Cytology, Histology and Histochemistry of Fruit Tree Diseases [1 ed.] 9780429265303, 9780429560323, 9780429564796, 9780429555855, 9780367210861

Table of contents :

1. Historical Aspects of the Pathological Anatomy of Fruit Tree Diseases


Alan R. Biggs






2. Anatomy and Histochemistry of Wound Responses in Bark Tissues and Implications for Management of Canker Diseases


Alan R. Biggs






3. The Cytology and Histology, and Histochemistry of Apple Scab


Christopher M. Becker






4. The Cytology and Histology of Pear Rust


Mitsuru Kohno and Hitoshi Kunoh






5. Cytology, Histology, and Histochemistry of Fruit Infection by Monilinia Species


Hayden J. Willetts and Suzanne Bullock






6. The Pathological Anatomy of Zygophiala jamaicensis on Fruit Surfaces


Hideo Nasu and Histoshi Kunoh






7. Pathological Anatomy of Fire Blight caused by Erwinia amylovora


Henk J. Schouten






8. Pathological Anatomy and Histochemistry of Leucostoma Canker on Stone Fruits and Other Selected Cankers, on Deciduous Fruit Trees


Alan R. Biggs






9. The Development and Ultrastructure of Gum Ducts in Citrus in Response to Pathogens


Esther Shedletzky






10. Pathological Anatomy of Root Diseases Caused by Phytophthora Species


Darren P. Phillips






11. Cytology, Histopathology, and Histochemistry of Citrus Blight


Ronald H. Brylansky






12. Cytology, Histology, and Histochemistry of MLO Infections in Tree Fruits


Sharon M. Douglas






13. Pathological Anatomy of Infection and Systemic Invasion of Deciduous Fruit Trees by Bacterial Pathogens


Isabel M. M. Roos, E. Lucienne Mansvelt, and M. J. Hattingh






14. Adaption and Response of Fruit Trees to Freezing Temperatures



Michael Wisniewski and Rajev Arora

Citation preview

HANDBOOK OF

Cytology, Histology, and

Histochemistry of

Fruit Tree Diseases

HANDBOOK OF

Cytology, Histology, and

Histochemistry of

Fruit Tree Diseases Edited by

ALAN R. BIGGS, Ph.D. Associate Professor University Experiment Farm West Virginia University Kearneysville, West Virginia

CRC Press Taylor & Francis Group Boca Raton London New York CRC Press is an imprint of the Taylor & Francis Group, an informa business

CRC Press Taylor & Francis Group 6000 Broken Sound Parkway NW, Suite 300 Boca Raton, FL 33487-2742 Reissued 2019 by CRC Press © 1993 by Taylor & Francis Group, LLC CRC Press is an imprint of Taylor & Francis Group, an Informa business

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PREFACE When I was asked initiall y b y the publisher to consider preparing a volume on cytology, histology, an d histochemistr y o f frui t tre e diseases , I recal l thinkin g tha t thi s woul d b e a difficult, althoug h achievable task, give n the abundance of literature that has been publishe d over th e years . Optimistically , I endeavored i n m y min d t o divid e a generic frui t tre e int o its component parts , i.e. , root s (primary an d secondary), shoot s (xylem, phloem , succulent , woody), leaves, flowers, and , of course, fruits. I then overviewed the literature to determine the mos t importan t disease s amon g th e various classe s o f disease-causin g organisms . Next , I chos e representativ e disease s foun d attackin g th e variou s plan t organs , wit h th e ide a i n mind tha t thi s volum e coul d present , i n essence , th e principle s o f cytology , histology , an d histochemistry o f frui t tre e disease s b y focusin g o n specifi c plan t organ-pathoge n typ e interactions. With diseases of fruit tree s being incited by fungi, bacteria , viruses, nematodes, mycoplasma-like organisms , fastidiou s prokaryotes , an d advers e environment , combine d with a minimum o f fiv e plan t organ s t o address , th e notio n o f 3 0 or mor e chapters seeme d forbidding a s wel l a s impractica l an d unnecessary . The process of narrowing the field o f candidate chapters down to manageable size turned out to hinge no t s o much upo n wha t I or others fel t wa s importan t t o include , bu t rathe r on the individuals i n our scientific communit y wh o were (1) willing, (2 ) able, and (3) available to present this information. Th e field o f practicing pathological anatomists , histologists, and cytologists i s comparatively smal l in the profession o f plant pathology a s a whole and, whe n this limite d fiel d mus t b e narrowe d t o thos e workin g wit h frui t trees , I discovere d tha t i f this boo k wa s goin g t o materialize , I woul d hav e t o eithe r writ e i t mysel f o r gai n th e cooperation o f those few frui t tre e pathologists wh o employ a n anatomical approac h t o their specific diseas e problems . Bein g daunte d b y the first option , I tried th e second . Durin g th e process o f securin g commitment s fro m potentia l authors , I realize d tha t I ha d gathere d together a uniqu e grou p o f peopl e fro m al l ove r th e world—uniqu e i n th e sens e that , fo r them, th e practic e o f plan t pathologica l anatom y an d histolog y i s no t onl y a science , no t only a n art , bu t rathe r a combination o f th e tw o fuele d b y a n extraordinary leve l o f fervor . I think thi s i s apparen t i n mos t o f th e chapter s i n thi s book . The literatur e abound s wit h paper s an d monograph s o n th e cytology , histology , an d histochemistry o f various diseases and disorders of deciduous and nondeciduous fruits. Man y of thes e studie s originate d i n the firs t decade s o f thi s centur y wit h th e belief tha t a n under standing o f diseas e proces s coul d lea d t o improved contro l o f th e disease i n question. Wit h the adven t o f effectiv e chemical s fo r controllin g suc h diseases , especiall y thos e cause d b y fungi, investigation s o n host-pathoge n relationship s becam e les s o f a priority. Thi s boo k i s not a compendiu m o f histopathologica l investigation s o n frui t trees . Instead , I hav e aske d the contributing author s t o focus o n disease problem s i n whic h researcher s remai n activ e in elucidating the host-pathogen interaction , examinin g systems for which no effective control s are available , investigatin g ne w disease s o f recen t importance , o r lookin g fo r nove l ap proaches t o ol d problems . Th e variou s approache s take n b y th e differen t author s ar e wid e ranging, reflectin g thei r varyin g area s o f interes t an d expertise , wit h topic s rangin g fro m cytological investigatio n o f pathogens (th e pear rust fungus, fo r example) t o studies of plant cell wal l fin e structur e i n relatio n t o cold temperatur e injury . Som e o f th e chapters addres s single diseas e problem s i n grea t dept h (th e chapter s o n appl e scab , citru s blight , an d fir e blight); wherea s other s ar e mor e comprehensiv e o f classe s o f pathogen s o r type-disease s (diseases cause d b y MLOs , Phytophthora spp. , Monilinia spp. , an d systemi c invasio n o f fruit tree s b y bacteria) . Th e focu s o f thi s boo k i s clearl y o n funga l an d bacteria l diseases , with additiona l chapter s o n MLOs , col d temperatur e injury , an d citru s blight , a disease o f unknown etiology .

The volum e i s incomplet e i n tha t representative s o f al l th e classe s o f disease-causin g organisms ar e no t included . Thi s i s due neithe r t o oversight no r design , bu t rathe r t o thos e factors liste d above . Specifi c chapter s o n nematod e disease s an d viru s disease s ar e no t included, eve n thoug h ther e are many excellen t scientifi c article s availabl e i n the literature. The reason s fo r puttin g thi s volum e togethe r wer e t o consolidat e muc h o f th e recen t histopathological literatur e int o on e well-illustrate d resource , t o provid e in-dept h o r com prehensive chapter s whic h coul d serv e a s springboard s fo r futur e investigations , an d t o demonstrate th e continuin g importanc e o f histopathologica l investigation s (an d th e skill s required in order to conduct such studies) to our discipline. It is hoped that all three objectives have bee n me t an d tha t th e presen t volum e wil l serv e t o increas e understandin g o f host pathogen interaction s an d also to stimulate new interests that inspire continued researc h int o important area s o f tre e frui t pathology .

THE EDITOR Alan R . Biggs , Ph.D. , i s Associat e Professo r o f Plan t Patholog y i n th e Colleg e o f Agriculture an d Forestr y an d Extensio n Specialis t i n Tree Frui t Patholog y a t West Virgini a University. Dr. Bigg s receive d hi s B.S . degre e i n Fores t Scienc e fro m th e Pennsylvani a Stat e University i n 1976 . He obtained his M.S. and Ph.D. degrees in 197 8 and 1982 , respectively, from th e Departmen t o f Plan t Patholog y a t th e Pennsylvani a Stat e University . H e wa s appointed t o the position o f Research Scientis t i n Plant Pathology a t the Agriculture Canad a Research Statio n i n Vineland, Ontari o i n 1983 , with primary responsibilities fo r disease s of fruit trees . In 1987 , he became Head of the Plant Pathology Section at the Vineland Research Station. Dr . Bigg s bega n hi s presen t positio n wit h Wes t Virgini a Universit y i n Septembe r 1989. Dr. Bigg s i s a membe r o f th e America n Associatio n fo r th e Advancemen t o f Science , the America n Phytopathologica l Society , th e Canadia n Phytopathologica l Society , an d th e International Associatio n o f Woo d Anatomists . H e serve d a s Associat e Edito r o f Phytopathology (198 8 t o 1990 ) an d wil l serv e a s Secretar y (1992 ) an d Chai r (1993 ) o f th e Deciduous Tre e Frui t Worker s o f th e America n Phytopathologica l Society . Dr. Bigg s ha s bee n th e recipien t o f researc h grant s fro m th e Natura l Science s an d Engineering Researc h Council , th e Ontari o Ministr y o f Agricultur e an d Food , th e Interna tional Societ y o f Arboriculture Researc h Trust, an d private industry. H e has published mor e than 5 0 researc h article s i n cytology , histology , physiology , biochemistry , epidemiology , tissue culture , an d genetic s o f frui t tre e diseases . H e recentl y co-edite d a boo k entitle d Defense Mechanisms of Woody Plants Against Fungi. Hi s curren t researc h interest s ar e i n defense mechanism s o f wood y plant s an d sustainabl e frui t production .

ACKNOWLEDGMENTS This volume is the fruit o f the labors of many individuals in addition to those who kindly authored th e variou s chapters . I exten d m y appreciatio n t o th e followin g individual s wh o provided eithe r review s o f chapter s o r helpfu l discussion s durin g th e cours e o f preparin g this book : C . Le e Campbell , Nort h Carolin a Stat e University ; Rober t E . Davis , U.S . De partment o f Agriculture-Agricultura l Researc h Service , Beltsville ; Edward Durner , Rutger s University; Willia m Merrill , Pennsylvani a Stat e University ; Willia m Olien , Clemso n Uni versity; Pau l Peterson , Nort h Carolin a Stat e University ; Davi d F . Ritchie , Nort h Carolin a State University ; Pau l W . Steiner , Universit y o f Maryland ; Wayn e F . Wilcox , Cornel l University; an d Keit h Yoder , Virgini a Polytechni c Institut e an d Stat e University .

CONTRIBUTORS Rajeev Arora , Ph.D . Plant Physiologis t Appalachian Frui t Researc h Statio n Agricultural Researc h Servic e U.S. Departmen t o f Agricultur e Kearneysville, Wes t Virgini a Christopher M . Becker , Ph.D . Research Associat e Department o f Plan t Patholog y Cornell Universit y New Yor k Stat e Agricultura l Experimen t Station Geneva, Ne w Yor k Ronald H . Brlansky , Ph.D . Professor an d Plan t Pathologis t Citrus Researc h an d Educatio n Cente r University o f Florid a Lake Alfred , Florid a Suzanne Bullock , M.Sc . Professional Office r School o f Biologica l Scienc e University o f Ne w Sout h Wale s Kensington, N.S.W . Australia Sharon M . Douglas , Ph.D . Research Plan t Pathologis t Department o f Plan t Patholog y an d Ecology Connecticut Agricultura l Experimen t Station New Haven , Connecticu t M. J . Hattingh, D.Sc . Professor Plant Patholog y Departmen t University o f Stellenbosc h Stellenbosch South Afric a

Mitsuru Kohno , Dr.Agr . Chief Researche r Department o f Developmen t an d Planning o f Researc h Mie Prefectura l Agricultura l Research Cente r Ichishi-gun Japan Hitoshi Kunoh , Ph.D. , Dr.Agr . Professor, Facult y o f Bioresource s Mie Universit y Tsu-city Japan E. Lucienn e Mansvelt , Ph.D . Plant Biotechnolog y an d Patholog y INFRUITEC Stellenbosch South Afric a Hideo Nasu , Dr.Agr . Researcher Division o f Plan t Protectio n Okayama Prefectur e Agricultura l Experiment Statio n Akaiwa-gun, Okayam a Japan Darren P . Phillips , Ph.D . Bureau o f Rura l Resource s Department o f Primar y Industrie s an d Energy Canberra Australia Isabel M.M . Roos , Ph.D . Plant Biotechnolog y an d Patholog y INFRUITEC Stellenbosch South Afric a

Henk J . Schouten , Ph.D . Center fo r Plan t Breedin g an d Reproduction Researc h (CPRO-DLO) Department o f Cultiva r Strateg y Wageningen The Netherland s Esther Shedletzky , Ph.D . Researcher Department o f Botan y Hebrew Universit y o f Jerusale m Jerusalem, Israe l

Haydn J . Willetts , Ph.D . Visiting Professo r School o f Biologica l Scienc e University o f Ne w Sout h Wale s Kensington, N.S.W . Australia Michael Wisniewski , Ph.D . Plant Physiologis t Appalachian Frui t Researc h Statio n Agricultural Researc h Servic e U.S. Departmen t o f Agricultur e Kearneysville, Wes t Virgini a

TABLE OF CONTENTS Chapter 1 Historical Aspect s o f th e Pathogica l Anatom y o f Frui t Tre e Disease s 1 Alan R . Bigg s Chapter 2 Anatomy an d Histochemistr y o f Woun d Response s i n Bar k Tissue s an d Implications fo r Managemen t o f Canke r Disease s 1 Alan R . Bigg s

3

Chapter 3 The Cytology , Histology , an d Histochemistr y o f Appl e Sca b 3 Christopher M . Becke r

5

Chapter 4 The Cytolog y an d Histolog y o f Pea r Rus t 9 Mitsuru Kohn o an d Hitosh i Kuno h

1

Chapter 5 Cytology, Histology , an d Histochemistr y o f Frui t Infectio n b y Monilinia Specie s 11 Hayden J . Willett s an d Suzann e Bulloc k

3

Chapter 6 The Pathologica l Anatom y o f Zygophiala jamaicensis o n Frui t Surface s 13 Hideo Nas u an d Hitosh i Kuno h

7

Chapter 7 Pathological Anatom y o f Fir e Bligh t Cause d b y Erwinia amylovora 15 Henk J . Schoute n

7

Chapter 8 Pathological Anatom y an d Histochemistr y o f Leucostom a Canke r o n Ston e Fruit s and Othe r Selecte d Canker s o n Deciduou s Frui t Tree s 16 Alan R . Bigg s Chapter 9 The Developmen t an d Ultrastructur e o f Gu m Duct s i n Citru s i n Respons e t o Pathogens 19 Esther Shedletzk y

9

1

Chapter 1 0 Pathological Anatom y o f Roo t Disease s Cause d b y Phytophthora Specie s 20 Darren P . Phillip s

5

Chapter 1 1 Cytology, Histopathology , an d Histochemistr y o f Citru s Bligh t 23 Ronald H . Brylansk y

7

Chapter 1 2 Cytology, Histology , an d Histochemistr y o f ML O Infection s i n Tre e Fruit s 25 Sharon M . Dougla s Chapter 1 3 Pathological Anatom y o f Infectio n an d Systemi c Invasio n o f Deciduou s Fruit Tree s b y Bacteria l Pathogen s 28 Isabel M . M . Roos , E . Lucienn e Mansvelt , an d M . J . Hatting h

3

1

Chapter 1 4 Adaptation an d Respons e o f Frui t Tree s t o Freezin g Temperature s 29 Michael Wisniewsk i an d Raje v Aror a

9

Index 32

1

Historical Aspects 1 Chapter 1

HISTORICAL ASPECTS OF THE PATHOLOGICAL ANATOMY OF FRUIT TREE DISEASES Alan R . Bigg s

TABLE OF CONTENTS I. Introductio

n2

II. Th

e Earl y Frui t Industr y an d Plan t Diseas e 2

III. Tre

e Frui t Disease s i n Nort h Americ a Prio r t o 188 0 3

IV. Th

e Birt h o f Microscop y an d Microtechniqu e 4

V. Th

e Adven t o f Stainin g an d Histochemica l Procedure s 5

VI. Th

e Daw n o f Plan t Anatom y an d Pathologica l Plan t Anatom y 6

VII. Th

e Arriva l o f Plan t Patholog y 7

VIII. Summar y 1

0

References 1

1

0-8493-2939-6/93/$0.00 + $.5 0 © 199 3 by CR C Press , Inc .

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Cytology, Histology, and Histochemistry of Fruit Tree Diseases

I. INTRODUCTIO N There are many excellent treatises on the histories of cytology, staining , histochemistry , plant anatomy , plan t pathology , an d frui t growing . Al l o f thes e ar e separat e an d distinc t monographs withi n discipline s o r o n particula r area s o f specialty . Thi s chapte r present s i n a short and very general form the many seemingly similar and disparate interest that ultimately converged an d gav e ris e t o th e specialt y are a addresse d b y thi s book . Wha t frui t disease s were prevalen t whe n Rober t Hook e publishe d "Micrographia " i n 1665 ? Wha t di d Gre w and Malpighi , th e founder s o f plan t anatom y i n th e 1680s , contribut e t o studie s o n th e pathological anatom y o f frui t trees ? Wh o wrot e th e firs t monograp h o n disease s o f frui t trees? Wh o lai d th e foundation s fo r investigation s int o pathologica l plan t anatomy ? Th e history o f frui t cultur e predate s recorde d history , wit h som e o f th e earlies t writing s fro m Theophrastus datin g prio r t o 28 5 B.C . Th e startin g poin t fo r thi s synopsi s i s the beginnin g of th e 17t h centur y an d coincide s wit h th e inventio n o f th e microscope .

II. TH E EARLY FRUIT INDUSTRY AND PLANT DISEASE At th e tim e o f th e settlemen t o f th e earlies t colonie s i n Nort h America , apple s wer e grown extensivel y fro m souther n Europ e northwar d t o th e Scandinavia n peninsula . Th e various waves of immigrants, the French to Canada; the English to New England, Maryland , Virginia, th e Carolinas , an d Georgia ; th e Dutc h t o Ne w York ; th e Swede s t o Ne w Jersey ; and people s fro m al l parts o f Europ e t o Pennsylvania, brough t youn g appl e trees, seeds , o r scions, fro m whic h wer e starte d th e earl y appl e tree s o f Nort h America . Th e earl y histor y of fungu s disease s o f frui t i n Nort h Americ a i s a mixtur e o f allegor y an d myth . I n 1639 , John Josselyn visited New England and enjoyed ' 'half a score of very fair pippins' ' harvested from trees planted by Governor Endicott on Governor's Island in Boston Harbor. In discussing disease problem s o n apple s h e reveale d tha t 'Trui t tree s ar e subjec t t o tw o diseases , th e measles which i s when the y are burned an d scorche d wit h the sun, an d lowsiness, whe n the woodpeckers jab hole s i n th e bark; th e wa y t o cure the m whe n the y ar e lowsi e i s to bore a hole i n th e mai n roo t wit h a auger , an d pou r i n a quantity o f Brandi e o r Rhum , an d the n stop it up with a pin mad e of th e sam e tree. " I t seem s plan t medicin e an d huma n medicin e were o n a simila r cours e i n th e Ne w World . I n th e Ol d World , th e tal k wa s o f resistan t varieties, when R. Austen, in 165 7 recorded that "cra b trees...are usually free fro m canker " (presumably Nectri a canker). 2 The fruit industr y gre w i n the 18t h century, albei t slowly , i n North America. Frui t wer e largely grow n fo r persona l o r loca l consumptio n o r fo r makin g beverage s o r anima l feed . However, larg e appl e plantings an d thriving nurser y businesse s wer e not uncommo n a t this time. Fo r example , mos t o f th e earl y plantation s i n Virgini a include d orchard s o f apple s and othe r fruits . I n Ne w York , th e Princ e Nurser y wa s activ e a s earl y a s 173 2 an d thei r catalogs, from 181 5 to 1850, were standard horticultural publications in the U.S. Commercial orcharding i n these early day s depended o n convenient proximit y t o markets o r satisfactor y transportation. Indeed , commercia l shipment s of apples from Pennsylvani a t o England wer e occurring a s early a s 1763 . Peter Kalm , perhap s unknowingl y portendin g th e future impor tance o f plan t disease s i n the fruit industry , wrot e i n 174 8 (while travelin g fro m Trento n t o Princeton, Ne w Jersey): "Nea r almos t ever y far m wa s a spacious orchard ful l o f peach an d apple trees , an d i n som e o f the m th e frui t ha d falle n fro m th e trees i n suc h quantitie s a s to cover nearl y th e whol e surface . Par t o f i t the y lef t t o rot..." . Cox e i n 181 7 wrot e th e firs t American boo k o n frui t growin g an d include d i n i t reference s t o peac h yellow s an d fir e blight o f pear. 3 I n th e unpublishe d secon d editio n o f thi s wor k (1829) , mentio n i s mad e of

Historical Aspects

3

a peac h variety , Morris' s Larg e Whit e Rareripe , a s bein g no t subjec t t o ro t (presumabl y brown rot) . A s som e growers bega n t o experiment wit h clona l rootstoc k an d scio n materia l in the firs t quarte r o f th e 19t h century, Th e American Pomologica l Societ y wa s founded i n 1848, although it was not formally recognize d by that name until 1852 . Fisher and Upshall's account of the fruit industr y on a state by state basis is fascinating readin g for those interested in th e histor y o f frui t cultur e i n Nort h America. 1

III. TREE FRUIT DISEASES IN NORTH AMERICA PRIOR TO 1880 Prior to 185 0 there wa s little concern abou t fruit tre e diseases in North America. I t was only aroun d 1860 , a s frui t bega n t o b e grow n fo r fres h marke t purposes , tha t blemishe s were no longer regarded as an inescapable act of "Providence" . Sprayin g orchards to control insects bega n aroun d 187 0 to 188 0 with th e us e o f Pari s Green , Londo n Purple , an d othe r arsenicals.4 Spraying for th e control of fungus disease s of fruit, mainl y appl e scab, in North America bega n sometim e betwee n 188 5 an d 1905 , followin g th e discover y o f Millardet' s mixture (Bordeaux mixture) in southern France in 1885 . Very little was known about insects and diseases at the time. Sharvelle et al.4 provide a concise account of the history of spraying for contro l o f frui t pests . One o f th e firs t disease s t o caus e seriou s economi c losse s i n th e U.S . wa s fir e blight , which began to devastate the apple and pear orchards of Pennsylvania an d New York befor e the en d o f th e 19t h century . Th e pathoge n sprea d rapidl y t o othe r states . Man y prominen t pioneers o f America n plan t patholog y worke d o n fir e blight , notabl y Thoma s J . Burril l a t the University o f Illinois , Erwi n F . Smit h a t the U.S. Departmen t o f Agriculture , an d J. C . Arthur, wh o worked i n New York and Indiana. Burril l unraveled the mysteries of fire bligh t and i s commonl y credite d wit h providin g th e firs t proo f tha t bacteri a ca n caus e diseas e i n plants. Onl y 2 year s earlier , Rober t Koc h i n Europ e ha d demonstrate d th e rol e o f bacteri a in causin g disease s o f animals . Two othe r diseases , peac h yellow s i n th e eas t an d th e grap e plagu e (late r know n a s Pierce's disease) in California, wreake d havoc by the end of the 19t h century. Peach yellows appeared i n New York around 180 1 and reached devastating proportions there by the 1840s . Kirtland, workin g i n northern Ohi o in 1855 , published a description o f plum brown ro t and state d tha t he had known th e disease for 3 0 years. 5 In his article, Kirtlan d describe s hi s microscopic observations , on e o f th e earlies t record s o f th e microscopica l observatio n o f diseased tissu e fro m a frui t tre e i n th e U.S . Th e fungu s wa s know n t o th e mycologist s a t the time as Torula fructigena, th e first description s havin g been given by Persoon i n 1796. 6 Prior t o an d fo r severa l year s afte r Kirtland , mos t mentio n o f ro t o f ston e fruit s referre d t o the curculi o insec t a s th e probabl e causa l agent . A s Kirtlan d wrote : The plu m crop , o f lat e years , ha s generall y faile d i n norther n Ohio . Th e resul t ha s bee n charged to the curculio, and in many instances correctly; but a fatal disease has been insidiously progressing amon g our fruit orchard s which has done more injury tha n that insect. The effec t of the two evils has not usually been discriminated one from the other. Indeed, few cultivators seem t o be awar e o f th e prevalenc e o f an y suc h disease .

Kirtland's observations at this period in history suggest that in the U.S. there were proponents of the germ theor y o f diseas e prior to de Bary's wor k on rusts an d smut s published i n 185 3 and hi s othe r importan t paper s o n potat o lat e bligh t publishe d i n th e 1860s . Kirtlan d no t only understoo d tha t brow n ro t wa s a disease, h e realize d i t wa s initiate d b y a fungus an d made a n effor t t o identif y it . I n a n addres s t o th e Ohi o Pomologica l Societ y publishe d i n 1867,7 h e said :

4

Cytology, Histology, and Histochemistry of Fruit Tree Diseases I have watched carefull y th e sudde n an d premature deca y o f ou r plum crop , a t the period of its ripening, fo r th e last fifteen years . Fro m hints offered b y the work of Prof. Mitchell , an d several microscopic observation s o f my own, I was induced t o publish a n article in the "Th e Florist" o f Philadelphia , i n th e yea r 1855 , i n whic h I impute d th e origi n o f th e diseas e t o the Torula o r some analogou s specie s o f parasite fungi . Th e disease stil l prevails amon g us, and it is sure to destroy al l the plums whic h escap e puncture by the curculio. I t is, however , generally overlooke d b y ou r pomologists , an d it s effects ar e charged t o the depredation s o f that insect . Simila r disease occasionall y impair s ou r peach an d apple crops, t o a less extent. Whenever i t occurs o n eithe r o f thes e varietie s o f fruit , th e spur s an d youn g woo d bligh t o r canker, an d ceas e t o be fruitfu l fo r severa l years .

The appl e bligh t mentione d her e wa s probabl y fir e bligh t cause d b y Erwinia amylovora. Kirtland's vie w o f brow n ro t a s a fungu s diseas e wa s unappreciate d unti l Pec k provide d proof o f pathogenicity i n 1877 . B. T. Galloway an d E. F . Smith of the U.S.D.A. publishe d brief accounts of the histology of the disease on cherry fruits i n 188 9 and peach twig cankers in 1891 , respectively. 89

IV. THE BIRTH OF MICROSCOPY AND MICROTECHNIQUE Hans and Zaccharias Jansse n invente d th e compound microscop e abou t 1590 . Seventy five year s later , Rober t Hook e (163 5 t o 1703) , wh o i n hi s wor k "Micrographia " (1665) , was th e firs t t o illustrat e i n detai l a pathogeni c microscopi c fungus , probabl y ros e rus t Phragmidium mucronatum. 10 H e was not aware of the pathogenic capabilities of this fungus , however, an d th e ide a o f spontaneou s generatio n persiste d fo r anothe r 2 centuries . Hi s discovery o f th e cell , however , change d th e wa y humankin d though t abou t life . Fro m th e "Micrographia": I too k a good clea r piec e o f cork , an d wit h a pen-knife sharpene d a s keen a s a razor, I cut a piec e o f i t off , an d thereb y lef t it s surfac e smooth ; the n examinin g i t diligentl y wit h th e microscope,... but that possibly, i f I could use some further diligence,.. . I, with the same penknife, cu t off fro m th e former smoot h surfac e a n exceedingly thi n piece of it : and placing i t on a black objec t plate , becaus e i t wa s itsel f a whit e body , an d castin g th e ligh t o n i t wit h a deep plano-conve x glas s I could exceedingl y plainl y see.. .

Hooke's simpl e microscop e possesse d n o stage . Instead , th e object s wer e mounte d o n a point attache d t o a pedestal a t th e base . Hook e wa s als o th e firs t t o moun t anima l o r plan t material in water or oil, although the technique did not come into general use until the 1820s. Leeuwenhoek i s credite d a s foundin g th e stud y o f microbiology . B y 1683 , the Dutc h len s maker ha d describe d protozoa , bacteria , an d othe r microbe s i n water , ora l bacteria , an d human sperm . H e usuall y mad e a microscop e fo r eac h specifi c objec t h e wante d t o vie w since the objects wer e generall y fixe d t o a point. Fo r specimen s tha t could no t be mounte d to a point, h e sprea d th e specime n ont o fine bit s of mic a tha t wer e later glued t o the objec t needle. Only one original Leeuwenhoek microscope remains today in Utrecht. Smith's article contains drawing s o f som e of thes e early microscope s an d specime n holder s an d include s a much mor e thoroug h histor y tha n tha t provide d here. 11 The microscopica l laborator y i n th e moder n sens e di d no t exis t i n th e 17t h an d 18t h centuries. Indeed , u p until th e beginning o f th e 19t h century, th e microscope wa s regarde d as a toy or a curiosity, rather than as a scientific instrument. Nelso n mentions Pepys in 166 4 paying £10 fo r a microscope an d thinkin g i t " a grea t pric e fo r a curious bauble". 12 During the 18t h century, almost all works on the microscope were written by microscope manufacturers wit h grea t detail s mad e o n thei r construction . Still , ther e wa s littl e seriou s use o f th e microscope . Objec t carrier s o r slide s wer e introduce d nea r th e beginnin g o f th e 18th century. I n 1742 , Henr y Baker 13 wrot e seve n page s o n th e subjec t o f preparation . H e

Historical Aspects

5

suggested th e us e o f bit s o f glas s t o cove r object s an d als o suggeste d th e us e o f glas s o f different color s t o mak e som e object s mor e distinguishable . Historians emphasiz e th e barrennes s o f th e 18t h century , a s compare d t o th e 17th , i n the development o f th e microscope . Wit h on e notable exception , Joh n Hil l (171 6 to 1775) , this i s als o true o f microtechnique . I n his wor k entitle d "Th e constructio n o f timber , fro m its early growth , explaine d b y the microscope", 14 Hil l utilized technique s tha t had not been employed u p t o tha t tim e an d tha t di d no t com e int o genera l us e unti l 5 0 year s later , an d then only as rediscovered b y others. He was the first t o use maceration in the study of wood. He pu t macerate d piece s o f woo d int o alu m an d the n spirit s o f win e t o harde n them , a procedure resemblin g ou r moder n fixatio n methods . Hil l wa s undoubtedl y th e firs t t o us e staining a s a n ai d i n th e stud y o f th e microscopica l anatom y o f plants . H e prepare d a n alcoholic tinctur e o f cochineal , i n which , afte r filtering , h e placed th e stem s o f plants . A n even mor e advance d techniqu e h e employe d wa s a mordanting o f th e tissue s befor e devel oping the color. Hil l used tw o different technique s o f clearin g tissues , using spirit s of win e for pin e tissue s an d turpentin e fo r othe r tissues . Thes e methods , obviousl y extremel y val uable, were not used by others until much later. Hill apparently was a disagreeable individual and becaus e o f thi s ha d considerabl y les s influenc e o n hi s fiel d a t th e time. 15 Hi s section s were cu t o n a microtom e (know n a s "cuttin g engines") , whic h wer e wel l know n a t th e time. Th e firs t microtom e i n th e moder n sens e wa s mad e b y Adam s aroun d 1770. 16 Generally, th e year s 183 0 to 183 5 sa w outstandin g wor k by th e English microscopists , including th e introductio n o f glas s slides , Canad a balsam , an d th e mountin g o f object s i n liquids (casto r oi l fo r fung i wa s use d i n 1849). n I n contras t t o th e investigator s o f othe r countries, the British microscopists maintained a great freedom an d oral interchange of ideas. Thus, a n individua l ma y hav e devise d a trul y origina l breakthrough , an d bee n conten t t o communicate i t onl y b y wor d o f mouth . Prio r t o the formatio n o f th e Royal Microscopica l Society of London in 1839, there was no formal meeting place for microscopists, and informal verbal communication s predominated . Thi s ma y hel p explai n wh y i t i s ofte n difficul t t o ascribe particular discoveries to single individuals. With a few notable exceptions, the English microscopists made only a few contributions to botany that were based on their microscopical observations. However , the y wer e largel y instrumenta l i n advancin g microtechnique . Th e great interes t i n microscopy tha t had built upon th e discoveries o f the 1830 s had it s heyday in th e perio d fro m 185 0 to 1860 . Wit h th e appearanc e o f fiv e treatise s o n th e microscop e and microtechnique, the methods that had been known to only a small group of microscopists became know n worldwid e an d wer e take n u p an d perfecte d outsid e England .

V. THE ADVENT OF STAINING AND HISTOCHEMICAL PROCEDURES The most importan t contributio n o f th e English microscopist s t o microtechnique durin g the year s 186 0 t o 187 5 wa s th e introductio n o f staining , especiall y fo r plan t tissues . Th e German botanist s di d no t avai l themselve s o f th e Englis h methods , largel y du e t o thei r philosophy o f utmost simplicit y i n preparing botanical material for study . Indeed, Hug o von Mohl ha s bee n quote d a s sayin g tha t th e microtom e wa s largel y superfluou s fo r scientifi c investigations.11 Generally , Britis h method s i n microscopy wer e not use d i n Germany unti l about 1850 , owin g t o the interest s o f vo n Mohl . Smith 11 believe s tha t Hil l shoul d b e give n recognition fo r the first us e of staining methods, whereas others believe that Theodor Hartig should b e give n credit . Cort i i s give n th e hono r o f havin g bee n th e firs t t o appl y stainin g methods t o th e content s o f th e cell , thu s givin g birt h t o th e stud y o f cytology. 17 Gerlac h (1820 to 1896) , althoug h no t th e firs t t o use stains , develope d method s o f stainin g bacteri a and anima l tissue s tha t cam e int o genera l use .

6 Cytology,

Histology, and Histochemistry of Fruit Tree Diseases

The beginning s o f histochemistr y a s a scienc e hav e bee n attribute d t o th e year s 183 0 through 1855 , and its origins were primarily botanical. For some decades, the whole practice of histochemistr y i n it s tru e sens e wa s i n th e hand s o f botanists . Lin k 180 7 performe d th e earliest microchemica l reaction s whe n h e use d iro n sulfat e fo r determinin g tanni c aci d i n leaves. Th e mai n wor k i n those earl y year s wa s that o f th e Frenc h botanis t Raspail , whos e "Essai d e Chemie Microscopique Appliqué e à la Physiologie" appeare d i n 1830. 18 The use of the starch-iodin e reactio n i n microscopy an d the introductio n o f method s fo r protei n an d cell wal l carbohydrate s hav e bee n attribute d t o Raspail . Th e discover y o f protoplas m an d chemical reaction s withi n cell s by von Mohl 19 led Schleide n (180 4 to 1881), 20 who worke d with plan t tissues , an d Schwan n (181 0 t o 1882) , wh o worke d independentl y o f Schleide n with anima l tissues , t o formulate (afte r persona l discussio n an d laborator y study ) the "Cel l Theory" i n 1838 . Hermann Schach t (182 4 to 1864 ) in 185 2 provided th e first collectio n o f microchemical method s fo r th e recognitio n o f man y differen t plan t products , includin g cellulose, xylogen , protein , starch , gums , an d dextrines. 21 Test s fo r sugar s an d fat s wer e not yet well developed. Smith, 11 writin g i n 1915 , indicated tha t by 185 0 the microchemica l determination o f the constituents of the plant cell was in a fairly satisfactor y state . The years 1875 throug h 191 5 sa w th e zoologist s develo p an d expan d th e earl y botanica l techniques . From this time through th e early 20th century, botanica l microtechnique develope d throug h refinements tha t zoologists made in the English microscopical method s and which were then adapted b y th e botanists . Althoug h th e earl y attempt s a t stainin g wer e firs t mad e b y mi croscopists o r botanist s o n botanica l material , th e developmen t o f stainin g i s du e almos t completely t o zoologists. None of the early zoologists cite John Hill's work . Fro m the years 1862 throug h 1882 , o f ove r 5 5 publishe d paper s o n staining , onl y 3 wer e publishe d b y botanists. I n discussin g th e developmen t o f botanica l histochemistr y afte r thi s period , th e problem i s to find ou t a t wha t particula r tim e method s devise d b y anima l histologist s wer e first use d i n botanica l applications . Fro m 187 5 t o 188 0 th e notabl e progres s i n botanica l microtechnique wa s th e adoptio n o f stainin g procedures . Th e America n microscopist s ar e generally credited with having developed double staining methods for stems and other tissues in the lat e 1870s . I t shoul d b e note d tha t man y o f th e earl y phytopathologica l studie s use d a triple stai n of safrani n (firs t use d by Ehrlich i n 1877) , gentian violet , an d orange G which was firs t combine d b y Flemmin g i n 1891. 22 Klebs first use d paraffin fo r embedding in 1869 , while celloidin was introduced i n 187 9 by Duval . I n botany , th e firs t us e o f paraffi n belong s t o Francott e (1886) , an d i t becam e well established i n the years 188 7 to 1892 . Fixation i s a relatively recent development, wit h most o f th e fixin g mixture s havin g bee n propose d i n the perio d fro m 188 0 to 1890 . Flem ming's variou s mixtures , develope d fro m 188 2 to 1884 , were used a s standard fixative s b y the earl y phytopathologists . Th e expansio n o f botanica l microtechniqu e int o th e variou s subdisciplines o f botany , includin g phytopathology , i s reflecte d b y publicatio n o f Staus burger's Botanische Prakticum, variou s edition s o f whic h appeare d fro m 188 4 throug h 1913. n Heslop-Harriso n an d Kno x provid e a goo d genera l historica l perspectiv e o n plan t histochemistry.23

VI. THE DAWN OF PLANT ANATOMY AND PATHOLOGICAL PLANT ANATOMY Theophrastus, regarde d a s th e fathe r o f botanica l science , mad e man y notabl e obser vations o n th e anatom y o f plant s i n th e 3r d centur y B.C . Wit h th e rise o f Christianit y an d the concomitant "Dar k Ages" in biological sciences , no significant interna l studies of plants occurred until the advent of magnification. Th e founders of plant anatomy, Nehemiah Grew 24 (1641 t o 1712 ) an d Marcell o Malpighi 25 (162 8 t o 1694 ) wer e stimulate d b y Hooke' s ob -

Historical Aspects

7

servations; however, the y lef t littl e record o f thei r working methods . Malpigh i say s nothing at al l abou t hi s methods , wherea s Gre w state d tha t i t i s importan t t o examin e a specime n from severa l views , includin g oblique , perpendicular , an d transverse , "al l thre e bein g req uisite". I t i s Grew , however , wh o i s generall y regarde d a s th e fathe r o f plan t anatomy . Eames an d MacDaniels ' accoun t o f th e histor y o f plan t anatom y illustrate s thes e earl y contributions i n mor e detai l tha n i s required here. 26 In the lat e 18t h century, J . J . P . Moldenhawe r (176 6 to 1827 ) introduce d th e techniqu e of maceration i n the stud y o f plant tissues, thus introducin g th e concept o f the individualit y of cells . Hug o vo n Mohl' s (180 5 t o 1872 ) attentio n t o th e livin g content s o f th e cel l wa s the initiatio n poin t fo r furthe r studie s o n th e fundamenta l natur e o f th e protoplast . Vo n Mohl's contribution s t o plant anatom y wer e numerou s an d varied , includin g th e nature an d method o f formatio n o f vessels, the structure of the epidermis an d nature of the cuticle, an d also th e natur e o f lenticel s an d th e formatio n o f bark . Th e firs t wor k devote d exclusivel y to plant histology was that of Schacht, whic h appeared in 1851. 27 This work contains minute directions fo r th e anatomica l observatio n o f differen t plants , an d thu s wa s o f grea t value . Theodor Harti g (180 5 t o 1880 ) studie d th e anatom y o f woo d an d phloe m an d discovere d and name d th e siev e tube. 28'29 Nagel i (181 7 t o 1891 ) firs t use d th e term s "xylem " an d "phloem", althoug h Theophrastu s ha d use d the m i n a slightly differen t form . In 1877 , de Bary wrote Comparative Anatomy of the Phanerogams and Ferns. Althoug h lacking a comprehensiv e presentatio n o f th e structur e o f th e plan t bod y a s a whole , thi s book wa s regarde d a s on e o f th e mos t usefu l an d usabl e referenc e book s i n plan t anatom y during th e nex t 5 0 year s afte r it s appearance. 26 Th e firs t basi c treatise s i n pathologica l anatomy, writte n b y Rober t Hartig , describ e th e attac k o f Arme liaria me Ilea an d Fome s annosus o n Prunus spp. , an d sho w ho w thes e fung i penetrate intac t roots. 3031 I n contrast , Hartig als o stated that many microbe s canno t penetrate directly or consume intact bark, an d thus man y pathogen s o f wood y plant s ar e woun d parasites . Harti g als o prove d tha t fung i cause stainin g an d deca y i n timber .

VII. THE ARRIVAL OF PLANT PATHOLOGY The unraveling of the theory of spontaneous generation can not be traced to one specifi c event. Severa l circumstances occurred over the period fro m th e late 18t h century to the time of d e Bar y an d Pasteur . I n 1729 , th e Italia n Michel i (167 9 t o 1737 ) describe d th e cultura l experiments tha t he used to demonstrate tha t several common mold s were distinct entities. 32 This wor k wen t unappreciate d unti l th e tim e o f Spallanzan i a t th e en d o f th e centur y an d Pasteur i n 1864 , who disproved spontaneou s generation . Th e study o f mycology develope d during thi s perio d a s th e identitie s o f differen t fung i wer e determined , eve n thoug h the y were regarded mostl y a s saprobe s rather tha n parasites . Th e stud y o f plant disease emerge d via th e examinatio n o f rus t an d smu t disease s o f cereal s an d potat o lat e blight . Majo r contributions towards understanding the nature of rust diseases were made by Felice Fontana (1730 t o 1805) , Josep h Bank s (174 3 t o 1820) , T . A . Knigh t (175 9 t o 1838) , an d N . P . Scholer (177 2 to 1851) . Heteroecism i n the rusts was not generally accepte d unti l de Bary' s work i n 186 5 to 1866 . For smuts, the works of M. Tillet (1714 to 1791) and B. Prévost are noteworthy. Prévost , whose wor k wen t unnotice d b y hi s contemporaries , prove d tha t smu t wa s funga l i n origi n and coul d b e controlle d wit h coppe r sulfat e see d treatments . Hi s work , publishe d i n 1807 , is regarded a s a landmar k i n biology , a s wel l a s i n microbiolog y an d plan t pathology. 33 I n the 1830s , the Italian Agostino Bassi (1773 to 1856 ) demonstrated that the muscardin disease of silkworm s wa s cause d b y a fungus . A fe w year s later , Davi d Grub y (181 0 t o 1898 ) described thrus h an d ringworm disease s o f human s a s cause d b y fungi . Thus , whe n potat o

8

Cytology, Histology, and Histochemistry of Fruit Tree Diseases

late bligh t swep t throug h Europ e i n th e 1840s , i t seem s surprisin g tha t ther e wa s s o muc h controversy abou t it s etiology . Again , i t wa s d e Bar y wh o finall y establishe d tha t Phytophthora infestans was the causal agent . Spontaneou s generatio n wa s stil l receivin g detaile d discussion i n books as late as 183 3 by the German, Fran z Unger (180 0 to 1970) . Berkeley' s series of articles in the Gardener's Chronicle from 185 4 to 185 7 may mark the turning point for th e ne w idea s emergin g o n th e funga l natur e o f plan t diseases . Hi s wor k an d tha t o f de Bar y an d Brefel d i n Germany , th e Tulasn e brother s i n France , an d Woroni n i n Russi a influenced th e approac h t o plan t diseas e ove r th e nex t 5 0 year s b y deepenin g ou r leve l o f knowledge o f th e parasitic fungi . A s the profession o f plan t patholog y emerge d wit h Juliu s Kuhn's textboo k i n 1858 , publication s fro m German y dominate d fo r ove r 4 0 years . No t until the turn o f the century di d w e se e textbooks fro m Franc e an d England. Th e year 190 6 marked th e publicatio n o f B . M . Duggar' s Fungous Diseases of Plants an d th e beginnin g of th e dominanc e o f Englis h languag e textbook s o n plant patholog y i n th e U.S . Thomas J . Burril l (183 9 to 1916 ) was appointed professo r o f botany an d horticulture a t the University o f Illinois i n 1869. 34 He began hi s microscopical observation s o n diseases of fruits i n 187 1 and was the first t o describe a bacterial disease of plants (1878 to 1884 ) when he showe d tha t fir e bligh t o f appl e an d pea r wa s cause d b y th e bacteriu m no w know n a s Erwinia amylovora. Credi t shoul d b e give n t o Burrill fo r th e firs t microscopica l stud y o f a bacterial frui t tre e disease. Burril l wa s a "self-made " phytopathologis t i n the sens e tha t h e had n o forma l connectio n wit h th e Europea n schoo l o f thought . H e graduated fro m Illinoi s Normal Universit y i n 186 5 and travelled wit h Powell' s firs t Rock y Mountai n Expeditio n i n 1867.34 Burrill' s interes t i n microscop y i s no t appreciate d widel y b y moder n phytopathol ogists; however, h e maintained membership s i n the Royal Microscopical Society , the American Societ y fo r Microscop y (President , 1885 ; Secretary , 1886-1889) , an d th e America n Microscopical Societ y (President , 1905) . H e taugh t course s i n microscop y an d pomolog y in which h e provided microscope s (o f both European an d American origin ) for hi s students. Two books, publishe d aroun d th e time that Burril l wa s finishing hi s training an d beginnin g his early research , ma y hav e bee n influential : One Thousand Things for the Microscope b y M. C . Cook e i n 1868, 35 and The Microscopic Dictionary: A Guide to the Examination of the Structure and Nature of Microscopic Objects, b y Griffit h an d Henfre y i n 1875. 36 E. F . Smit h establishe d th e stud y o f bacteri a a s an importan t branc h o f phytopatholog y and is perhaps especially note d fo r hi s studies on crown gall. 38 It should be noted, however , that Smit h wa s involve d i n man y researc h endeavors , an d hi s 189 1 publicatio n o n brow n rot canker s wa s on e o f th e firs t histologica l studie s o f a fungus diseas e o f frui t trees. 38 At abou t th e tim e o f Burrill' s classi c work , Pec k i n Ne w Yor k stat e describe d brow n rot a s on e o f th e mor e commo n disease s o f fruits. 39 Pec k wa s th e firs t t o demonstrat e th e pathogenicity of the fungus by means of inoculations. Just 2 years earlier, Sorauer in Germany (1879) ha d writte n th e firs t monograp h o n disease s o f frui t trees. 40 One of th e most importan t concept s emergin g fro m th e early studie s i n plant patholog y is tha t th e majorit y o f plant s ar e resistan t t o infection . Mycologist s an d plan t pathologist s before the time of de Bary appear to have been oblivious to the importance of this phenomenon until d e Bar y publishe d hi s illuminatin g works , d e Bar y elucidate d th e factor s responsibl e for susceptibilit y o r resistanc e i n connection wit h th e lif e o f facultativ e parasites . H e dem onstrated tha t som e fungu s spore s germinat e onl y whe n i n contac t wit h hos t plants , an d called attentio n t o th e fac t tha t th e myceli a o f som e obligat e parasite s ar e confine d t o th e immediate poin t o f attack , whil e other s sprea d widel y fro m th e poin t o f infectio n ove r o r through th e host. Resistance i n plants ha s bee n investigate d wit h referenc e t o th e morphology , genetics , cytology, an d physiolog y o f hosts . Th e mos t obviou s typ e o f hos t resistanc e i s associate d with som e morphologica l o r anatomica l characte r an d wa s earlie r assume d t o b e th e mos t

Historical Aspects

9

important type. 41 For example, Cobb 42 advanced th e theory tha t plants possessing a heavily cutinized o r waxy epidermis, o r a corky stem , wer e resistant to invasion by fungi. Thi s idea was supporte d b y Bolley 43 an d Anderson, 44 wh o attempte d i n 188 9 an d 189 0 t o correlat e resistance i n cereal s wit h certai n morphologica l characters . Thi s ide a wa s supporte d b y Sappin-Trouffy wh o discovere d i n 189 6 tha t th e myceliu m o f Puccinia graminis wa s re stricted t o th e chlorophyllou s parenchym a an d di d no t develop i n th e collenchyma. 41 Th e growth o f th e fungu s wa s correspondingly restricte d wit h th e amoun t o f parenchym a bein g small i n relatio n t o th e amoun t o f collenchyma . J . C . Arthu r confirme d thi s ide a b y hi s observation i n 190 2 tha t m y celia o f certai n rust s ma y b e limite d i n thei r developmen t b y the anatomica l natur e o f th e tissue s o f Spartina. I n contrast , War d (1902 ) conclude d th e morphological character s o f Bromus ha d littl e effec t o n rust resistance , an d Cob b reporte d in 1892 42 that the relative hairiness o f resistant whea t varietie s wa s not sufficien t t o explain their resistanc e t o rust . In th e are a o f frui t pathology , Vallea u (1915) 45 lai d th e basi s fo r studie s o f hos t mor phology i n pathoge n resistanc e wit h hi s researc h o n th e brow n ro t fung i o f plum s (se e Chapter 5) . Som e o f Valleau' s conclusions : Varieties sho w grea t differenc e i n resistanc e t o infectio n owin g t o th e productio n o f paren chymatous plugs which fill the stomatal cavity and to lenticels made up of corky cells through which the hyphae are unable to penetrate. Cork y cells lining the stomatal cavity merely delay infection.

In addition to the obstacles to penetration give n above, Valleau state s that after hypha e have gained entrance to the fruit, variation s in cultivar resistance are correlated with skin thickness and frui t firmness . Higgins46 published the first report (1914)46 that described for a woody plant the defensive responses o f foliag e t o funga l infection , describin g th e abscissio n layer , o r cicatrice , fo r cherry "sho t hole " lea f spot . Wiltshire, 47 wa s th e firs t (1922 ) t o describe woun d woo d i n xylem rays and how they acted to restrict the lateral movement of Nectria galligena in apple wood. Th e lack of effective fungicid e control s fo r man y fruit disease s partially explain s the plethora o f anatomica l an d cytologica l studie s i n th e 5 0 year s followin g th e acceptanc e o f the ger m theor y o f disease . It i s no t clea r ho w o r fro m who m plan t pathologist s prio r t o th e tur n o f th e centur y obtained thei r workin g knowledg e o f botanica l microtechnique . A t th e time , th e scientifi c community was relatively smal l and most of the individuals actively involved in pathological investigations kne w eac h othe r personall y o r throug h correspondence . Mos t coul d rea d German o r had som e link wit h th e German phytopathologists . Indeed , eve n int o the 1920s , E. C . Stakma n require d a year of study in Germany fo r ne w faculty member s a t Minnesota. Humphreys 189 3 translation o f Zimmerman' s boo k o n botanical microtechnique 48 wa s sig nificant, a s wa s C.J . Chamberlain' s book , Methods in Plant Histology, firs t publishe d i n 1901.49 Bot h o f thes e text s undoubtedl y advance d th e cours e o f phytopathologica l investi gations i n th e U.S . B y 1932 , th e latte r wor k ha d bee n revise d fiv e times . Within th e U.S.D.A. , whic h wa s establishe d i n 1862 , Thoma s Taylo r (182 0 t o 1910 ) was appointe d microscopis t i n 187 1 t o begi n a systemati c stud y o f disease s o f plants. 37 I n that firs t year , Taylor , wh o wa s originall y fro m Scotland , publishe d illustrate d report s o n fungus disease s o f grapes , pears , an d peaches . Ove r th e next 5 years, h e published report s on fire blight , peac h yellows, black knot of plum, an d cranberry diseases. During this time, Taylor, regarde d b y his peers a s a self-taught amateu r i n phytopathology, studie d medicin e at Georgetow n University , receivin g th e M.D . i n 1882 . Hi s mai n interes t wa s i n th e are a of edibl e an d poisonou s mushroom s an d hi s Student's Handbook wa s publishe d i n 1897 . Few citation s o n histologica l method s ar e give n i n mos t o f th e earl y papers , althoug h

10

Cytology, Histology, and Histochemistry of Fruit Tree Diseases

Flemming an d Haidenhai n ar e name d i n associatio n wit h th e technique s employed . I n on e of Keitt' s earl y paper s o n peac h scab, 50 h e provide d a more thoroug h descriptio n tha n di d many o f hi s colleagues . In killing material fo r histologica l work , severa l standar d fixin g agent s were employed, viz. , Flemming's weak , medium , an d stron g fluids , picro-formal , an d chrom-acetic . Th e stain s used were Flemming's triple, Haidenhain's iron alum-haematoxylin, Durand' s haematoxylin eosin, gentia n violet , an d safranin . Fo r mos t purposes , Flemming' s mediu m solutio n an d triple stai n gav e th e bes t results .

(He continued , describin g th e actio n o f th e stai n o n th e variou s tissues. ) Nixon, usin g paraffin-embedded material , als o found Flemming' s tripl e stai n useful fo r observing th e fir e bligh t bacteriu m i n hi s histologica l stud y o f fir e blight. 51 Fo r fixing , Flemming's stron g solutio n wa s use d fo r succulen t materia l an d Benda's solutio n wa s use d for cankere d tissue . Nixo n als o provided caution s abou t overstainin g an d recommende d a n optimum sectio n thicknes s o f 7 |xm . Rosen , als o studyin g fir e blight , seem s t o hav e ha d extensive experienc e i n botanica l microtechnique , judgin g fro m al l th e differen t method s tested, although , lik e th e previou s authors , h e provide d fe w citation s fo r microtechniqu e other tha n thos e paper s dealin g specificall y wit h fir e blight. 52 The early phytopathologists recognized the importance that natural plant resistance could play i n th e managemen t o f orchar d diseases . Th e studie s an d example s cite d abov e ar e by no means a n exhaustive revie w o f th e literature fro m a historical perspective ; th e example s do reveal, however , th e major event s that shape d th e evolution o f the histology o f tre e frui t diseases. We have seen that histological studie s can reveal mechanisms o f pathogenesis an d host resistance and thus make substantial contributions to our understanding of host/pathogen interaction. A s w e continu e t o see k ne w method s fo r controllin g establishe d diseases , an d as we encounter ne w disease s o f unknow n origin , histologica l studie s wil l continue t o play a ke y rol e i n advancin g th e professio n o f phytopathology .

VIII. SUMMARY The histor y o f botanica l microtechniqu e bega n wit h th e developmen t o f method s o f microscopy i n Englan d fro m th e tim e o f Hook e an d continuin g int o th e earl y 1800s . Th e English microscopist s continue d t o refin e microscop e techniqu e throug h 1875 , althoug h botanical applications were based largely on the methods of the German botanists until about 1875. Afte r thi s date , botanica l microtechniqu e owe s a larg e deb t t o th e rise o f zoologica l applications fro m whic h th e botanists borrowe d heavily . Th e us e of th e microscop e i n tre e fruit patholog y bega n wit h th e observation s o f th e earl y mycologist s i n th e lat e 18t h an d early 19t h centuries . Followin g th e adven t o f th e ger m theor y o f disease , Rober t Hartig' s studies o f th e pathologica l anatom y o f fores t tre e species , an d th e translatio n o f Germa n works on botanical microtechnique into English, fruit pathologists provided the first glimpse s into th e cytology , histology , an d histochemistr y o f frui t tre e diseases . I n th e U.S. , th e observations of Thomas Taylor, Beverl y T. Galloway , an d Erwin F. Smith of the U.S.D.A . and Thoma s J . Burril l o f th e Universit y o f Illinoi s ar e som e o f th e earlies t anatomica l accounts of tree fruit diseases . Brow n rot of ston e fruits i s an excellent example o f the long evolution o f histologica l studie s o f funga l disease s o f tre e fruits ; beginnin g wit h th e my cological observation s o f Persoon i n 1796 , followed b y Kirtland's early observations o f th e disease i n 1855 , succeede d b y Peck' s proo f o f pathogenicit y i n 1877 , an d culminatin g i n the histologica l observation s o f Gallowa y an d Smit h i n 188 9 an d 1891 , respectively. Th e evolution o f histologica l studie s i n phytobacteriolog y i s trace d fro m th e earl y observation s of fir e bligh t i n Ne w Yor k an d Pennsylvani a an d i s abbreviated , i n compariso n t o brow n rot, owin g t o the intellec t an d skill s o f Thoma s J . Burrill .

11

Historical Aspects

REFERENCES 1. Fisher , D . V . an d Upshall , W . H. , Eds. , History of Fruit Growth and Handling in the United States of America and Canada, Regatt a Cit y Press , Kelowna , B . C , Canada , 1976 , 302 . 2. Austen , R. , A Treatise o f Fruit-Trees , Shewin g th e Manne r o f Grafting , Planting , Pruning , an d Orderin g of Them , London , 1657 , 54 . 3. Coxe , W. , A Vie w o f th e Cultivatio n o f Frui t Trees , an d th e Managemen t o f Orchards an d Cider ; wit h Accurate Descriptions of the Most Estimable Varieties of Native and Foreign Apples, Pears, Peaches, Plums, and Cherries , Cultivate d i n th e Middl e State s o f America , Philadelphia , 1817 , 253. 4. Sharveile , E. G., Welsh , M. F., and Mcintosh, D. L., Diseases of fruit plant s and development of control methods, i n History of Fruit Growth and Handling in the United States of America and Canada, Fisher , D. V . an d Upshall , W . H. , Eds. , Regatt a Cit y Press , Kelowna , B . C , Canada , 1976 , 302 . 5. Kirtland , J . R. , Prematur e deca y o f th e plum , Florist Horticult. J., 4 , 34 , 1855 . 6. Pe r soon, C . H. , Synopsis Methodica Fungorum, Vol . 2 , Gottingan , 1796 . 7. Warder , J . A. , American Pomology. Apples, Ne w York , 1867 , 744 . 8. Galloway , B . T. , Brow n ro t o f th e cherry , Monilinia fructigena Pers. , USDA Rep., 1888/1889 . 9. Smith , E . F. , Peac h bligh t (Monilinia fructigena, Persoon) , J. Mycol., 7 , 36 , 1891 . 10. Hooke , R. , Micrographia, London , 166 5 (origina l no t seen , citatio n fro m Smith , 1915 11). 11. Smith , G . M. , Th e developmen t o f botanica l microtechnique , Trans. Am. Microsc. Soc, 34 , 71 , 1915 . 12. Nelson , E . M. , Wha t di d ou r forefather s se e i n a microscope?, J. R. Microsc. Soc, All , 1910 . 13. Baker , H. , The Microscope Made Easy, 1s t éd., London , 1742 . 14. Hill , J., Th e Construction o f Timber, fro m It s Early Growth, Explaine d b y the Microscope, Etc. , London , 1770 (original no t seen , citatio n fro m Smith , 1915 11)15. Clark , G . an d Kasten , F . H. , History of Staining, 3r d éd. , William s & Wilkins, Baltimore , 1963 . 16. Adams , H. , Essays on the Microscope, 2n d éd. , F . Kammacher , London , 1798 . 17. Baker , J . R. , Cytological Technique, 2n d éd. , Methuen , London , 1945 . 18. Raspail , F . V. , Essa i d e Chimi e Microscopique Appliqué e à la Physiologie , Paris , 1830 . 19. vo n Mohl, H. , Einig e Beobachtunge n iibe r die blaue Fàrbung der vegetabilischen Zellmembra n durc h Jod , Flora, 23 , 609, 1840 . 20. Schleiden , M . J. , Eingi e Bemerkunge n übe r de n vegetabilische n Faserstof f un d sei n Verhaltnis s zu m Stàrkemehl, Ann. Phys. Chem., 43 , 391 , 1838 . 21. Schacht , H. , Die Pflanzenzelle der inner eBau und das Leben des Gewâchse, Berlin , 1852 . 22. Flemming , W. , Neu e Beitrãg e zu r Kenntnis s de r Zelle , Arch. Mikrosk. Anat., 37 , 685 , 1891 . 23. Heslop-Harrison , J . an d Knox , R . B. , Plan t histochemistry , i n Histochemistry, The Widening Horizons of its Applications in the Biomedical Sciences, Stoward , P . J. an d Polak, J . M. , Eds. , John Wiley & Sons, New York , 1981 , 1 . 24. Grew , N. , The Anatomy of Plants, London , 1682 . 25. Malpighi , M. , Opera Omnia, 2 vol., 1687 . 26. Eames , A . J . an d MacDaniels , L . H. , An Introduction to Plant Anatomy, 1s t éd. , McGraw-Hill , Ne w York, 1925 , 364. 27. Schacht , H. , Da s Mikroskop und seine Anwendung, insbesonder e fur Pflanzen-Anatomi e un d Physiologie, Berlin, 1851 . 28. Hartig , Th. , Chlorgen , Bot. Z . 12 , 553 , 1854 . 29. Hartig , Th. , Übe r di e Functione n de s Zellenkerns , Bot. Z., 12 , 574, 1854 . 30. Hartig , R. , Wichtige Krankheiten der Waldbaüme. Beitrãge zur Mykoligie und Phytopathologie fur Botaniker und Forstmànner, J . Springer , Berlin , 1874 . 31. Hartig , R. , Die Zersetzungserscheinungen des Holzes der Nadelholzbàume und der Eiche in Forstlicher, Botanischer und Chemischer Richtung, J . Springer , Berlin , 1878 . 32. Micheli , P . A. , Nova Plantarum Genera, Florence , 1729 . 33. Prévost , B. , Memoi r o n th e immediat e caus e o f bun t o r smu t o f wheat , translate d b y G . W . Keitt , Phytopathol. Classic, 6 , 1939 . 34. Trelease , W. , Thoma s J . Burrill , portrait , Bot. Gaz., 62 , 153 , 1916 . 35. Cooke , M . C , One Thousand Things for the Microscope, Frederic k Warn e an d Co. , London , 1869 , 12 3 pp. 36. Griffith , J . W . an d Henfrey , A. , The Microscopic Dictionary — A Guide to the Examination and Investigation of the Structure and Nature of Microscopic Objects, 3r d éd. , Va n Voors t Paternoste r Row , London, 1975 , 84 5 pp. 37. True , A . C , A Histor y o f Agricultura l Experimentatio n an d Researc h i n the Unite d States , 1607—1925 , U.S. Dept. Agrie.IMisc. Pub., No . 251 , 1937 , 321. 38. Smith , E . F. , Peac h blight , Monilia fructigena Persoon , J. Mycol., 7 , 36 , 1891 .

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Cytology, Histology, and Histochemistry of Fruit Tree Diseases 39. Peck , C . H. , Repor t o f th e botanist , N.Y. State Mus. Nat. Hist. Annu. Rep., 34 , 24 , 1881 . 40. Sorauer , P . C . M. , Die Obstbaumkrankheiten, Berlin , 1879 . 41. Reed , H . S. , A Short History of the Plant Sciences, Chronic a Botánica , Waltham , MA , 1942 , 323. 42. Cobb , N . A. , Contribution s t o a n economi c knowledg e o f th e Australia n rusts , Agrie. Gaz. NSW 3 , 181 , 1892. 43. Bolley , H . L. , Whea t rust , Indiana Agrie. Exp. Stn. Bull., No . 26 , 1889 . 44. Anderson , H . C . L. , Rus t i n wheat . Experiment s an d thei r objects , Agrie. Gaz. NSW, 1 , 81 , 1890 . 45. Valleau , W . D. , Varieta l resistanc e o f plum s t o brown rot , J. Agrie. Res., 5 , 365 , 1915 . 46. Higgins , B . B. , Contributio n t o th e lif e histor y an d physiolog y o f Cylindrosporium o n ston e fruits , Am. J. Bot., 1 , 145 , 1914 . 47. Wiltshire , S . P. , Studie s o n th e appl e canke r fungus . II . Canke r infectio n o f appl e tree s throug h sca b wounds, Ann. Appl. Bot., 9 , 275 , 1922 . 48. Zimmerman , A. , Botanical Microtechnique, translate d b y E . J . Humphrey , Ne w York , 1893 . 49. Chamberlain , C . J. , Methods in Plant Histology, Universit y o f Chicago Press , Chicago , 1901 . 50. Keitt , G . W. , Peac h sca b an d it s control, U.S. Dept. Agrie. Bull., No . 395 , 1917 . 51. Nixon , E . L. , Th e migratio n o f Bacillus amylovorus i n appl e tissu e an d it s effec t o n th e hos t cells , Pa. State Coll. Agrie. Bull., No . 212 , 1927 . 52. Rosen , H . R. , Th e lif e histor y o f th e fir e bligh t pathogen , Bacillus amylovorus, a s relate d t o th e mean s of overwinterin g an d dissemination , Univ. Ark. Agrie. Exp. Stn. Bull., No . 244 , 96 , 1929 .

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Wound Responses in Bark Tissues Chapter 2

ANATOMY AND HISTOCHEMISTRY O F WOUND RESPONSES IN BARK TISSUES AND IMPLICATIONS FOR MANAGEMENT OF CANKER DISEASES Alan R . Bigg s

TABLE OF CONTENTS I. Introductio

n1

4

II. Anatom y o f Bar k 1 A. Tissue s o f th e Inne r Bar k 1 B. Natura l an d Woun d Periderm s 1

4 4 6

III. Anatom y o f Woun d Response i n Bar k 1 A. Ligh t Microscop y 1 B. Electro n Microscop y (TEM ) 2

7 7 1

IV. Externa l Factor s Influencin g th e Generatio n o f Ne w Bar k Tissue s 2 A. Temperatur e2 B. Epiphyti c Microbe s 2 C. Wate r Stres s 2 D. Relativ e Humidit y 2 E. Miscellaneou s Factor s 2

1 3 5 7 7 7

V. Regeneratio

7

VI. Pathoge VII. Heritabilit

n o f Vascula r Cambiu m an d Woun d Closur e 2 n Resistanc e i n a Dynamic Infectio n Cour t 2 y o f th e Woun d Reactio n 3

8 0

VIII. Concludin g Remark s 3

1

References 3

1

0-8493-2939-6/93/$0.00 + $.5 0 © 199 3 by CR C Press , Inc .

14 Cytology,

Histology, and Histochemistry of Fruit Tree Diseases

I. INTRODUCTIO N Many importan t an d seriou s disease s o f frui t tree s ar e caused b y pathogens tha t initiat e infections a t wound s cause d b y insects , humans , wind , hail , animals , an d nutritiona l an d physiological disorders . Researc h o n woun d response s o f frui t tree s i s required i n orde r t o understand th e processes tha t favor o r impede the development o f funga l infection s i n fruit , leaves, shoots, and roots. It is possible that more precise information abou t wound responses could lead to innovative control measures based on a better understanding o f the chronology of the wound response, how wound response may be influenced b y external factors , o r how the woun d respons e coul d b e modifie d fo r improve d diseas e control . In trees , i t i s plausibl e t o vie w th e tissu e regeneratio n proces s followin g woundin g a s being coincident with or closely allied to the defense process , given that structural response s often coincid e wit h physiological processe s that contribute to the biochemical foundatio n o f resistant structures . I n thi s context , th e effor t t o distinguis h betwee n structura l an d physi ological response s become s unnecessary . An y distinction s betwee n physiologica l processe s and structura l woun d responses i n this chapter are made merely fo r convenience an d for th e purpose of discussion. It is the goal of this chapter to discuss recent findings on the anatomical and epidemiological consequence s of wounding i n peach trees and to draw attention to other investigations tha t ar e relevan t t o host-pathoge n interactions . Thi s chapte r focuse s o n ana tomical aspect s o f woun d respons e a s i t occur s i n reactio n t o injurie s o f th e livin g tissue s of the inner bark. Fo r literature on the wound reaction i n fruits, reader s are referred t o other articles.13

II. ANATOM Y OF BARK Studies of the defense system s of trees in general hav e concentrated primaril y o n xylem tissues becaus e o f thei r economi c importanc e t o the forestr y industry . Becaus e bar k tissue s shield th e xyle m fro m th e environment , containmen t o f mechanica l injurie s an d infectiou s microorganisms b y bar k tissue s i s critica l t o tre e health . Th e integrit y o f norma l perider m and th e abilit y o f plant s t o for m ne w periderm s a t wound s o r injurie s ar e essentia l charac teristics for normal plant growth and development. However, i n comparison to xylem tissues, responses of periderm and other bark tissues to injury an d infection ar e defined inadequately . The ter m "bark " i s use d mos t ofte n i n a nontechnical contex t an d refer s t o al l tissue s external t o the vascula r cambium. 4-5 Accordingly , th e bar k i s an aggregatio n o f tissue s an d organs tha t include s phloe m an d secondaril y thickene d tissue s fro m th e secondar y plan t body, a s well a s epidermis, cortex , an d phloe m derive d fro m th e primary plan t body. 5 Th e living portio n o f the bark consist s o f the phloem an d the tissues o f the innermos t periderm , the phelloge n an d phelloderm . Al l livin g tissue s hav e bee n collectivel y terme d th e "inne r bark".6 Trockenbrodt 7 ha s provide d a n informativ e surve y an d discussio n o f terminolog y used i n th e bar k anatom y literature . Most pathogens of fruit tree s are unable to penetrate directly the corky, suberize d tissue s of mos t oute r bar k tissues . Thes e oute r layer s represen t constitutiv e defense s o r preforme d anatomical barrier s t o pathoge n ingress . Give n tha t th e cutinize d epidermi s o r suberize d periderms o f tree s ar e th e firs t tissue s tha t potentia l pathogen s encounter, 8 an d give n tha t the majorit y o f tree s remai n aliv e fo r decade s (o r fo r centurie s wit h fores t trees) , thes e barriers apparentl y ar e ver y effective . A. TISSUE S O F TH E INNE R BAR K The livin g inne r bar k o f a tree i s composed o f secondar y phloe m an d perider m tissue s (Figures 1 and 2). I n addition, cortica l tissue s fulfil l a n important rol e in the bark of youn g

FIGURES 1 and 2. Ligh t micrographs o f longitudinal an d transverse section s of 2-year-old nonwounde d peach bark showin g first perider m (P) , cortex (C) , primary phloem fiber s (PPF) , secondar y phloe m (SP) , an d cambia l regio n (CM) . Ba r = 2 0 |xm .

Wound Responses in Bark Tissues

15

16

Cytology, Histology, and Histochemistry of Fruit Tree Diseases

stems. Periderm, th e term first use d by von Mohl, 9 is a protective tissue of secondary origi n which replace s th e epidermi s i n stem s an d root s tha t hav e continua l secondar y growth . Detailed description s o f perider m formatio n ar e available. 4510 Roots , stems , an d branche s of gymnosperms , mos t dicotyledons , an d a fe w monocotyledon s develo p periderm. 5 Her baceous dicotyledons may form periderm, usually in the roots or oldest portions of the stem. In mos t coniferou s an d dicotyledonou s trees , a perider m replace s th e epidermi s a s th e protective laye r withi n th e firs t yea r o f growth . A s tree s age , sequen t periderm s ma y aris e at successivel y greate r depths , thu s causin g a n accumulatio n o f dea d tissue s o n th e surfac e of th e stem or root an d contributing t o the formation o f rhytidome o n rough-barked specie s or simpl y oute r bar k o n smooth-barke d species . The perider m consist s o f th e followin g tissues , a s originall y describe d b y d e Bary: 11 phellogen (cor k cambium) , th e latera l meriste m whic h produce s th e periderm ; th e phelle m (cork), th e suberize d protectiv e tissu e forme d outwardl y b y th e phellogen ; an d th e phello derm, a living parenchyma formed inwardl y by the phellogen. Phelloge n cells usually appear oblong i n transvers e an d radia l section s an d appea r polygona l o r irregularl y shape d whe n examined i n tangentia l section. 4-512 Phelloge n cell s ar e characteristicall y thi n walled , hav e protoplasts, ar e vacuolated t o varying degrees , an d may contain variou s substances , starch , and chloroplasts. Th e phellogen ma y consis t o f onl y on e layer or as a zone of meristemati c cells.13 Phelloderm cell s resemble cortica l parenchym a cell s i n shap e an d content, althoug h their radial arrangemen t make s them easily distinguishe d fro m cortica l cells . Walls of phelloderm cell s ma y b e thickene d an d intercellula r space s ma y b e abundant . There ar e tw o mai n type s o f phelle m cells , suberize d cor k cell s an d lignifie d phelloi d cells. Cor k cell s ar e radiall y shortened , wit h relativel y thic k walls , an d phelloi d cell s ar e usually thi n walle d an d radiall y elongate. 14 Bot h cell s ar e dea d a t maturit y an d generall y lack intercellula r spaces. 5 Th e arrangemen t o f phelle m cell s i n bar k varie s accordin g t o species.5 Cortical tissue s ar e foun d primaril y i n th e bar k o f youn g stems . Th e firs t perider m i n the stem s o f mos t specie s arise s i n th e cortex , whic h i s ultimatel y she d a s ne w periderm s arise. Phloe m tissue s ar e intimatel y involve d i n th e developmen t o f bar k structure. 131517 The inne r bar k o f smooth-barke d specie s consist s largel y o f phloe m tissues . I n addition , patterns o f phloe m elemen t depositio n i n conjunctio n wit h particula r pattern s o f perider m development are responsible for the structure of ring, scale, and furrowed barks. 18 The living cells o f th e oute r phloe m giv e rise t o deep-seate d periderms . B. NATURA L AN D WOUN D PERIDERM S According to Esau,5 natural (including first and sequent periderms) and wound periderms are basically alik e i n metho d o f origi n an d growth. Th e differenc e betwee n the m i s mainl y in timin g o f origi n an d restrictio n o f woun d perider m t o th e plac e o f injury . Also , woun d periderm i s believe d t o diffe r fro m natura l periderm s i n tha t th e forme r i s induce d b y a stimulus o r injury , o r b y factor s othe r tha n thos e responsibl e fo r th e inductio n o f natura l periderms. 41921 In peach and other angiosperms that the author has studied, wound periderms are distinct from th e first periderm s base d on their dissimilar histochemical reaction s t o lignin reagents 22,23 and th e formatio n o f a ligno-suberized boundar y fro m cell s extan t a t the tim e o f woundin g as a prerequisite to periderm differentiation.2224 2 6 Mullick24 provided an extensive anatomical and biochemical analysi s o f first , sequent , an d woun d periderm s i n gymnosper m bark . Hi s terminology (i.e. , exophylactic periderm for first an d sequent periderms, and necrophylacti c periderm fo r al l woun d periderm s an d rhytidome ) i s beginnin g t o fin d favo r amon g re searchers.

Wound Responses in Bark Tissues

17

III. ANATOM Y OF WOUND RESPONSE IN BARK A great dea l i s known abou t woun d anatom y i n fruit tree s an d ther e i s ample, althoug h not exhaustive, documentation in the literature on the anatomical events that lead to boundary zone an d woun d perider m formatio n i n wood y plants. 24-2733 A. LIGH T MICROSCOP Y In peac h bark , th e firs t observabl e respons e t o mechanica l woundin g ma y b e viewe d with th e ligh t microscop e a s earl y a s 2 4 h afte r wounding , althoug h ther e ma y b e cultiva r differences i n th e timin g o f event s an d othe r specifi c feature s o r ther e ma y b e difference s due t o environmental effect s o n plant response . I n injure d peac h bar k tissues , considerabl e degradation o f starc h granule s occur s withi n th e firs t 1 2 t o 2 4 h . B y 9 6 h thes e granule s will have disappeared altogethe r i n the area of tissue undergoing dedifferentiation , althoug h they ma y stil l b e eviden t i n th e desiccate d are a nea r th e woun d surfac e an d i n th e interna l tissues some distance from the wound.22 Cells in the incipient boundary zone undergo changes in th e nucleu s an d th e abilit y o f th e cytoplas m t o tak e u p morphologica l stains . I n mos t cells o f th e incipien t boundar y zone , metaphas e nucle i ar e easil y see n an d th e nucleol i ar e prominent 4 8 h afte r woundin g (Figur e 3) . In Prunus pérsica an d other Prunus spp. , depositio n o f a polysaccharide substanc e ha s been observe d wit h periodi c acid-Schif f 's (PAS ) reagen t i n th e wall s o f cell s locate d i n a zone abou t 30 0 |x m fro m th e woun d surface. 22 Polysaccharid e depositio n occurre d prio r t o the formatio n o f a visibl e lignifie d zone . Althoug h increase d ligni n ca n b e detecte d wit h biochemical method s withi n th e firs t 2 4 h afte r wounding, 34 th e firs t sign s o f lignificatio n detectable with histochemical reagents (phloroglucinol/HCl) ar e apparent within 72 h (Figure 4) an d occu r interna l t o the are a of polysaccharid e depositio n (i f th e latter occurs) . Report s of lignificatio n a s a respons e t o woundin g i n tre e bar k ar e numerous. 24 26'28'31'33-3537 Th e first lignifie d cell s can be detected in areas of the wound in closest proximity to the vascular cambium. Althoug h mos t cel l type s exhibi t lignificatio n i n respons e t o wounding , th e par enchymatous cells in phloem ray tissue often ar e the first to stain visibly with phloroglucinol/ HC1. Suberin depositio n i n lignifie d cell s occur s withi n 2 4 to 48 h after visibl e lignification . Results o f studie s o n bot h angiosperm s (includin g peach , apple , an d swee t cherry ) an d gymnosperms hav e show n tha t th e impermeabilit y o f th e imperviou s laye r tha t i s forme d from extan t cell s prio r t o periderm regeneratio n i s closely relate d t o the formatio n o f intra cellular suberi n lining s i n cell s presen t a t th e tim e o f wounding , an d whic h hav e becom e lignified followin g wounding. 22,37 Simila r processes occur in wounded xyle m parenchyma 38 and wounde d tissue s o f variou s herbaceou s an d wood y plan t specie s an d organs. 23 The ligno-suberize d boundar y zon e i s most ofte n locate d approximatel y 0. 8 t o 1. 0 m m internal t o th e woun d surface . Initia l cell s o f thi s tissu e ca n b e detecte d withi n 4 t o 7 d in wounds o n activel y growin g tree s i n midsumme r (Figur e 5), 33-39 an d usuall y occu r i n a n area o f th e woun d wit h closes t proximit y t o th e vascula r cambium . A meristemati c laye r forms immediatel y interna l to and abutting th e primary ligno-suberize d tissu e and is usually detected 2 4 t o 4 8 h afte r th e formatio n o f th e latte r tissu e (Figure s 6 t o 8) . Woun d (nec rophylactic) perider m ma y b e wel l forme d b y 1 0 d postwounding . Complet e formatio n o f the boundar y zon e an d ne w perider m aroun d th e entir e woun d ma y tak e u p t o 2 8 d unde r ideal condition s (Figure s 9 an d 10) ; however, a s boundar y tissue s continu e t o for m i n a n outward direction, new phellogen cells form immediately internal to the established boundary tissue. A s phelle m i s produce d i n a n outwar d direction , th e ligno-suberize d boundar y i s crushed an d diminishe s i n thickness. 39 It i s importan t t o not e tha t th e presence o f tissue s i n various stage s o f woun d respons e

18 Cytology,

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FIGURES 3 to 7. Ligh t micrograph s o f longitudina l section s throug h tissu e undergoing dedifferentiatio n durin g generation o f ne w phelloge n afte r mechanica l woundin g o f bar k o f health y 2-year-ol d peac h bark . Outsid e bar k in al l figure s i s oriente d towar d to p o f plate . Ba r = 1 0 fjim . Figur e 3 . Sectio n throug h th e secondar y phloe m 48 h postwounding showin g mitoti c activit y associate d wit h tissu e redifferentiation . Cell s t o the lef t o f the arrow s participate i n phellogen generation; cells to the right are becoming hypertrophied an d are developing lignifie d wall s and intracellula r suberi n linings . Figur e 4. Sectio n throug h perider m an d corte x 7 2 h postwounding showin g tw o distinct tissu e region s i n th e transitio n zon e betwee n health y (left ) an d necroti c tissu e (right) . Tissu e t o th e right of th e arrow s show s firs t positiv e reactio n wit h phloroglucinol/HCl , bu t i s no t ye t imperviou s t o flui d diffusion . Figure 5. Sectio n of differentiated phelloge n (PG ) and incipien t ligno-suberized imperviou s tissue (IT and arrows ) 96 h postwounding. Figure s 6 and 7 . Bar k tissu e staine d wit h phosphine , unde r brigh t fiel d an d th e sam e unde r UV excitation, respectively , showin g imperviou s tissu e (IT) , first phelle m (PH) , an d newl y regenerate d phelloge n (PG) 7 d postwounding. Not e in Figure 7 the thin suberi n lining of the cells in the impervious zone (small arrows). (Figures 3 through 7 from Biggs , A . R. , Can. J. Bot., 62 , 2814 , 1984 . Wit h permission. )

Wound Responses in Bark Tissues

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FIGURE 8 . Ligh t micrograp h o f staine d transvers e sectio n o f differentiate d phelloge n (PG ) showin g phelle m (PH), phelloder m (PD) , an d imperviou s tissu e (IT) , 7 d postwounding . Th e woun d i s oriente d towar d th e right side of th e micrograph, health y tissu e on the left, firs t perider m a t top. Ba r = 1 0 ^m. (Fro m Biggs , A . R. , Can. J. Bot., 62 , 2814 , 1984 . Wit h permission. )

can be observed by sampling wounded tissues at any one time, i.e., 7 to 24 d after wounding , depending upo n species , environmenta l conditions , an d inheren t regenerativ e capacity . Ligno suberized cell s d o no t for m synchronousl y int o a distinc t boundar y zone. 33 Th e cell s an d subsequent tissue s for m firs t betwee n th e wound surfac e an d the vascular cambium an d last in the region o f th e original phellogen . On e can ofte n vie w woun d tissue s rangin g fro m n o visible reactio n t o thos e exhibitin g complet e perider m regeneratio n withi n th e sam e histo logical section. 33

FIGURES 9 and 10 . Fluorescenc e an d ligh t micrographs, respectively , o f transverse section s of 2-year-ol d peac h bar k at 28 d postwounding staine d wit h phosphin e GN showin g necrophylacti c perider m (NP ) forme d interna l t o a ligno-suberize d boundar y (IT) . Not e necroti c tissu e (NT) , livin g tissu e (LT) , exophylacti c perider m (EP), primar y phloe m fiber s (F) , an d woun d callu s tissu e (CL) . Ba r = 2 0 |xm . (Fro m Biggs , A . R. , Phytopothology, 76 , 905 , 1986 . Wit h permission. )

20 Cytology, Histology, and Histochemistry of Fruit Tree Diseases

Wound Responses in Bark Tissues

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B. ELECTRO N MICROSCOP Y (TEM ) At th e ultrastructura l level , dramati c change s ar e eviden t withi n th e firs t 2 4 h i n cell s adjacent t o th e wound. Generally , thes e change s reflec t th e subcellula r alteration s visibl e with ligh t an d fluorescence microscop y (describe d above) . Th e mos t noticeabl e subcellula r modifications ar e in the nucleus and include changes in nucleolar fine structure and chromatin organization, bot h o f which sugges t rRNA synthesi s and active production o f ribosomes. I n the cytoplasm , increase s occu r i n roug h endoplasmi c reticulu m (ER) , fre e ribosomes , an d polysomes. Cells in the region of activity of the new phellogen show increases in the amount of cytoplasm , smoot h ER , an d dictyosomes. 40-41 Al l thes e change s reflec t th e intensifie d transcriptional, translational , an d secretor y activitie s o f th e respondin g cells . Afte r 2 4 h , ultrastructural change s ar e limited t o those cells either undergoing dedifferentiatio n t o for m the ligno-suberize d boundar y zon e o r thos e undergoin g dedifferentiatio n t o for m th e ne w phellogen an d it s derivatives . Ultrastructural evidenc e fo r cel l wal l suberizatio n i n wounded peac h bark wa s observe d at 8 d afte r woundin g (Figure s 1 1 to 18) . Boundar y zon e cel l wall s wer e completel y line d on th e insid e wit h a n electron-luci d materia l correspondin g t o cel l wal l lining s wit h th e histochemical an d autofluorescenc e characteristic s o f suberin. Th e suberi n portio n o f th e cell wall s appeared , a t first , electro n lucid , followe d b y th e formatio n o f man y ligh t an d dark lammelations . Th e suberi n linin g i n individua l cell s appeare d unifor m i n thickness , although thicknes s o f the linin g varie d fro m cel l to cell (ca . 4 0 to 12 0 nm). Suberize d cell s in th e boundar y zon e containe d senescin g cytoplas m wit h fragment s o f undifferentiate d dense materia l tha t forme d a thin, discontinuou s granula r deposi t insid e th e suberi n layer . The granular, electron-dense materials likely resulted from the disintegration of the cytoplasm and th e variou s cel l organelles . Betwee n 8 and 1 2 d postwounding, th e primar y wall s an d the middle lamell a i n the boundary zon e exhibited a n increase i n electron density , probabl y due t o th e depositio n o f phenoli c polysaccharid e materia l i n th e wall . I n peach , thes e substances, usuall y referre d t o as gum, ar e produced nonspecificall y i n response t o wounds or infections (se e Chapter 11) . Examination o f the boundary zone with transmission electro n microscopy (TEM ) revealed that suberin linings were discontinuous over pit areas 41 (Figures 19 an d 20) . Therefore , i t i s likel y tha t th e impermeabl e natur e o f thes e primar y ligno suberized boundarie s i s due, to some extent, t o both ligni n an d suberin. Vesicle s of varyin g proportions frequently wer e associated wit h the periphery of the senescent cytoplasm (Figur e 21). Cells o f th e ne w necrophylacti c phelle m posses s dense , granula r cytoplas m wit h fe w distinct organelle s (Figure s 2 2 to 24). I n mature phellem, cel l contents appea r a s a compact mass o f electron-dens e amorphou s materia l intersperse d wit h electron-luci d deposit s an d dark bodie s o f variou s dimensions . Numerou s vesicula r element s wer e observed, th e membrane element s appearin g t o b e embedde d randoml y throughou t th e granula r matrix . Th e plasmalemma typically was separated from the cell wall. Phellem cells possessed a compound middle lamell a wit h a n amorphous fin e structure . Thi s portion of the cell wall appeare d re d when stained with ohloroglucinol/HCl. Suberin comprised the largest portion of the secondary wall and displayed fine ligh t and dark lammelations. The thickness of phellem suberin layers (ca. 6 0 t o 35 0 nm ) increase d wit h distanc e fro m th e phellogen . Cel l wal l pit s an d plas modesmata canal s wer e no t observe d i n th e phellem . Phelle m cell s wit h intac t organelle s were detected infrequentl y and , whe n detected (Figur e 24) , were characterized b y abundan t mitochondria, roug h ER , dictyosomes , an d associate d vesicles .

IV, EXTERNA L FACTOR S INFLUENCIN G TH E GENERATIO N OF NE W BAR K TISSUE S Treatises o n th e rol e o f woun d healin g i n th e resistanc e o f plant s t o pathogens ofte n

22

Cytology, Histology, and Histochemistry of Fruit Tree Diseases

FIGURES 1 1 to 18 .

Wound Responses in Bark Tissues

23

neglect th e importanc e o f externa l factor s a s determinant s o f woun d respons e rat e and , indirectly, the host/pathogen interaction . Environmental factors may influence an y or several of the cascad e o f event s tha t ar e initiated followin g woundin g (se e Reference 42 ; Table 1) , and thereby alte r quality an d quantity of wound-related events . Additional factor s othe r than those discusse d belo w probabl y affec t th e woun d respons e eithe r directl y o r indirectly , including plan t nutrient status , level of herbivory an d history of defoliation, carbon/nitroge n ratio a s influence d b y roo t an d shoo t pruning , aci d rain , an d ai r pollution . A. TEMPERATUR E Temperature has a strong influence o n the rate of wound healing in woody plant species, including frui t trees. 20-35'4345 A significan t correlatio n exist s betwee n temperatur e an d rat e of boundar y zon e an d perider m regeneratio n i n wounde d bar k o f apple , swee t cherry , an d peach.44 Tree s wounde d a t variou s time s durin g th e growin g seaso n wer e examine d fo r complete formatio n o f th e primar y ligno-suberize d zon e an d ne w periderm . Althoug h th e tissues coul d b e detecte d wit h a 7 - t o 21- d tim e perio d afte r wounding , degree-day s (bas e = 0° C provided the best fit o f data among the five degree-day bases examined) accumulate d during th e postwoundin g perio d explaine d ove r 80 % o f th e observe d variatio n i n woun d response. Fo r peach , complet e lignificatio n an d suberizatio n wer e observe d afte r 25 6 an d 411 degee-days , respectively . Swee t cherr y an d appl e wer e lignifie d afte r 21 2 an d 19 2 degree-days, respectively , an d suberize d afte r 32 7 an d 39 7 degree-days , respectively . Tre e phenological stag e di d no t appea r t o exer t significan t influenc e o n th e woun d response s measured i n thi s study . In experiment s t o determin e th e formatio n o f th e primar y ligno-suberize d laye r an d phellogen followin g lea f abscissio n i n peach , Bigg s an d Northover 43 reporte d tha t potte d trees maintaine d i n growt h chamber s a t 7.5 , 12. 5 an d 17.5° C showe d th e firs t indication s of th e primar y ligno-suberize d laye r a t 18 , 9, an d 6 d, respectively . Subsequen t generatio n of phelloge n an d th e appearanc e o f th e firs t phelle m cell s wer e observe d a t 30 , 18 , and 1 2 d, respectively . Earlie r researc h o n th e influenc e o f temperatur e o n wound-induce d cel l division ha s show n tha t withi n limits , th e time required fo r th e first cel l division i s linearl y and inversely related to temperature.46 Maximum and minimum temperature limits for wound responses have not been established for any tree species, however, in potato tubers, maximum FIGURES 1 1 to 18 . Transvers e section s o f peach bar k examined wit h UV epifluorescence illuminatio n (Figure s 11 to 15 , ba r = 1 0 jxm ) an d TE M (Figure s 1 6 to 18 , ba r = 1 |Jim). I n Figure s 1 2 to 15 , th e woun d surfac e i s approximately 80 0 \x,m abov e th e note d cellula r changes . Not e th e presenc e o f phloe m fiber s (F ) i n Figures 1 2 to 14. Figure 11. Nonwounded control tissue showing ray parenchyma in the region of primary and secondary phloem. Note th e lac k o f autofluorescence . Figur e 12 . Tissu e 6 d postwounding . Firs t trace s o f ligni n autofluorescenc e are i n the cell corner-middl e lamell a regio n o f boundar y zon e cells (arrows ) immediatel y externa l t o living tissue s (LT). Figur e 13 . Tissue s 8 d postwounding . Not e depositio n o f suberi n lining s (arrows ) i n boundar y zon e cell s and th e dedifferentiatio n o f interna l tissue s i n th e proces s o f formin g ne w phelloge n (LT) . Figur e 14 . Tissu e 12 d postwounding showin g a completely differentiate d necrophylacti c perider m wit h phelloderm (PD ) and two to four layer s o f phelle m cell s (NP) . Figur e 15 . Tissue s 1 4 d postwounding , showin g a completel y differentiate d necrophylactic perider m wit h 3 to 5 phellem cel l layer s (NP ) an d newl y differentiate d phelloder m (PD) . Figure s 16 to 18 . TEM s o f transverse section s o f peac h bar k boundar y zon e tissues. Figur e 16 . Portio n o f boundar y zon e cell 8 d postwounding wit h suberi n linin g (S ) adjacen t t o a nonsuberize d cel l (NSC) . Plasmodesmata l canal s (P ) in share d cel l wal l (CW ) appea r mor e electro n dens e tha n suberin . Suberi n appear s onl y slightl y lamellat e an d i s relatively electron lucid. Figure 17. Share d cell wall of two adjacent cell s in the boundary zone 1 2 d postwounding. The compoun d wal l (CW ) i s ver y electro n dense . Suberi n lining s (S ) ar e o f unifor m thicknes s withi n individua l cells an d var y i n thicknes s amon g cells . Not e th e fin e ligh t an d dar k lamellation s o f th e suberize d wal l an d th e cytoplasmic debri s (CD ) appresse d t o the wall of th e upper cell. Figur e 18 . Hig h magnificatio n vie w of suberize d cell wall in the boundary zone showing the fine ligh t and dark lammelations characteristic o f suberin. (Fro m Biggs, A. R . an d Stobbs , L . W. , Can. J. Bot., 64 , 1606 , 1986 . Wit h permission. )

24

Cytology, Histology, and Histochemistry of Fruit Tree Diseases

FIGURES 1 9 to 24.

Wound Responses in Bark Tissues

25

suberization occurs at 20° to 25°C.45'47 49 Krahmer35 found that periderm could not be detected microscopically in leaf scars of apple at temperatures . cambivora (Petri)Buism.,22'32'37/*. megasperma Drechs.32 38 and P. parasitica Dastur. 30,39 Th e Phytophthora spp . that cause root diseases of fruit tree s ar e nonspecifi c pathogen s wit h relativel y broa d hos t ranges . Phytophthora species usually experience a soilborne phase during part or all of their lif e cycle,40,41 causing decay in plant roots, and they may form cankers around the base of trunks (Figure 1 A to D).33,42"51 The life cycle of P. cinnamomi, on e of the most successful member s of th e genus , i s representative o f heterothalli c soilborn e form s (Figur e 2). 52 P. cinnamomi, like most other soilborne Phytophthora spp. , penetrate plant root s preferentially i n the zone of elongation behind the root tip and wounds (Figures 3 and 4A to D).1 9 4 5 5 3 58 After infection , mycelium ma y produc e necroti c lesion s an d sprea d throughou t th e roo t syste m an d collar , destroying tissu e a s i t progresse s (Figure s 4 E an d 5 B an d 5D). 20-42-59 Thes e ar e primar y disease symptoms. Disease caused by P. cinnamomi is also characterized by the development of secondar y symptom s suc h a s lea f chlorosis , diebac k o f twig s an d oute r branche s o f th e plant, an d lea f microphylly . Secondar y symptom s suc h a s thes e ar e ofte n th e firs t sign s observed when Phytophthora spp. attack root and crown tissues, whether it be P. cinnamomi infecting avocado s (Figur e ID) 4248 or Australian nativ e plan t communities, 6061 severa l dif ferent Phytophthora spp . attackin g walnut s (Figure s 5 A an d C), 20 or P. cactorum attackin g apples.62 Concomitant wit h th e invasio n o f roo t tissu e b y hypha e ar e th e man y change s i n hos t anatomical structur e an d physiologica l processes. 6366 Th e followin g discussio n center s o n such postinvasive changes produced by plant hosts in response to infection b y Phytophthora spp. Th e interactio n betwee n P. cinnamomi an d th e commerciall y importan t frui t tree , avocado pea r {Persea americana var . drymifolia Mill.) , i s use d a s a cas e stud y o f th e * However , recen t wor k tha t group s Oomycete s wit h th e kingdo m Protoctist a rathe r tha n th e kingdom Fungi 4 is now bein g accepte d b y som e researchers. 5

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FIGURE 1 . (A ) A coco a tree whic h ha s undergon e sudde n diebac k du e t o sever e attac k b y Phytophthora palmivora. Not e extensiv e lea f senescenc e an d absenc e o f coco a pod s o n th e branches . (B ) Inse t o f (A) . Canke r (arrow) ha s circumvente d th e whol e trunk , destroyin g al l sof t oute r tissue s an d cambium . (Photograph s take n b y author in capacity a s research scientis t based a t the Cocoa Black Pod Research Trust of Papua New Guinea (PNG), Kar Kar Island, PNG.) (C) A P. cinnamomi-infected peac h tree showing extensive trunk canker (arrow). (Photograph courtesy of Dr. F. Greenhalgh, Plant Research Institute, Burnley, Victoria, Australia.) (D) An avocado tree attacked by P. cinnamomi i n an orchard a t Mt. Tamborine , Queensland , Australia . Not e the dieback o f th e outer branches , wilted leave s (commo n secondar y symptom s o f th e disease cause d b y P. cinnamomi), an d th e exposed remainin g fruit (arrow) . (Fro m Phillips , D . P. , Response s o f Susceptibl e an d Resistan t Avocad o Cultivar s t o Infectio n b y Phytophthora cinnamomi, Ph.D . thesis , Universit y o f Melbourne , Melbourne , Australia , 1989. )

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FIGURE 2 . Th e lif e cycl e o f Phytophthora cinnamomi, illustratin g alternativ e stage s in the life cycl e b y whic h the fungus adapt s to environmental changes . (Redraw n fro m Weste , G. , i n Plant Diseases: Infection Damage and Loss, Blackwel l Scientific , Oxford , 1984 , 273 . Original drawin g b y H . J . Swart . Wit h permission. )

pathological anatom y o f root diseases caused by Phytophthora spp . throughout th e chapter , because littl e informatio n i s availabl e fo r mos t othe r interaction s involvin g Phytophthora spp. an d frui t trees .

II. INFECTIO N PROCESS IN SUSCEPTIBLE HOSTS Where host s hav e co-evolve d wit h funga l pathogens , susceptibilit y t o diseas e i s th e exception rathe r tha n th e rule. 67 Plant s ar e continually expose d t o attac k b y a vast range of air- an d soilborn e fungi , viruses , insects , nematodes , an d bacteria . Yet , a s i s commonl y noted i n review s o f hos t defens e mechanisms , th e majorit y o f plant s remai n health y an d grow vigorousl y mos t o f th e time. 67-68 Thi s phenomeno n ma y b e a n indirec t reflectio n o n the capacit y o f either : 1. Th

e plant's first lin e of defense — th e cuticle of leaves, cork of stem, an d exodermi s of root s — t o resist infectio n 2. Th e abilit y o f th e pathoge n t o first reach , germinat e on , an d penetrat e th e hos t If infectio n i s successful , plant s ma y als o resis t subsequen t invasio n o f a pathogen b y presenting a larg e rang e o f bot h nonspecifi c an d specifi c defens e reaction s which , whe n fully differentiated , ma y b e a combinatio n o f physiological , biochemical , o r physica l re sponses. The defense mechanisms themselves are not considered to bear any particular degree of specificity, bu t this may be denoted by the manner in which they are induced. Preforme d

Root Diseases Caused by Phytophthor a

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FIGURE 3. Variatio n i n encystment pattern , diagrammati c representation . (Redraw n fro m Hinch , J . an d Weste, G., Aust. J. Bot., 27 , 679 , 1979 . Wit h permission. ) (Pattern A) Th e cells a t the tip of th e root represent th e root cap cells that wer e present o n practically al l root s studied. Thes e cell s ar e sloughe d of f a s th e plan t grows . Thi s represent s a common typ e o f encystmen t pattern ; most zoospore s ar e attracted t o the region immediatel y behin d th e root tip , forming severa l layers . Th e rest of the root, excep t th e cut end , attracte d onl y smal l number s o f zoospores ; mos t zoospore s accumulate d nea r th e center . Examples: Themeda triandra (syn . T. australis), Acacia spp . (withou t sheath) . (Pattern B) This represent s th e mos t commo n typ e of patter n found , wit h mos t zoospore s accumulatin g i n the region behin d th e roo t tip . Fe w zoospore s wer e foun d o n th e roo t hairs . Examples : Isopogon ceratophyllus, Leptospermum juniperinum, Banksia spp . (excep t B. spinulosa), Eucalyptus spp. , Pittosporum undulatum. (Pattern CI) Thi s represents th e accumulation o f zoospores i n the tip-most region of the root and in the region behind th e roo t tip ; the res t o f th e roo t attracte d fe w zoospores . Examples : Melaleuca spp. , Hakea spp. , Banksia spinulosa. (Pattern C2) This i s a similar pattern t o CI, bu t with fe w zoospore s attracte d to the root hair region. Example : B. spinulosa. (Pattern D) I n case s i n whic h th e roo t hai r regio n starte d immediatel y behin d th e roo t tip , mos t zoospore s accumulated i n the region of maturation. Onc e again, fe w zoospore s accumulated i n the root hair region. Example : Hakea spp . (Pattern E) With this type, characterized b y Xanthorrhoea australis, X. resinosa, andX . minor, larg e numbers of zoospore s encyste d i n th e extensive , highl y pigmente d roo t ca p sheath . (Pattern F) In some plants with root cap sheaths, mostly Acacia spp., most zoospores encysted in the maturation region immediatel y behin d th e sheath .

fungitoxic compounds , secondar y metabolite s suc h a s oxidize d phenolic s (quiñones) , an d phytoalexins ar e example s o f physiologica l responses . Preexistin g o r induce d structura l barriers suc h a s tylose s an d gums , tissu e lignificatio n an d o r suberization , papilla e o r cel l wall appositions , hypha l encapsulation , an d meristematic barrier s suc h as wound periderm s and cor k tissu e ar e example s o f physica l barriers. 67-69 72 Over t diseas e expressio n (suscep tibility) only occurs when the pathogen circumvents a number of the host defense responses , or th e hos t fail s o r i t to o slo w i n expressio n o f defens e reactions . The histolog y o f pre - an d earl y postpenetratio n event s b y Phytophthora spp . ha s bee n reviewed i n recen t years. 55'73 76 Suc h event s ar e summarize d fo r P. cinnamomi (Tabl e 1) .

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Cytology, Histology, and Histochemistry of Fruit Tree Diseases

FIGURE 4 . (A ) Ligh t micrograp h o f a sectio n o f Zea mays roo t take n i n th e zon e o f elongatio n 9 0 mi n postinfection. Th e fungal cysts have germinated and hyphae are penetrating the root mucilage layer and the epidermal cell walls (arrows). (Magnificatio n x 140. ) (A, C, an d D from Hinch , J. M . e t al., Protoplasma, 126 , 178 , 1985. With permission. ) (B ) Scanning electro n micrograp h o f zoospore s o f P. cinnamomi that hav e encysted o n th e root surface o f a Lupinus angustifolius L . seedling . Th e ger m tub e o f eac h cys t penetrate s th e epiderma l cell s inter cellularly. (Photograp h courtes y o f Dr . G . Weste , Schoo l o f Botany , Universit y o f Melbourne , Parkville , an d Dr . J. Hinch , Plan t Researc h Institute , Burnley , Victori a Australia. ) (C , D ) Ligh t an d fluorescenc e micrograph s o f serial sections , take n 2 h postinfection , showin g a singl e hyph a penetratin g th e hos t epiderma l laye r throug h th e middle lamella . Callós e deposit s o f varyin g thicknes s (smal l arrows ) hav e forme d behin d th e hypha l ti p (larg e arrow). (Magnificatio n x 512. ) Th e section s i n th e ligh t an d fluorescenc e micrograph s ar e staine d i n toluidin e blue an d 0.1 % decolorized anilin e blue , respectively . (A , C , an d D from Hinch , J . M . e t al. , Protoplasma, 126 , 178, 1985 . With permission.) (E) A lesioned root in avocado clonal plant, rootstock 'Duk e 7' (moderately resistant), 12 d postinoculation a t it s tip wit h a droplet containin g 8 0 zoospores o f P. cinnamomi. Th e lesio n i s 6.2 c m lon g and stoppe d extendin g (arrow ) o n da y 1 0 postinoculation. Th e fungu s wa s readil y reisolate d fro m necroti c tissu e at th e tim e o f harves t an d th e plan t wa s hel d a t a soil-roo t temperatur e o f 25°C . (Par t E fro m Phillipa , D . P. , Responses of Susceptibl e an d Resistant Avocad o Cultivars to Infection b y Phytophthora cinnamomi, Ph.D . thesis, University o f Melbourne , Melbourne , Australia , 1989. )

Root Diseases Caused by Phytophthor a 21

1

FIGURE 5 . Top s an d root s o f 6-month-ol d seedling s o f Norther n Californi a blac k walnu t (Juglans hindsii) ( A and B ) an d Parado x (J. hindsii x Englis h walnu t (J. regia)), ( C an d D ) grow n fo r 3 months i n noninfeste d soi l (a) and artificially infeste d soi l with Phytophthora cactorum (b), P. cinnamomi (c), Phytophthora sp . (isolate 1029 ) (d), an d P. megasperma (e) . Not e tha t al l fou r Phytophthora spp . (b , c , d , an d e ) cause d visibl e damag e t o J. hindsii (A and B), whereas only P. cinnamomi (c) severely affected 'Paradox ' seedlings (C and D). (From Mircetich, S. M . an d Matheron , M . E. , Phytopathology, 73 , 1481 , 1983 . With permission. )

Attraction t o and penetration o f roots by P. cinnamomi and other Phytophthora spp . is rapid and simila r irrespectiv e o f th e susceptibilit y o r compatibilit y o f th e host . Thi s ha s bee n clearly show n i n P. cinnamomi diseas e of avocado 45 and eucalypt roots, 77,90 P. megasperma var. sojae attac k o f soybea n hypocotyls, 91 P. megasperma f.sp . glycinea attac k o f soybea n roots,14 P. infestans attac k o f potat o tubers, 10 an d P. fragariae Hickma n infectio n o f root s of bot h susceptibl e an d resistan t strawberr y cultivars. 92 A differenc e i n attractio n o f zoospore s t o roo t tip s wa s reporte d betwee n rabbitey e (resistant) an d highbus h (susceptible ) blueberrie s infecte d wit h P. cinnamomi, 97, but no t b y Hinch and Weste 53 for Australia n nativ e specie s (Figur e 3) . However, i t was also noted tha t young rabbitey e rootlet s wer e equall y susceptibl e t o penetratio n an d subsequen t infectio n compared wit h highbus h rootlets. 93 Primary roots are most frequently invade d by P. cinnamomi19^13 an d other Phytophthora spp. i n th e zon e o f elongation , an d thi s regio n provide s littl e o r n o mechanica l resistanc e to penetratio n (Figur e 4A) . Th e epidermi s i s youn g an d thi n walle d an d th e exodermi s i s undifferentiated an d endodermi s unthickened, 55-73 compare d wit h mor e matur e roo t tissu e further bac k fro m th e roo t tip . However , specie s suc h a s P. cinnamomi ar e know n t o als o infect majo r root s possessin g tissu e tha t ha s bee n secondaril y thickened. 50-9495 Germ tubes fro m encyste d zoospore s preferentially penetrate the outer epidermal layer s of primary root s intercellulary , i.e. , betwee n th e anticlinal cel l walls (Figure s 4B to D). 5455 In one study of P. megasperma f.sp. glycinea on soybean, up to 94% of germ tube penetrations were reported t o be through anticlina l cel l walls, wit h only 5 % forming swolle n germ tubes closely appresse d t o th e roo t surface. 14 Appressori a d o no t appea r t o b e a featur e o f ger m tube penetratio n fo r zoospore s of Phytophthora spp . Evidenc e exist s o f th e middl e lamell a of epiderma l cell s bein g partiall y hydrolyze d b y invadin g ger m tube s an d hyphae. 5586 P. cinnamomi has been found to demonstrate some capacity to produce enzymes that dephenolize

212 Cytology,

Histology, and Histochemistry of Fruit Tree Diseases TABLE 1 Pre- an d Earl y Postpenetratio n Event s b y P. cinnamomi

Time afte r inoculation (h) Even

t Ref

e releas e an d preferentia l attractio n t o roo t ti p and/o r cu t o r ex - 19 posed wound s Attraction i s nonspecifi c fo r hosts , an d irrespectiv e o f thei r susceptibil - 45 ity Attraction ca n b e chemotaxi c fo r sugars , amin o acids , an d ethanol , and / 19 or electrotaxi c 0.5 Zoospor e encystmen t a t roo t surfac e 55 Adheres, recognitio n bein g mediate d b y L-fucos e receptor s o n zoospor e 80-8 and possibl y pectin-base d materia l o n roo t Cyst germinatio n Prealignment o f emergin g ger m tub e 8 0.5-1 Penetratio n o f epidermis , preferentiall y intercellula r alon g anticlina l cel l 45 walls throug h middl e lamella e Evidence o f bot h enzymi c an d mechanica l penetratio n 54 2-4 Infectio n establishe d i n cortex; hypha e ar e seale d of f fro m empt y cyst s b y 5 amorphous plug s a t cys t bases ; mainl y intercellula r developmen t i n oute r cortex Microsporangia presen t 4 16 Hypha l penetratio n o f endodermi s an d stel e 7 18-24 Lesio n readil y apparen t an d necrosi s i s widesprea d 45 Sporangia o n roo t surfac e 7 Hyphae i n stele , concentrate d i n phloem an d xyle m lumen ; largel y intra - 8 cellular Papillae/wall apposition s ma y occu r i n corte x 54 Extensive cel l wal l hydrolysi s i n roo t ti p 55 4-6 d Hypha l ramificatio n extensiv e throughou t stel e an d cortex; hypha l vesicle s 45 and swelling s abundan t i n corte x Small haustorial-lik e structures/sid e branche s fro m intercellula r hypha e i n 84 cortex

.

0 Zoospor

, 53 , 55 , 7 7 , 78 , 7 9 , 77 2 3

5 5 3 3 4

, 55 , 6 4 , 77

, 54 , 7 3

, 85 , 86 , 73 , 87-8 9 , 85

lignin,96 97 and partially disintegrat e wall s of xylem vessels with secondary thickening i n the susceptible avocad o rootstoc k 'Top a Topa', 42 a s does P. capsici i n infected tissu e of peppe r

roots. 98

A. HOST-SPECIFI C RESPONSE S Once infectio n hypha e hav e gaine d entr y int o th e oute r layer s o f hos t tissue , whethe r inter- o r intracellulary , host-specifi c Phytophthora spp . characteristicall y for m haustoria , which penetrat e th e cel l wall s int o th e cytoplasm , bu t no t th e vacuole . Thi s i s tru e fo r infections cause d b y P. infestans, 10 P. parasitica var . nicotianae (Dastur ) Waterh., 74 an d P. megasperma var. sojae on soybean hypocotyl, 16-91 but not for P. megasperma f.sp. glycineainfected soybea n roo t tissue. 14 Haustoria l siz e ma y change . However , the y al l hav e a cel l wall continuou s wit h tha t o f th e intercellula r hypha e an d ar e generall y large r tha n thos e formed i n resistan t host s (Figure s 6 A an d B). 10 The haustori a ma y als o b e surrounde d b y an extrahaustoria l matrix. 10 Beside s th e extensiv e developmen t o f haustoria , cel l wal l ap position developmen t i n associatio n wit h the m may 16'98 o r ma y not 10 b e commonplace . It is reported tha t no discernible difference s exis t i n the ultrastructure o f both inter - an d intracellular hypha e o f P. infestans whe n invadin g tuber s o f bot h susceptibl e an d resistan t cultivars of potato.10 Furthermore, in P. parasitica var . nicotianae infection o f tobacco roots,

Root Diseases Caused by Phytophthor a

213

FIGURE 6. Summar y diagram of haustorial apparatus of Phytophthora infestans in a resistant (A) and a susceptible (B) potato cultivar. Extrahaustoria l matri x (ema) , extrahaustorial membran e (erne) , fungal lipi d bodie s (fl) , funga l plasma membrane , (fp) , funga l cel l wall , (fw) , hos t cel l dictyosom e (hd) , hos t cel l microbod y (hm) , hos t cel l plasma membran e (hp) , hos t cel l tonoplas t (ht) , hos t cel l vacuol e (hv) , hos t cel l wal l (hw) , wal l appositio n (wa) , wall o f haustoriu m (wh) . (Fro m Hohl , H . H . an d Stõssel , P. , Can. J. Bot., 54 , 900 , 1976 . Wit h permission. )

little or no change in cell ultrastructure was observed in cells of the susceptible host-pathogen race combinations i n advanc e o f th e pathogen, 74 unlik e tha t whic h occurre d i n the resistan t host/pathogen rac e combinatio n (se e Sectio n III.A) . Th e lac k o f hos t cel l necrosi s unti l 2 4 to 48 h postinfection i n suspects has also been noted for P. infestans on potato.75 Cell necrosis eventually followed , bu t di d no t preven t continue d growt h an d developmen t o f funga l my celium or subsequent sporulatio n a s would have occurred in biotrophs such as rust. Mycelia l development wa s usuall y sever e an d systemi c throughou t hos t tissue .

214

Cytology, Histology, and Histochemistry of Fruit Tree Diseases

B. NONSPECIFI C HOS T RESPONSE S The process of infection by a nonspecific pathogen such as P. cinnamomi in a susceptible host i s summarized i n Table 1 . In a moderately resistan t rootstoc k suc h a s the avocado host 'Duke 7\ lesion s progresssed into well-differentiated primar y host tissue containing vascula r cambium an d lignifie d stela r elements , bu t wa s the n arreste d (Figur e 4E). 42,89 I n th e sus ceptible rootstock 'Top a Topa', th e pathogen wa s not arrested an d infection wa s extensive. 42 The histology of lesioned root in both rootstocks revealed three zones of tissue with distinctive anatomy in association with the lesion boundary.4289 The changes that resulted from infectio n are summarize d i n Table 2 (see Sectio n III.B) . I n most cases, th e infection o f a susceptibl e host i s characterized b y extensive an d intensiv e inter - an d intracellular hypha l developmen t and ramification throughou t both cortical and stelar tissues, as depicted i n light micrograph s of lesione d tissu e o f th e moderatel y resistan t rootstoc k 'Duk e 7 ' (Figure s 7 A t o C). 89 Cell lysis an d degradatio n i s ofte n extensiv e an d accompanie d b y accumulatio n o f materia l tha t stains dee p blue-blac k and/o r blue-gree n i n toluidin e blu e O , indicatin g th e presenc e o f polyphenols and tannins (Figure 7C).55 ' 73 ' 76 ' 89 " This is confirmed b y strong autofluorescenc e of infecte d tissue. 7389 Smal l haustorial-lik e structures , o r sid e branches , ar e sometime s observed suc h a s for host s infecte d b y P. cinnamomi 42-73 and P. capsici. 98

III. POSTINFECTION CHANGES IN THE ANATOMY OF RESISTANT HOSTS Plant defense s includ e a large range o f nonspecifi c mechanism s suc h a s cell wal l mod ification, whic h appea r t o function a s physical barrier s an d ma y b e closel y correlate d wit h the preventio n o r restrictio n o f disease . Whethe r suc h response s pla y a genera l rol e i n successful hos t defens e i s controversial , an d eac h cas e mus t b e carefull y considere d indi vidually. Som e example s o f physica l defens e mechanism s resultin g i n anatomica l change s are discusse d below . A. HOST-SPECIFI C RESPONSE S In resistan t host s o f host-specifi c pathogen s haustoria l structure s ar e als o forme d upo n infection o f hos t tissue , bu t diffe r fro m thos e forme d i n susceptibl e hosts . The y ar e ofte n much smalle r an d surrounde d b y a thinne r extrahaustoria l matri x wit h well-develope d ap positions typically encasin g th e entire haustorium (Figur e 6A). 1011 The wall apposition s ar e usually characterize d b y electron-translucen t materia l embedde d wit h smal l electron-dens e patches.10 A vita l respons e normall y observe d i n resistan t host s i s th e hypersensitive response . Hypersensitivity, th e rapi d collaps e an d deat h o f hos t cells , i s generally considere d t o be a specific defens e response that is a critical factor in preventing the establishment of a biotroph early o n i n infectio n i n man y resistan t hosts. 70 Th e rapi d collaps e o f hos t cell s lead s t o cessation o f growth an d development o f th e invading pathogen i f this shoul d be a biotroph, i.e., a parasite requiring livin g hos t tissue fo r growth . Th e response i s formed i n epiderma l and oute r cortica l cell s immediatel y adjacen t t o invadin g hypha e withi n a fe w hour s o f infection, suc h as in roots of resistant lines of tobacco infected b y P. parasitica var. nicotianae 14 and soybea n hypocoty l infecte d b y P. megasperma var . sojae. 16 I n infectio n o f resistan t lines of strawberry b y P. fragariae, hypha l penetration als o did not extend beyond the outer layer o f roo t ti p cells. 92 Postinvasive sporulatio n i n suc h interaction s i s usuall y reporte d a s being eithe r greatl y reduce d o r altogethe r absent. 75 A secon d majo r hos t defens e respons e associate d wit h host-specifi c resistanc e i s physiological — th e production o f phytoalexins . Phytoalexin s ca n be defined a s "antimicrobia l compounds of low molecular weight synthesized by and accumulated in plants after exposur e to microorganisms".100 These compounds produced quickly enough, and in sufficiently hig h

215

Root Diseases Caused by Phytophthor a TABLE 2 Summary o f Histologica l Observatio n o f Uninfecte d Roo t Tissu e an d P. cinnamomi-lnfected Tissu e o f 'Duk e T (moderatel y resistant ) an d 'Topa Topa ' (susceptible ) Clona l Rootstoc k Material 42 Character

Infected 'Duk e T 'Top

a Topa '

Uninfected control s (both rootstocks )

Zone 1/Lesio n (infecte d tissue ) Absent Non-necrotic Intact Turgid Red-purple/blue

Hyphae Cell Walls Contents Stain wit h toluidin e blue O Phenolics/tannins

Inter- an d intracellula r Inter - an d intracellula r Necrotic Necroti c Lysed/fragmented Lysed/fragmente d Plasmolyzed Plasmolyze d Blue-black/dark blue - Blue-black/blue-gree n green Accumulated/widespread Accumulated/widesprea d

Autofluorescence Xylem Discoloration Vessel Wall s % Blockag e b y tyloses/detritus Starch grain s

Strong

Absent Absen t Lignification reduce d Lignificatio n reduce d Up to 40-45% U p t o 40%

Absent Lignification complet e 3. 0 c m in advance of lesio n boundaries, th e percentag e o f xyle m element s containin g tylose s wa s