reducing nematode populations, but the strategy often requires an impractical 6e10 years. The long requisite fallow period probably results from nematode survival on undislodged roots. Roots occasionally can remain viable for up to 8 or more years following vine uprooting (McKenry and Buzo, 1996). Leafroll Leafroll is another globally distributed, debilitating grapevine virus. It is associated with infection by one or more, of up to 10, GLRaVs (Almeida et al., 2013). They are all ssRNA viruses and members of two subgroups of closteroviruses, GLRaV-3 being the most widespread and economically important. Leafroll may be induced singly, or in combination with any of these strains. Two vitiviruses (GVA and GVB) have also been associated with leafroll, although they are more commonly associated with Kober stem grooving and corky bark, respectively. Symptomatic expression varies considerably, but generally does not lead to vine degeneration. Many scion and rootstock cultivars are symptomless carriers of the infectious agent(s). As with fanleaf degeneration, the agents probably originated in Europe or the Near East. Most feral North American grapevines have yet to be found to carry any strains. Spread of the causal agent(s) depends primarily on graft transmission. Nevertheless, insect vectors may occasionally be involved. For example, GLRaV-1, -3, and -5 can be transmitted by phloem feeders, such as mealybugs or soft scale insects (Sforza et al., 2003). In the phloem, the virus induces callose accumulation. This can disrupt translocation of nutrients from the leaves to other parts of the vine. Several species of mealybugs also have been reported to transfer GVA and GVB.

Disease, pest, and weed management

The disease complex derives its name from a marked, basal leaf downrolling. Symptoms most frequently develop late in the season. The interveinal areas of leaves may also turn yellow-chlorotic (white cultivars) or deep red (red cultivars). In contrast, the main veins remain distinctly green. Infected vines can occasionally be detected by their retention of leaves much longer than adjacent healthy vines. In addition, leaf blades may fall, leaving petioles still attached to the cane. Whole vines, as well as individual shoots and leaves, are dwarfed in comparison to healthy plants. Disrupted photosynthesis can seriously delay fruit ripening and reduce yield (w40%). Other influences are reduced sugar content, enhanced titratable acidity, and altered pigmentation. The productive life span of a vine is markedly reduced. Disease expression is often more obvious and debilitating in cool climates. The discovery that the mealybug Planococcus ficus can feed on grapevine roots, and the virus remains viable on vine root remains for extended periods after roguing, is of considerable concern. Because control measures have been directed at above-ground feeding, eliminating above-ground parts is insufficient, leaving viruliferous insects to survive on root remnants, infecting replacement vines. P. ficus is the key vector in South Africa, the Mediterranean, Argentina, and now some parts of California. Effective insecticides are available (Daane et al., 2012), but virus transmission can occur before the vector is killed. Thus, coccinellid predators and parasitic wasps (e.g., Anagyrus) are valuable adjuncts in vector control. However, their effectiveness is limited by ants feeding on mealybug honeydew, deterring predator/parasite attack. Thus, ant stem barriers may be required to increase the efficacy of biological control (Addison, 2002). Additional measures include the application of pheromones to disrupt mealybug mating, weed control (to remove alternative host plants), and extended fallowing before replanting (Pietersen et al., 2013). Where the incidence of the disease is comparatively low, roguing is often limited to the affected area. Disease-free nursery stock may be generated by thermotherapy or by micropropagation. Because the identity of all causal agents still remains unestablished, confirmation of elimination of the infectious agent(s) from source plants is performed by grafting to sensitive cultivars (indexing). The economic implications of the various control options are discussed by Atallah et al. (2012). Yellow speckle Yellow speckle is a widespread, but relatively minor, viroid disease of grapevines. Other viroids occur in grapevines, but their economic significance and relationship to recognized grapevine diseases remain unclear. Symptoms of infection by grapevine yellow speckle viroids (GYSVd1 and GYSVd2) are often temporary, and

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develop only under special climatic conditions. Foliar symptoms generally develop at the end of the summer, and consist of leaf spotting. When sufficiently marked, the scattered chlorotic spots may resemble the veinbanding symptom of fanleaf degeneration. Studies suggest that shoot growth is slightly curtailed and grape acidity is reduced (Wolpert et al., 1996). Control is dependent on planting viroid-free vines. Elimination of the causal agent can be achieved by micropropagation. Thermotherapy is ineffective (Barlass and Skene, 1987).

Nematode pathogens Nematodes are a large group of microscopic, unsegmented roundworms that live predominantly as saprobes in soil. However, some are parasitic on plants, fungi, and animals. Those attacking grapevines are restricted to feeding on roots. They derive their nutrition from extracting the cytoplasmic fluids from root cells. This is accomplished with a spearlike stylet that punctures host cells. Feeding may be restricted to the surface of roots or, following burrowing, may occur in the root cortex. In addition to the direct damage caused by feeding, and the resultant root disruption, nematodes may transmit viruses and facilitate infection by other pathogens. Active dispersal by nematodes in soil is both slow and limited, with most long-distance movement being through the action of wind and water or vineyard machinery, or by the translocation of infested plants. Reproduction occurs via egg production, with or without the interaction of males. The multiyear survival of eggs in a dormant state often markedly reduces the effectiveness of fallowing in nematode control. The degree of damage caused by nematodes (including that of the viruses they transmit) was poorly understood until the effects of applying preplant soil fumigants became common. Combined with fallow, fumigants can dramatically reduce nematode populations in shallow, sandy soils. Fumigants have more limited efficacy in deep or clayey soils, where penetration is restricted. Effective sterilant action requires penetration to a depth of 40e120 cm (the zone of highest root densityd depending on soil structure). The most effective and widely used nematicide, methyl bromide, is scheduled for deregulation (but with exceptions). Methyl iodide appears to be an effective alternative (Ohr et al., 1996) and is not a stratosphere ozone depleter. Other potential options include sodium methyldithiocarb (Vapam), chloropicrin þ iodomethane, propargyl bromide, dimethyl disulfide, sulfuryl fluoride, and possibly sodium and potassium azide (Schneider et al., 2006; Cabrera et al., 2011). Application of specific yeasts (Hashem et al., 2008) and/or compost (Weckert et al., 2009) has shown some

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4. Vineyard practice

promise, and can be used in functioning vineyards. Nonetheless, effective nematode control in the future may require integrated nematicide applications, increased use of resistant/tolerant rootstock, and modified cultural practices (Zasada et al., 2010). Because of the perennial nature of grapevines, most long-term control measures entail the use of resistant or tolerant rootstocks. Although no current rootstock cultivar is resistant to all grapevine-attacking nematodes, some are relatively resistant or tolerant to one or more of the important pathogenic genera. Therefore, determination of both the actual and the potential nematode pests (and viruses they could transmit) can be a critical component of rootstock selection in vineyard establishment (Walker, 2009). For example, 10-17A provides broad nematode resistance, but not for fanleaf degeneration (GFLV), whereas O39-16 supplies better protection in situations where both X. index and GFLV are present. Problems in selecting the “best” rootstock are illustrated in McKenry et al. (2004). Before replanting an infested vineyard, the old vines are usually cut off at just above the soil, the trunks are painted with a systemic herbicide to kill the roots, and the field is left fallow for 1 year. Without resistant rootstock or fumigation, fallow periods of at least 7e10 years are recommended. Where ground covers are used, planting nematoderesistant crops is an important factor. For example, Cahaba white vetch (Vicia sativa), barley, and Blando brome grass have an advantage in sites known to harbor parasitic nematodes. Conversely, nematodefavorable hosts, such as peas, lupins, clovers, and most vetch crops, should be avoided. Mulching of underrow cover crops, such as biofumigant Brassica spp., reduces nematode populations (McLeod and Steel, 1999; Rahman and Somers, 2005). Brassicas (e.g., mustard, arugula, horseradish, capers) produce glucosinolates that remain inactive while in the living plant. Upon mulching, though, these compounds are hydrolyzed by myrosinase, releasing volatile isothiocyanates. These help to biofumigate vineyard soils (Kruger et al., 2013). Saponins from Gypsophila paniculata (baby’s breath) are also a potential bionematocide, and one that does not inactivate mycorrhizal spores (Pensec et al., 2013). For more on bionematicides see Ntalli and Caboni (2012). The detection and identification of nematode problems usually require microscopic examination of the root system. Above-ground symptoms are insufficiently diagnostic. Many nematode species can attack grapevine roots, but few induce significant damage. The most serious are the root-knot (Meloidogyne) and dagger (Xiphinema) nematodes. They can both cause significant root damage, as well as transmitting several serious grapevine viruses. Other nematodes occasionally found feeding on grapevine roots include species of

Pratylenchus (lesion nematodes), Tylenchulus (citrus nematodes), and Criconemella (ring nematodes). Root-knot nematodes Root-knot nematodes (Meloidogyne spp.) are sedentary endoparasites that penetrate young feeder roots. After penetration, adjacent cells are stimulated to divide and increase in size, producing gall-like swellings (Fig. 4.66). Where multiple infections occur, knot formation may give the root a chain-of-beads appearance. Reproduction commonly occurs during the spring and fall root-growth periods (de Klerk and Loubser, 1988). Each female may produce up to 1500 eggs. Second-stage juveniles have been shown to travel up to 30 cm in 3 days through sandy loam (Bettiga, 2013), movement often being directed toward compounds (semiochemicals) released by vine roots. The most important species are Meloidogyne incognita, M. javanica, M. arenaria, and M. hapla. Infection results in a decline in vigor and yield, as well as increased susceptibility to water and nutrient stress. Symptomatic severity can be limited by increasing the water supply and pruning (yield limiting). Damage is typically most marked in young developing vines planted in highly infested, lighttextured soils. Older vines with deep root systems seem to be less affected by root-knot nematodes. Dagger nematodes In contrast to root-knot nematodes, dagger nematodes (Xiphinema spp.) are migratory and feed on epidermal cells near the root tip (van Zyl et al., 2012). Concentrated feeding may initiate root bending, followed by lesion darkening. Extensive attack causes root death and induces the production of tufts of lateral roots. In addition to destroying roots, X. index is a principal vector of GFLV. The combined action of both nematode and viral infections can quickly make a vineyard commercially unproductive. Other species of Xiphinema may transmit a range of other, but less serious, grapevine viruses (see Walter, 1991).

Insect and mite pests Insects and mites can cause extensive grapevine damage. Although most infest only a single part of the vine, all components are attacked by one or more species. Control measures have largely been based on pesticides, but greater emphasis is currently being placed on cultural and biological controls. Insects and mites are both distinguished by a hard exoskeleton. This is shed several times during growth. Mites and some insects pass through several immature, adult-like stages before becoming reproductive, whereas most insects pass through several wormlike (larval) stages and a pupal stage before becoming a short-lived adult.

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Disease, pest, and weed management

Late II stage larvae feeding on giant cells. Root begins to from gall

III Stage larvae 3rd molt

2nd molt

IV Stage larvae 4th molt Adult nematodes Male leaves root

II Stage larvae invade rootlet and cause formation of giant cells

II Stage larvae attack rootlets

Small galls appear on recently infected roots

Emerging II Stage larve infect new roots

Egg sac

1st molt II Stage larva free in soil II Stage larva

I Stage larva

Galls at various stages of development on roots of infected plant

Egg

Female lays eggs into egg sac

Old galls may contain many egg laying females and new infections

FIGURE 4.66 Disease cycle of root-knot nematodes. From Agrios, G.N., 1997. Plant Pathology, fourth ed. Academic Press, New York, NY, reproduced by permission.

Insects are distinguished by possessing three main body parts (head, thorax, and abdomen), sensory antennae, three pairs of legs attached to the central thorax, and a segmented abdomen. In contrast, mites possess a fused cephalothorax (broadly attached to an unsegmented abdomen), have four pairs of legs (except in the initial, immature stage), and lack antennae. Two pairs of legs are attached to the cephalothorax and the other pairs are located on the abdomen. Both groups reproduce primarily by egg production. Although some insects have evolved intimate relationships with grapevines, notably phylloxera, others are nonspecialists, infesting a wide range of hosts. As with fungal pathogens, the degree of infestation does not necessarily correlate with severity. Severity depends more on the specific genetic properties of the cultivare pest interaction, macro- and microclimatic conditions, soil properties, and resistance to control agents. Phylloxera Of all grapevine pests, the obligate parasite phylloxera (Daktulosphaira vitifoliae) has had the greatest impact on viticulture and wine production. In North America, it is a minor endemic pest of Vitis species,

east of the Rocky Mountains. Most species of the Euvitis are variously affected by either root or leaf galls (Table 4.14). Feeding by the insect activates genes normally associated with fertilized flowers initiating carpel formation (Schultz et al., 2019). However, no North American species experiences the destructive swollen galls (tuberosities) that form on V. vinifera roots. Some Vitis species, such as V. berlandieri, are relatively resistant to both effects, but full resistance occurs only in the Muscadinia (notably V. rotundifolia). Most Vitis species, growing beyond the original range of phylloxera (North America), are susceptible to root galling and occasionally both root and leaf galling. It seems likely that importation of rooted V. riparia stock from the United States introduced the pest to Europe, initiating a progressive devastation of European vineyards, commencing in the 1860s (Downie, 2002). V. vinifera was particularly susceptible to root galling. Subsequent inadvertent introductions have led to phylloxera spreading almost worldwide. Californian and South African strains appear to have been derived from populations found on V. vulpina in the southern Gulf states (Downie, 2002). Strains in Australia, New Zealand, and Peru seem to have

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TABLE 4.14

Susceptibility of Vitis species to root and leaf expressions of phylloxera.

Host reaction

Resistant (bearing no to few galls on roots)

Tolerant (bearing many galls on roots)

Vitis (V.) species already exposed to phylloxera (eastern North America) Resistant (bearing no to few galls on leaves)

V. rotundifolia

V. aestivalis

V. berlandieri

V. girdiana

V. candicans

V. labrusca

V. cinerea

V. lincecumii

V. cordifolia

V. monticola

V. rubra Tolerant (bearing many galls on leaves)

V. riparia (V. vulpina)

None

V. rupestris Vitis species not previously exposed to phylloxera (western North America, Asia, Europe, and Middle East) Resistant (bearing no to few galls on leaves)

V. coignetiae

V. arizonica V. californica V. davidii V. romanetti V. ficifolia (¼V. flexuosa) V. vinifera (incl. V. sylvestris)

Susceptible (bearing many galls on leaves)

V. betulifolia

V. amurensis

V. reticulata

V. piazeskii

From Wapshere, A.J., Helm, K.F., 1987. Phylloxera and Vitis: an experimentally testable coevolutionary hypothesis. Am. J. Enol. Vitic. 38, 216e222, reproduced by permission.

originated in California. There is concern that indigenous Chinese Vitis spp., which are susceptible to phylloxera, may be endangered by its introduction in China (Du et al., 2009). It has been the general view that phylloxera provoked a similar decimation of the wild (sylvestris) populations in Europe. However, destruction of its natural habitats (along flood plains and riverbanks), combined with damage caused by the introductions of two earlier devastating diseases (powdery and downy mildews), may have been more instrumental in their decline. Remnant populations in central and southern Europe show few signs of phylloxera infestation (Ocete et al., 2011), despite their sensitivity when inoculated under laboratory conditions. This anomaly is hypothesized to be due to the anoxic conditions generated by the annual flooding and/or gravity-induced sedimentary accumulations in the vine’s natural riverine habitats. Both are unfavorable to pest infestation. At the height of its spread in Europe, phylloxera destroyed more than 2 million hectares, and almost brought European wine production to a halt in severely affected areas. It also demonstrated the urgency for laws regulating the importation of vine material (still

inadequately appreciated, or at least applied, in some countries), and provided the first clear example of the effectiveness of biological control. Grafting sensitive V. vinifera cultivars to resistant rootstocks was so successful that the danger posed by phylloxera was partially forgotten. The predominant use of a single resistant rootstock cultivar (AR#1) in California resulted in considerable hardship when a new biotype (biotype B, a mixture of genetically different strains) appeared that seriously infested AR#1 (previously unaffected by biotype A). It necessitated replanting about 5000 ha in Napa and Sonoma between 1988 and 1995. Biotypes other than the two main current variants have also been identified in California (De Benedictis et al., 1996). The need to replant vineyards on a massive scale has apparently permitted several relatively minor vascular pathogens of older grapevines to make their presence known. Both Phaeomoniella chlamydospora (Petri disease) and Cylindrocarpon (black foot disease) caused significant young vine decline in replanted vineyards (Scheck et al., 1998). This expression of a replant problem is distinct from other forms associated with soil infested with fungi (e.g., Armillaria mellea and Pythium spp.), bacteria (e.g.,

Disease, pest, and weed management

fluorescent pseudomonas), or parasitic nematodes (and the viruses they can transmit). The complexities of the aphid-like phylloxera life cycle are detailed in Forneck and Huber (2009), and illustrated in simplified form in Fig. 4.67. Several asexually reproduced generations may occur per year. Despite a winged stage that can arise from root galls, longdistance dispersal without human involvement is slow. For the sexual stage to typically occur, the insect must pass sequentially through both leaf- and root-galling phases. However, many Vitis spp. are comparatively resistant to one of these gall phases (Table 4.14). Thus, absence of a leaf-galling phase (Fig. 4.68) probably explains the absence of the sexual cycle in California. This also applies to most other viticultural areas where phylloxera is not indigenous. Occasionally, grafted cultivars may develop leaf galls. These are thought to develop from nymphs that migrate from leaf galls on shoots that sporadically arise from rootstocks (Remund and Boller, 1994). Phylloxera biotypes differentially pathogenic on grapevine cultivars, such as the one that arose in California, appear to be common (King and Rilling, 1991; Forneck et al., 2000). This occurs despite the rarity of the sexual stage in most infested areas. Differences in

Winter egg

Sexual forms

Wander to other leaves

Cycle on LEAVES

Fall to ground, attack roots

Winged form

Wander to roots of other vines

Nymph

Cycle on ROOTS

FIGURE 4.67 Life cycle of grape phylloxera. From FergussonKolmes, L., Dennehy, T.J., 1991. Anything new under the sun? Not phylloxera biotypes. Wines Vines 72 (6), 51e56, reproduced by permission.

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pathogenicity may be expressed in the ability of phylloxera biotypes to feed, stimulate gall formation, or reproduce rapidly. Susceptible hosts respond to root feeding by directing nutrients to the damaged sites, favoring gall formation. This is probably induced by IAA, a constituent of phylloxera saliva (Scha¨ller, 1968). Feeding also induces the formation of distinctive hook-shaped bends and swellings on young roots called nodosities (Plate 4.12). These soon succumb to secondary infection by fungi, such as Fusarium, Pythium, and Cephalosporium spp. (Granett et al., 1998). In contrast, more mature roots produce semispherical swellings termed tuberosities. These give the root a roughened, warty appearance. Their development is the more serious manifestation of infestation. They markedly disrupt water and nutrient flow in the root’s vascular tissues. The insect population is maintained primarily on tuberosities. Rapid nodosity decay, and the seasonal production of new feeder roots, can limit the development of dense populations of feeding insects (Williams and Granett, 1988). Resistant rootstock cultivars show limited, if any, galling, followed by rapid healing. In Bo¨rner, for example, the roots react to feeding with localized hypersensitivity and necrosis (Dietrich et al., 2010); regrettably, the cultivar is susceptible to lime-induced chlorosis. In contrast, tolerant rootstocks show galling. Nonetheless, vigorous root growth normally compensates for any damage, and nodosities support only limited insect populations (Boubals, 1966). Adverse cultural conditions may, however, lead to progressive damage resulting in vine decline, termed phylloxera “comeback.” Above-ground symptoms of root infestation are relatively indistinct, and are primarily expressed as a progressive vine decline. The first clear indications of infection usually appear as stunted growth and premature leaf yellowing. With sensitive cultivars, these effects annually become more marked, finally culminating in vine death. Phylloxera root infestation can be confirmed only by root observation. The distinctive lemon-yellow eggs (Plate 4.13) and clusters of yellowish-green nymphs and adults on young roots are diagnostic. Tuberosities isolated from dying vines usually possess few phylloxera. Nevertheless, the presence of the tyroglyphid mite Rhizoglyphus elongatus appears to be an indicator of past phylloxera presence. The mite lives on the decaying cortical tissues of tuberosities. Many environmental conditions can affect the severity of vine attack. It is well known that phylloxera infestation is much less significant on sandy soils. It has been suggested that this results from the higher silicon content, either in the soil solution or in vine roots

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FIGURE 4.68

Vine leaf infested by phylloxera. Inset: gall in cross section showing phylloxera. From The Gardener’s Chronicle, reproduced in Barron, A.F., 1900. Vines and Vine Culture, fourth ed.. Journal of Horticulture, London.

PLATE 4.13

PLATE 4.12 In vitro-generated phylloxera nodosity on “Cabernet Sauvignon” (3 days old). From Forneck, A., Kleinmann, S., Blaich, R., Anvari, S.F., 2002. Histochemistry and anatomy of phylloxera (Daktulosphaira vitifoliae) nodosities on young roots of grapevine (Vitis spp.). Vitis 41, 93e98, reproduced by permission.

Close-up of colony of phylloxera adult female, eggs, and nymphs. Photo by Jack Kelly Clark, from Larry J. Bettiga, Grape Pest Management, University of California, Agriculture and Natural Resources, Copyright © 2013 The Regents of the University of California. Used by permission.

(Ermolaev, 1990). The higher temperatures (>32
C) that may occur in sandy soils are also unfavorable to phylloxera and limit its damage (Foott, 1987). Irrigation and fertilization can occasionally diminish the damage, whereas drought increases severity (Flaherty et al., 1982).

Quarantine is a vital component limiting phylloxera spread to noninfested areas. Even where the pest is present, quarantine may prevent the importation and distribution of new biotypes. In nurseries, vines can be disinfected by placing the washed root system in hot water (52
Ce54
C) for about

Disease, pest, and weed management

5 min. Nursery soils can be disinfected by pasteurization or fumigation. If the vineyard soil is already infested, the major control measure remains grafting to resistant rootstocks. Typically, rootstocks with some V. vinifera parentage should be avoided (Granett et al., 1996). Nevertheless, 1202C and O39-16 seem exceptions to this rule, at least in California. Whether rootstocks of pure North American Vitis parentage will continue to remain resistant to phylloxera under vineyard monoculture is unknown. Reports of decline have been noted in Germany. It is hoped that advances in categorizing phylloxera strains via DNA fingerprinting will assist in understanding the occasionally conflicting worldwide data on rootstock resistance or tolerance to phylloxera. Although chemical agents such as aldicarb (Temik) (Loubser et al., 1992) have shown some promise against phylloxera, control will remain dependent on quarantine and grafting for the foreseeable future. Leafhoppers (sharpshooters) Specialized xylem-feeding insects, notably leafhoppers and related forms, are not only harmful pests in their own right, but also critical vectors in the transmission of several destructive pathogens. In many grapegrowing regions of North America, the western and eastern grape leafhoppers (Erythroneura elegantula and E. comes, respectively) are the most important species; in southern California, the variegated leafhopper (E. variabilis) and the nonindigenous glassy-winged sharpshooter (Homalodisca coagulata) are the major species, whereas the potato leafhopper (Empoasca fabae) is particularly significant in the southeastern United States. About 20 species of sharpshooters and leafhoppers are vectors of Xylella fastidiosa, the causal agent of Pierce’s disease. In Europe, Empoasca leafhoppers tend to be particularly damaging, especially E. vitis. Scaphoideus littoralis (synonym S. titanus) is the primary vector of flavescence dore´e. In South Africa, Acia lineatifrons is the prominent species. Most of these insects have fairly similar life cycles and are controlled by similar techniques. Depending on the species and prevailing conditions, the insects may go through one to three generations per year. Those species passing through several generations per year are generally more serious, as their numbers can increase dramatically throughout the season. Leafhoppers typically lay their eggs on the underside of leaves. After hatching, the nymphs pass through five molts before reaching the adult stage (Fig. 4.69). All stages feed on the cytoplasmic fluid of leaf and fruit tissue. Feeding results in the formation of white spots, which on heavily infested leaves leads to a marked loss in color. Growth of one or more hyphomycetes on escaped sap and insect honeydew can produce a sooty

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appearance on plant surfaces. Pronounced damage is usually caused only when infestations reach >10e15 leafhoppers/leaf. Severe infestation can lead to leaf necrosis, premature defoliation, delayed berry ripening, and reduced fruit quality. Some varieties, notably latematuring cultivars, tend to sustain greater damage than early-maturing varieties. Such cultivars not only endure leafhopper infestation for a longer period, but may also suffer from the migration of leafhoppers from early-maturing varieties. Most leafhopper species affecting grapevines do not infest grapes exclusively. Thus, in the fall and early spring months, they often survive and multiply on other plants. Adult overwintering can occur under leaves, weeds, or debris, in and around vineyards. Control measures have been increasingly based on enhancing the population of indigenous parasites and predators. In California, the wasp Anagrus epos is an effective parasite on the eggs of the western grape leafhopper (less so on the variegated leafhopper). Their short life cycle permits up to 10 wasp generations per year. By July, the parasite population may reach levels sufficient to destroy 90%e95% of leafhopper eggs. The numbers of A. epos can be augmented by selective habitat diversification, such as planting prune trees upwind from vineyards (Murphy et al., 1996). The windbreak produced by the trees further enhances parasite concentration. Another parasitic wasp, Aphelopsus cosemi, attacks nymphs, resulting in their sterilization. Several predatory insects, such as lacewings and ladybugs, as well as the general predatory mite Anystis agilis, attack leafhoppers. The release of commercially reared green lacewings, Chrysoperla spp., can be both effective and economically feasible if the timing is correct. Release should coincide with egg hatching, and at a population of about 15e25 leafhoppers per leaf (Daane et al., 1993). Of cultural practices, basal leaf removal can be useful, as most first-generation leafhoppers occur on basal leaves. Members of the genus Gonatocerus (Pilkington et al., 2005) are the principal parasitoid wasps feeding on eggs of the glassy-winged sharpshooter. They can infest 10%e50% of the eggs during the first generation, and up to 90%e100% on the second late-summer population. Anagrus epos may also prove effective. One of the problems associated with this, or any other control measure against these insects, is that its major significance derives from their being vectors for Xylella fastidiosa, the agent of Pierce’s disease. As such, just a single feeding by a carrier can result in effective X. fastidiosa transmission. The feeding damage caused by the sharpshooter is itself relatively insignificant. Of potential interest is the use of Alcaligenes. This is a symbiotic bacterium that limits the multiplication of X. fastidiosa in the insect vector (Bextine et al., 2004). Symbiotic control is also under investigation to control Scaphoideus titanus, the major leafhopper carrier

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4. Vineyard practice

Summer 3

4

About May 1 to June 25 vine growth is rapid.

2

Eggs, third brood.

Eggs, Second brood.

Warm summer season. Wingless young hoppers About (nymphs) appear. Apr. 15 to May 15 Overlapping broods with adult hoppers, eggs, first brood. Vine eggs and nymphs in most Shoots 10ʺ to 20ʺ. JUNE JULY untreated vineyards. About Mar. 15 AUG MAY to Apr. 15 vines budding.

Spring

APR

SEPT

MAR

OCT

About Feb. 15 to Mar. 15 vines still dormant.

FEB

Vines become dormant. Leaves fall.

NOV JAN

Fall

First warm days. Overwintering adult hoppers become active and move to green feed.

DEC Adult hoppers seek protection in leaves and other refuse.

Overwintering hoppers active on warmest days. Dec. 1 to Feb. 15 vines dormant; pruning season.

1

Adult leafhoppers winter over in weeds and refuse offering protection.

5

Winter

FIGURE 4.69 Life cycle of grape leafhoppers in California. From Flaherty, D.L., Jensen, F.L., Kasimatis, A.N., Kido, H., Moller, W.J. 1982 Grape Pest Management. Publication No. 4105. Cooperative Extension, University of California, Oakland, CA, reproduced by permission.

of the phytoplasma causing flavescence dore´e (Marzorati et al., 2006). Chemical control of leafhoppers has shifted from synthetic pesticides, such as organophosphates, to “softer,” nicotine-based compounds (e.g., imidacloprid). When required, they have the advantage of causing minimal disruption to beneficial biocontrol agents.

lay egg clusters on or close to flowers and grape bunches. Those less specialized to grapevines lay eggs on leaves. The eggs hatch into pale-colored (A)

Tortricid and other moths Grapevines are attacked by a wide diversity of tortricid moths. In southern regions of Europe Lobesia botrana is the major pest species, whereas in more northern regions Eupoecilia ambiguella is the principal agent. In much of eastern and central North America, the most damaging tortricid is the grape berry moth, Paralobesia (Endopiza) viteana (Fig. 4.70A). In California the omnivorous leafroller Platynota stultana (Fig. 4.70B) and the orange tortrix Argyrotaenia citrana are the notable forms in warmer and cooler regions, respectively. In Australia the light-brown apple moth Epiphyas postvittana is the major tortricid of significance. It has recently been found in California, and the species is now naturalized throughout much of New Zealand. Because of their taxonomic affinity, tortricid moths possess relatively similar life cycles. Adult females

Adult

Larva

(B)

Adult

Larva

FIGURE 4.70 Diagrams of the adult and larval stages of (A) the grape berry moth (Endopiza viteana) and (B) the omnivorous leafroller (Platynota stultana).

Disease, pest, and weed management

larvae that feed predominantly on flowers and developing fruit or on leaves, depending on the species. Following several molts, the larvae form a weblike cocoon in which they metamorphose into pupae. After a variable period, adult moths emerge and mate, initiating the next generation. Adults are small, relatively inconspicuous brown moths. They generally possess a bell-shaped wing profile, and develop projecting, snoutlike mouthparts. Depending on the species, and prevailing climatic conditions, two to four generations develop per year. Although tortricid moths possess many properties in common, significant differences occur in their control. For example, European berry moths form cocoons under rough bark and in the crevices and cracks on trellis posts. American berry moths form folds in leaves in which they spin their cocoons. These eventually fall to the ground. Although tortricid moths affecting grapes in California and Australia form cocoons, the insects do not hibernate as pupae, as do other American and European berry moths. The Californian tortricid moths survive as larvae in web nests formed in mummified grape clusters, or on other vine and vineyard debris. These hibernating characteristics influence the type and success of sanitation used in reducing their overwintering success. Second- and third-generation larvae are the most damaging, due to their increase in numbers throughout the season. In addition to direct-feeding damage, larvae produce wounds subsequently infected by bunch-rot fungi, as well as spoilage yeasts and bacteria. Their action can further attract infestations by fruit flies (Drosophila spp.). Not only can tortricid larvae produce wounds, facilitating bunch rot, but several species are carriers of Botrytis. The details of transmission are most well established for the European berry moth (Lobesia botrana), but the light-brown apple moth has the same potential in Australia (Bailey et al., 1997). Adult females of L. botrana preferentially select Botrytis-infected berries on which to lay their eggs, while the larvae selectively feed on infected berries (Mondy et al., 1998b). In addition, females raised on Botrytis-infected grapes produce more eggs (Mondy et al., 1998a). As with other insect pests, increased emphasis is being placed on maintenance by endemic predatory and parasitic agents. The diminished use of pesticides and the establishment of habitats for sustaining populations of indigenous control agents are essential to sustained biological control (Sengonca and Leisse, 1989). In addition, synthetic pesticides are being replaced by commercial preparations of Bacillus thuringiensis, or by pheromone applications to disrupt mating success. The effectiveness of pheromones in disorientating male Lobesia botrana is affected by the height of the applicators and development of the leaf canopy (Sauer and Karg,

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1998). Grape leaves partially absorb pheromones, slowly releasing them into the atmosphere (Schmitz et al., 1997). In many instances, the release of artificially reared egg parasites, such as Trichogramma minutum and T. embryophagum (Plate 4.14), has been successful in controlling several tortricid pests. Another species, Trichogramma carverae, appears to provide effective control of the light-brown apple moth (Epiphyas postvittana). Green lacewings (Chrysoperla spp.) and many spiders are also active predators. Egg and pupal parasites kill the insect before damage can be done, whereas larval predators and parasitoids limit population buildup by preventing reproduction. Frequently, biological agents provide adequate control. However, the availability of several selective pesticides provides an alternative option when other control measure are inadequate (Varela et al., 2010). Where insects overwinter on the ground, row cultivation to bury hibernating pupae or larvae can be useful. This can be effective as it may take several years for tortricid populations to build up to damaging numbers (Bettiga, 2013). For species not specialized to grapevines, such as Lobesia botrana, the use of grass vs. broadleaf cover crops (which the pest prefers) can diminish their incidence on vines. Other moth species damaging grapevines include the garden tiger moth (Arctia caja), the turnip moth (Agrotis segetum), and the dark sword-grass moth (Agrotis ipsilon). Mealybugs Mealybugs are a group of small (150 mm total available water in the root zone (water extracted by roots < 0.15 MPa) • >75 mm of readily available water in the root zone (water extracted by roots < 0.2 MPa) • >15% air-filled pore space •