The Mycorrhizal Symbiosis in Mediterranean Environment: Importance in Ecosystem Stability and in Soil Rehabilitation Strategies: Importance in Ecosystem Stability and in Soil Rehabilitation Strategies [1 ed.] 9781620813416, 9781620812785

Citation preview

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved. The Mycorrhizal Symbiosis in Mediterranean Environment: Importance in Ecosystem Stability and in Soil Rehabilitation Strategies : Importance in

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved. The Mycorrhizal Symbiosis in Mediterranean Environment: Importance in Ecosystem Stability and in Soil Rehabilitation Strategies : Importance in

ENVIRONMENTAL SCIENCE, ENGINEERING AND TECHNOLOGY

THE MYCORRHIZAL SYMBIOSIS IN MEDITERRANEAN ENVIRONMENT

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

IMPORTANCE IN ECOSYSTEM STABILITY AND IN SOIL REHABILITATION STRATEGIES

No part of this digital document may be reproduced, stored in a retrieval system or transmitted in any form or by any means. The publisher has taken reasonable care in the preparation of this digital document, but makes no expressed or implied warranty of any kind and assumes no responsibility for any errors or omissions. No liability is assumed for incidental or consequential damages in connection with or arising out of information contained herein. This digital document is sold with the clear understanding that the publisher is not engaged in rendering legal, medical or any other professional services. The Mycorrhizal Symbiosis in Mediterranean Environment: Importance in Ecosystem Stability and in Soil Rehabilitation Strategies : Importance in

ENVIRONMENTAL SCIENCE, ENGINEERING AND TECHNOLOGY Additional books in this series can be found on Nova‘s website under the Series tab.

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

Additional e-books in this series can be found on Nova‘s website under the e-book tab.

The Mycorrhizal Symbiosis in Mediterranean Environment: Importance in Ecosystem Stability and in Soil Rehabilitation Strategies : Importance in

ENVIRONMENTAL SCIENCE, ENGINEERING AND TECHNOLOGY

THE MYCORRHIZAL SYMBIOSIS IN MEDITERRANEAN ENVIRONMENT IMPORTANCE IN ECOSYSTEM STABILITY AND IN SOIL REHABILITATION STRATEGIES

MOHAMED HAFIDI Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

AND

ROBIN DUPONNOIS EDITORS

Nova Science Publishers, Inc. New York The Mycorrhizal Symbiosis in Mediterranean Environment: Importance in Ecosystem Stability and in Soil Rehabilitation Strategies : Importance in

Copyright © 2012 by Nova Science Publishers, Inc. All rights reserved. No part of this book may be reproduced, stored in a retrieval system or transmitted in any form or by any means: electronic, electrostatic, magnetic, tape, mechanical photocopying, recording or otherwise without the written permission of the Publisher. For permission to use material from this book please contact us: Telephone 631-231-7269; Fax 631-231-8175 Web Site: http://www.novapublishers.com NOTICE TO THE READER The Publisher has taken reasonable care in the preparation of this book, but makes no expressed or implied warranty of any kind and assumes no responsibility for any errors or omissions. No liability is assumed for incidental or consequential damages in connection with or arising out of information contained in this book. The Publisher shall not be liable for any special, consequential, or exemplary damages resulting, in whole or in part, from the readers‘ use of, or reliance upon, this material. Any parts of this book based on government reports are so indicated and copyright is claimed for those parts to the extent applicable to compilations of such works.

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

Independent verification should be sought for any data, advice or recommendations contained in this book. In addition, no responsibility is assumed by the publisher for any injury and/or damage to persons or property arising from any methods, products, instructions, ideas or otherwise contained in this publication. This publication is designed to provide accurate and authoritative information with regard to the subject matter covered herein. It is sold with the clear understanding that the Publisher is not engaged in rendering legal or any other professional services. If legal or any other expert assistance is required, the services of a competent person should be sought. FROM A DECLARATION OF PARTICIPANTS JOINTLY ADOPTED BY A COMMITTEE OF THE AMERICAN BAR ASSOCIATION AND A COMMITTEE OF PUBLISHERS. Additional color graphics may be available in the e-book version of this book. Library of Congress Cataloging-in-Publication Data The mycorrhizal symbiosis in Mediterranean environment : importance in ecosystem stability and in soil rehabilitation strategies / editors, Mohamed Hafidi and Robin Duponnois. p. cm. Includes bibliographical references and index.

ISBN:  (eBook)

1. Mycorrhizas--Mediterranean Region. 2. Mycorrhizas--Ecology--Mediterranean Region. 3. Biotic communities--Mediterranean Region. 4. Soil restoration--Mediterranean Region. 5. Plant-soil relationships--Mediterranean Region 6. Mediterranean Region--Environmental conditions. I. Hafidi, Mohamed. II. Duponnois, Robin. QK604.2.M92M9647 2012 627'.5--dc23 2012007270

Published by Nova Science Publishers, Inc. † New York The Mycorrhizal Symbiosis in Mediterranean Environment: Importance in Ecosystem Stability and in Soil Rehabilitation Strategies : Importance in

Contents Preface

vii

Topic 1: Mycorrhizal Symbiosis and Performance of the Eco- and Agro Systems Chapter I

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

Chapter II

Chapter III

Chapter IV

Chapter V

Chapter VI

Natural Diversity of Ectomycorrhizal Fungi in Moroccan Forest Woodlands Bakkali Yakhlef Salah Eddine and Duponnois Robin Ectotrophic Mycorrhizal Symbioses Are Dominant in Natural Ultramafic Forest Ecosystems of New Caledonia Y. Prin, M. Ducousso, J. Tassin, G. Béna, P. Jourand, V. Dumontet, L. Moulin, C. Contesto, J. P. Ambrosi, C. Chaintreuil, B. Dreyfus and M. Lebrun Mycorrhizal Status and Arbuscular Mycorrizal Fungal Diversity of Endangered Plant Species in the Sierra Nevada National Park Concepción Azcón-Aguilar, Javier Palenzuela, Nuria Ferrol, Fritz Oehl and José Miguel Barea Ectomycorrhizal Fungal Communities in Quercus Suber Ecosystems E. Lancellotti and A. Franceschini Reclamation Strategies of Semiarid Mediterranean Soil: Improvement of the Efficiency of arbuscular mycorrhizal fungi by Inoculation of Plant Growth Promoting Microorganisms and Organic Amendments Almudena Medina and Rosario Azcón The Use of Mychorrhiza in Soilless Grown Vegetables Ozlem Ikiz, H. Y. Dasgan and H. Okkaoglu

The Mycorrhizal Symbiosis in Mediterranean Environment: Importance in Ecosystem Stability and in Soil Rehabilitation Strategies : Importance in

1 3

25

49

71

87

107

vi Chapter VII

Chapter VIII

Contents Implications of Mycorrhizal Symbioses in the Trajectory of Plant Invasion Process: How Do They Matter? Sanon Arsene, Ndoye Fatou, Ramanankierana Heriniaina and Duponnois Robin Changes in Ectomycorrhizal Community Structure of Coccoloba Uvifera L Mature Trees and Regenerating Seedlings at Two Levels of Salinity Avril Raymond, Abdala G. Diédhiou, Clémence Chaintreuil, Sandrine Bessard, Seynabou Séne, Abdennebi Omrane, Régis Courtecuisse, Samba Sylla, Robin Duponnois and Amadou M. Bâ

129

151

Topic 2 : Biotechnology and Controlled Mycorrhization

169

Chapter IX

171

Mycorrhizal Industry in Modern Agriculture Silvio Gianinazzi and Sylvie Masquelier

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

Index

The Mycorrhizal Symbiosis in Mediterranean Environment: Importance in Ecosystem Stability and in Soil Rehabilitation Strategies : Importance in

185

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

Preface The role of mycorrhizal symbiosis has been usually related to its impact on the plant mineral nutrition and consequently on the development of the plant species. However, it has been demonstrated that this symbiotic process has a key role in ecosystem stability. Numerous recent studies have outlined the major role of mycorrhizal symbioses in ecosystem functioning and the necessity to manage this soil microbial component in order ensure the productivity of plant ecosystems and to maintain plant diversity. The main objectives of this book are to present recent results showing the expected benefits in managing the mycorrhizal symbiosis in order to ensure the conservation of endemic plant diversity and to rehabilitate degraded soils in Mediterranean areas. Chapter I - In Morocco, the forest woodlands undergo a worrying regression in spite of the intensive plantation programs. The success of these programs with a good plant establishment after transfer to the field is related among others to the cultural techniques used in nurseries. The controlled mycorrhization in nurseries, by selected ectomycorrhizal fungi, improves survival, establishment, and growth of seedlings after out planting. The aboveground surveys of sporocarps are usually poor indicators of the community structure below ground. This is because sporocarp production is triggered by specific environmental conditions. A classical approach for identifying mycorrhizas is therefore to trace hyphal connections between sporocarps and mycorrhizal sheaths. However, special skills are required when root density is so high that hyphae emerging from the stipe base cannot be attributed unambiguously to a single mycorrhizal. An alternative promising way to identify mycorrhizas consists of comparing specific DNA regions of mycorrhizas and sporocarps. Polymerase Chain Reaction coupled with Restriction Fragment Length Polymorphism analyses (PCR/RFLP) and sequencing have been applied in mycorrhizal research to identify strains of naturally occurring mycorrhizal fungi and also to differentiate and identify mycorrhizal symbionts unambiguously. Chapter II - Insularity, geological history and biogeography have made from NewCaledonia a hot spot of biodiversity where extremely diversified ecosystems occupies ultramafic terrains with drastic edaphic conditions in terms of fertility and metallic toxicity. In the framework of the mine project of the Koniambo Massif, a large nickel deposit, we tried to explore the diversity of ectomycorrhizal symbioses within these poorly explored natural ultramafic ecosystems.

The Mycorrhizal Symbiosis in Mediterranean Environment: Importance in Ecosystem Stability and in Soil Rehabilitation Strategies : Importance in

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

viii

Mohamed Hafidi and Robin Duponnois

Floristic inventories along an altitudinal gradient ranging from 700 to 900 m evidenced 4 different plant communities. The 2 lower plant communities, 3 and 4, were dominated by 2 endemic tree genera, Tristaniopsis (Leptospermoideae) and Nothofagus (Nothofagaceae) respectively, whose ectomycorrhizal (ECM) status was shown and explored through molecular methods on sporocarps, mycorrhizae and soil mycelium. We evidenced a diversified fungal community in the basal plant community dominated by two tree species of the genus Nothofagus. The molecular characterization of these ECM fungi was established on the total ribosomal inter transcribed spacer (ITS) by PCRsequencing and BLASTn analysis, revealing the relative abundance of the Cortinariaceae among our samples. Samples belonging to this fungal family were phylogenetically analyzed on the same ITS, in reference to sequences of samples with geographically different origins, including countries derived from the Gondwanaland fragmentation. If no clear phylogenetical relationships were evidenced, our study confirmed the same relative dominance of ECM Nothofagaceae, as well as the relative abundance of associated Cortinariaceae, in New Caledonia as in several of the Gondwanaland-originating countries. Chapter III - Mycorrhizas have played a key role in plant evolution on Earth as well as on the development of the structure and diversity of terrestrial ecosystems. Most plants depend on mycorrhizas to thrive, particularly in fragile and stressed environments, as those in certain areas of the high Mediterranean mountains of the Sierra Nevada National Park (Granada, Spain). Sierra Nevada constitutes an exceptional refuge for the flora and one of the enclaves with higher biodiversity levels of the European continent. It presents about 2100 plant species and 80 exclusive endemisms, some of them threatened with extinction. With the objective of ascertaining the impact of mycorrhizal associations at facilitating the conservation of species from the threatened flora of Sierra Nevada a research programme was initiated aiming at (i) determining the mycorrhizal status of selected species of the endangered flora of Sierra Nevada, (ii) analysing the diversity of the mycorrhizal fungi associated with the selected species, and (iii) establishing a mycorrhizal fungi germplasm bank. Thirty four plant species belonging to 22 different botanical families were selected. All of them belong to the endangered and/or endemic flora of Sierra Nevada. The results showed that six out of the 34 selected species had no mycorrhizal colonization. All the other 28 species (about 80 % of the studied plant species) showed arbuscular mycorrhizal (AM) colonization and one of them (Salix hastata) also ectomycorrhizas. In most mycorrhizal plants, the typical structures characteristic of the AM symbiosis, mainly arbuscules and vesicles, could be observed. Arum, Paris and intermediate type AM were detected, produced by the colonization with coarse endophytes in most cases, although fine endophytes were also evident in some plant species. Approximately one third of the studied plants were colonised by dark septate endophytes. The density of AM fungal spores in the soil around the selected plants was relatively low, except for certain plant species, such as the fern Ophioglossum vulgatum. By using morphological and molecular criteria most of these spores were identified up to species level. AM fungal species richness was, however, quite high. More than 60 different AM fungal species were detected, belonging to the genera Glomus, Acaulospora, Entrophospora, Gigaspora, Scutellospora, Pacispora, Diversispora, Ambispora and Paraglomus. The most frequent genera in Sierra Nevada are Glomus and Acaulospora. Acaulospora species are mainly found at the highest altitudes and in acidic soils, being A. laevis the most common species. Glomus species predominate at lower altitudes and in neutral and alkaline soils, with G. constrictum and G. etunicatum as the most frequent species. A new AM fungal species (Entrophospora

The Mycorrhizal Symbiosis in Mediterranean Environment: Importance in Ecosystem Stability and in Soil Rehabilitation Strategies : Importance in

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

Preface

ix

nevadensis) was described recently. Some other spore types do not correspond to any of the species described so far. A germplasm collection of autochthonous AM fungi from Sierra Nevada has been established with the isolated fungi that could be grown as monospecific cultures. This collection is being used to ascertain the effect of a tailored mycorrhizal inoculation, with autochthonous mycorrhizal fungi, on the nursery production of target plants to be reintroduced in their natural habitats according to the conservation initiatives in the National Park. Chapter IV - The cork oak (Quercus suber L.) is an important resource from an ecological and socio-economic point of view for all the countries bordering on the Mediterranean basin. However, for decades, a gradual reduction in productivity and consistency of the forests of this species throughout its distribution area has been observed. While the degradation of these forests depends on many factors, a prominent role is played by the growing spread of the so-called ―cork oak decline‖. This non-specific phenomenon is caused by the combined action of several adverse factors of environmental and / or anthropogenic origin, which alter the balance between the biotic and abiotic components of ecosystems and induce physiological stress in the cork oak trees, often with a fatal outcome. In particular, the plants under stress undergo a reduction in the accumulation of carbohydrates in the roots and changes in the composition of root exudates. The indirect effect of the modified plant metabolism is the rearrangement of the microbial component of the rhizosphere, especially that represented by ectomycorrhizal fungi, whose species are known to contribute actively to plant nutrition as well as having an important ecosystem function. For these reasons, several studies of the dynamics of ectomycorrhizal communities in cork oaks affected by the phenomena of decline have been conducted in recent years in Sardinia (Italy). This research initially focused on defining the most appropriate methodological approach for the specific case study, and subsequently on checking the changes in ectomycorrhizal communities in relation to the health status of the cork oak trees on a local scale. Currently, large scale investigations are being conducted in order to identify the parameters of ectomycorrhizal communities that might allow their use as bio-indicators of the health of cork oak forests in general predictive models. Finally, the handling of the huge volume of data collected during this research project was carried using "eMyCo", a webbased database that permits the comparison of results of studies of ectomycorrhizal communities in different environments and the extrapolation of data to be subjected to metaanalysis Chapter V - Plant growth is limited in arid sites due to the adverse conditions coming from water stress. Moreover, soils from these areas are generally characterized by poor soil structure, low water-holding capacity, lack of organic matter and nutrient deficiency. In order to carry out a successful re-afforestation, it is necessary to improve soil quality and the ability of plants species to resist this harsh environment. Inoculation of plants with beneficial microorganisms such as Arbuscular Mycorrhizal Fungi (AMF) and others plant-growth promoting microorganisms (PGPM) may increase drought tolerance of plants growing in arid or semiarid areas. On the other hand, addition of organic amendments to the soil can reverse degradation of soil properties. Agro-waste residues such as dry olive cake (DOC) and sugar beet waste (SB) supplemented with rock phosphate (RP) can be used as organic amendments after fermentation by Aspergillus niger. it has been reported that the application of A. nigertreated DOC and/or SB to semi-arid soils increases aggregate stability, soil enzymatic activities, water soluble C and water soluble carbohydrates as well as nutrient availability,

The Mycorrhizal Symbiosis in Mediterranean Environment: Importance in Ecosystem Stability and in Soil Rehabilitation Strategies : Importance in

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

x

Mohamed Hafidi and Robin Duponnois

especially phosphorus (P). AMF inoculation, using autochthonous endophytes, has been shown to be more efficient with respect to increasing plant nutrition and growth as well as plant tolerance to drought than less adapted fungi. The combined treatments involving AMF and/or PGPM inoculation and addition of the amendments into the soil can be proposed as a successful revegetation strategy and a low-input biotechnology for plant performance in Pdeficient soils under semiarid Mediterranean conditions. The analysis of co-operative microbial activities is addressed to improve the understanding of the significance of rhizosphere microbial populations. Chapter VI - Mycorrhiza means ―fungal root‖. The basic principle of this association is the nutrients that are taken up from the soil are exchanged with sugar. The infected ecosystem normally contains a mixture of types of mycorrhizal associations. Lots of microorganisms form symbiosis with plants that range a continuous scale from parasitic to mutualistic. A typical example of this widespread mutualistic symbiosis is the arbuscular mycorrhiza formed between AMF and vascular flowering plants. Many scientists and mycologist research the relations (associations) between mychorrhiza and the plants biology and their inoculation methods. This relation includes the structure of the root and mychorrhizal inoculation. Mycorrhizas are complex symbiosis and the fungi involved produce a variety of structures within the root. The most common mycorrhizal types are the arbuscular mycorrhizas named by ―arbuscular is derived from characteristic structures‖. The arbuscules occur within the cortical cells of many plant roots and also some mycothalli colonized by AM fungi. An arbuscular mycorrhiza has 3 important components; the root itself, the fungal structures within and between the cells of the root and an extraradical mycellium in the soil. Chapter VII - The composition, structure, and persistence of plant communities are governed by the extent to which the physical limitation of the environment and biotic interactions promote or hinder individual plant growth performance and reproduction. One especially important biotic interaction is the symbiosis that forms between plants and soil mycorrhizal fungi. The ability of such mutualistic relationships to influence the performance of exotic invasive plants in their introduced range has received increasing attention. Although research is still in progress to determine in more detail how mycorrhizal association directly benefits these two partners (i.e. the invader and its mutualists), it is clear from the current literature that the invaders are capable, through a positive feedback loop, to take advantage from the presence and abundance of mycorrhizal propagules or to transform fungal community in such a way to favor themselves in the expense of native species. Contrastingly, though negative feedback responses are less common in the invader-mycorrhizal fungal association, such responses may lead to depressive effects on the exotic invader. This chapter is dedicated to present and discuss some of the relevant research work that has investigated the possible role that mycorrhizal symbioses play in directing the trajectory of exotic plant invasions. Chapter VIII - We tested the hypothesis that salinity could affect the species diversity and composition of ectomycorrhizal (ECM) fungi colonizing Coccoloba uvifera L. mature trees and seedling naturally regenerating in a sampling plot of 450 m2 where soils salinity is either low (2‰), farther from the sea, or high (15‰), closer to the sea, at Bois Jolan‘s Beach (16°14‘ N, 61°23‘ W) in Guadeloupe (Lesser Antilles). For this, we identified 234 sporocarps fruiting from under mature trees and seedlings, morphotyped 10 571 root tips of 6 mature trees and 30 seedlings, ITS-typed 6 sporocarps and 362 ECM morphotypes (MTs), and

The Mycorrhizal Symbiosis in Mediterranean Environment: Importance in Ecosystem Stability and in Soil Rehabilitation Strategies : Importance in

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

Preface

xi

sequenced at least 3 representatives of each RFLP type. In areas of salted soil at 2‰, mature trees were well developed with an abundant seedling recruitment, whereas in areas of salted soil at 15‰, mature trees near the sea were shunted and seedlings were absent. Among the 6 identified sporocarps as Cantharellus cinnabarinus, Amanita arenicola, Russula cremeolilacina, Inocybe littoralis, Inocybe xerophytica and Scleroderma bermudense fruiting in salted soil at 2‰, only one of them, S. bermudense, fructified also in salted soil at 15‰. Several sporocarps were only occasionally detected (e.g. C. cinnabarinus and I. xerophytica) or absent (e.g. A arenicola and I. littoralis) on root tips. This sporocarp survey weakly reflected the belowground assessment of the ECM fungal community. Six fungal taxa were identified by molecular analysis from 9 distinguished ECM MTs. Of the 6 fungal taxa, 2 were identified as Thelephorales and 4 as S. bermudense, R. cremeolilacina, C. cinnabarinus and I. xerophytica, respectively, by sequencing of the ITS region. One species of Thelephoraceae and S. bermudense dominated all ECM communities colonizing seedlings and mature trees in salted soil at 2‰ and 15‰, respectively. Salinity in the coastal forests may be an important factor structuring the ECM communities of seagrape. Thus, S. bermudense could form the potential ECM networks between mature trees and their seedlings, and play a major role in the regeneration and maintenance of C. uvifera coastal forests. Chapter IX - The United Nations Millennium Ecosystem Assessment assembles for the first time, in an exhaustive and integrated way, knowledge about services that humans can draw from nature, how society interacts with them and what impacts anthropogenic activities have on the evolution of ecosystems. Different ecosystem services are grouped into categories, each of which includes services dealing with agricultural activities. Consequently, modern agriculture should be based on the implementation of ecological management practices that deliberately maintain resilience of ecosystem services and reduce the risk of large, costly, or irreversible changes. This implies in particular to eliminate soil degradation practices, breed for new varieties well adapted to their environment, increase crop diversity, reduce chemical inputs and water supply, and manage beneficial microbes. Arbuscular mycorrhizal (AM) fungi form a very important multifunctional community of symbiotic plant microbes, and their positive effect on plant growth and health is a relevant example of an ecological service that can be provided by nature for promoting productivity in agro-ecosystem. However, their exploitation has been hampered in the last 50 years by the increasing use of crop varieties recalcitrant to mycorrhizal fungi, decreasing implementation of crop rotation systems and excessive chemical inputs. The developing mycorrhizal industry is faced with these problems which limit its contribution to ecosystem services involved in plant production and ecosystem preservation. The development of an industrial activity producing mycorrhizal inocula is a complex procedure for companies, as it involves not only the development of the necessary biotechnological know-how but also the ability to respond to specifically related legal, ethical, educational and commercial requirements. Legal requirements are particularly important for a newly developing mycorrhizal industry because they can be determinant for the success of this new biotechnology, which is one of the few using natural microbes in plant production. In this chapter, we will discuss how growing knowledge in the field of mycorrhizal biotechnology is determinant for implementing the contribution of the mycorrhizal industry to ecosystem services and modern agriculture.

The Mycorrhizal Symbiosis in Mediterranean Environment: Importance in Ecosystem Stability and in Soil Rehabilitation Strategies : Importance in

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved. The Mycorrhizal Symbiosis in Mediterranean Environment: Importance in Ecosystem Stability and in Soil Rehabilitation Strategies : Importance in

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

Topic 1: Mycorrhizal Symbiosis and Performance of the Eco- and Agro Systems

The Mycorrhizal Symbiosis in Mediterranean Environment: Importance in Ecosystem Stability and in Soil Rehabilitation Strategies : Importance in

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved. The Mycorrhizal Symbiosis in Mediterranean Environment: Importance in Ecosystem Stability and in Soil Rehabilitation Strategies : Importance in

In: The Mycorrhizal Symbiosis in Mediterranean Environment ISBN: 978-1- 62081-278-5 Editors: Mohamed Hafidi and Robin Duponnois © 2012 Nova Science Publishers, Inc.

Chapter I

Natural Diversity of Ectomycorrhizal Fungi in Moroccan Forest Woodlands Bakkali Yakhlef Salah Eddine1, and Duponnois Robin2,3 1

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

Centre de Recherche Forestière, Charia Omar Ibn Khattab, B.P. 763 Agdal-Rabat, Maroc 2 IRD, Laboratoire des Symbioses Tropicales et Méditerranéennes (LSTM), UMR 113 CIRAD/INRA/IRD/SupAgro/UM2, Campus International de Baillarguet, TA A-82/J, Montpellier, France 3 Laboratoire Ecologie and Environnement (Unité associée au CNRST, URAC 32), Faculté des Sciences Semlalia. Université Cadi Ayyad. Marrakech. Maroc

Abstract In Morocco, the forest woodlands undergo a worrying regression in spite of the intensive plantation programs. The success of these programs with a good plant establishment after transfer to the field is related among others to the cultural techniques used in nurseries. The controlled mycorrhization in nurseries, by selected ectomycorrhizal fungi, improves survival, establishment, and growth of seedlings after out planting. The aboveground surveys of sporocarps are usually poor indicators of the community structure below ground. This is because sporocarp production is triggered by specific environmental conditions. A classical approach for identifying mycorrhizas is therefore to trace hyphal connections between sporocarps and mycorrhizal sheaths. However, special skills are required when root density is so high that hyphae emerging from the stipe base cannot be attributed unambiguously to a single mycorrhizal. An alternative promising way to identify mycorrhizas consists of comparing specific DNA regions of mycorrhizas and sporocarps. Polymerase Chain Reaction coupled with Restriction Fragment Length Polymorphism analyses (PCR/RFLP) and sequencing have been applied in mycorrhizal research to identify strains of naturally occurring 

E-mail: [email protected] Tel/Fax: +212 665388686.

The Mycorrhizal Symbiosis in Mediterranean Environment: Importance in Ecosystem Stability and in Soil Rehabilitation Strategies : Importance in

4

Bakkali Yakhlef Salah Eddine and Duponnois Robin mycorrhizal fungi and also to differentiate and identify mycorrhizal symbionts unambiguously. The main objective of this chapter is to present and discuss some of the relevant research work on ectomycorrhizal fungi diversity in Moroccan forest ecosystems to select fungal isolates and producing fungal inocula adapted to local conditions in controlled mycorrhization programs.

Keywords: Ectomycorrhizal fungi, Diversity, Molecular tools, Moroccan forest ecosystems

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

Introduction The Moroccan forest is a mosaic of natural Mediterranean or Sahara ecosystems. The area of the forest domain is estimated at about 5.7 million hectares which are covered by deciduous forests (cork oak, holm oak, argan tree and acacia) and coniferous forest (cedar, arar, fir, juniper and pine trees). These areas harbor over to 4500 species and subspecies of vascular plants of which 800 are endemic. The contribution of different forest ecosystems to the economy and society is extremely important in terms of employment, goods and services, resources and environment. Moroccan forests provide each year about 600 000 m3 of timber and industrial wood, 30% of national needs. They also offer about 10 million m3 of firewood. Other services and Non Timber Forest Products are also associated with these habitats. Despite their valuable social functions, economic and ecological forests in Morocco are subject to a worrying degradation and biodiversity loss, on average 31 000 hectares lost annually. This is due to several factors, such as drought, overgrazing, overharvesting of timber and fires. Degradation of biodiversity, compaction and erosion of watersheds resulting in siltation of dams, the advancement of desertification and lack of natural regeneration are among the consequences of this degradation. To remedy this situation, the Moroccan state has developed a policy of reforestation in the first National Reforestation Plan (NRP) (AEFCS 1970) and then after the Master Plan for Afforestation (PDR) (AEFCS 1996), which provides over 10 years of planting 500 000 hectares. To date, the impact of this program is insufficient to maintain an environmental balance. A deficiency in the adaptation of plants to the stress of transplantation, during the transfer from the nursery to the site chosen for reforestation and conditions of that environment can indeed cause significant mortality or growth reduction unacceptable (Birot 1991). The performance of the plants after planting can be enhanced by the selection of species to plant, optimization of reforestation techniques and especially by the production of seedlings in nursery quality (Mousain et al. 1994). The latter is based on genetic criteria, morphological and physiological and remains dependent on farming techniques used from planting to planting date (Birot 1991). Controlled mycorrhization in recent decades has been a remarkable tool in the production of quality seedlings, characterized morphologically, by a well-branched root system bearing a large number of short roots and absorbing the physiologically by a root system that can to make early contact with the ground within which it is introduced during reforestation (Kropp and Langlois 1990). The positive results on gains in survival and growth achieved in forest plantations through the use of mycorrhizae are

The Mycorrhizal Symbiosis in Mediterranean Environment: Importance in Ecosystem Stability and in Soil Rehabilitation Strategies : Importance in

Natural Diversity of Ectomycorrhizal Fungi in Moroccan Forest Woodlands

5

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

relatively numerous (Shemakhanova 1962; Marx and Hatchell 1986; Le Tacon et al. 1992; Grove and Le Tacon 1993). For this reason, several researchers of the Mediterranean area have conducted research on mycorrhizal status of the main tree species and indigenous shrub species associated with them, with a view to determining its role in revegetation programs (Herrera et al. 1993; Requena et al. 1996; 1997; Azcona-Aguillar et al. 2003; Caravaca et al. 2003 a and b; Ouahmane 2007; Abourouh 2000; Bakkali et al. 2008). Morphological descriptions (Agerer 1991) or allozymes (Sen 1990) have provided useful data for identifying the mycorrhizal fungi below ground, but most species have not been described by these methods. Today a wide range of molecular techniques can be used to detect DNA sequence variation in ectomycorrhizal fungi (Gardes et al. 1991a, b; Henrion et al. 1992). Detection of polymorphism using PCR-RFLP analyses of the ribosomal DNA of the internal transcribed spacer (ITS) region has been successfully used for identifying several species of fungi (Amicucci et al. 1996; Gardes et al. 1996; Di Battista 1997; Karen et al. 1997; Pritsch et al. 1997). This simple technique requires only minute amounts of DNA and two specific primers flanking the ITS region. In Morocco, numerous studies reported phenotypic and molecular diversity of ectomycorrhizal (ECM) fungi associated with oak, eucalyptus, cedar, pine and rockrose scrubs (Abourouh 2000; Bakkali et al. 2008, 2009, 2011). If our purpose is limited to mycorrhizal fungi, it is interesting to note that the type of a biological species is not fixed once and for all. A fungus may begin its existence as a saprophyte before meeting its specific partner, with whom he will establish a mycorrhizal relationship, it can give way under certain conditions, a parasitic life style. On the other hand, mycorrhizal species are also able to exploit the forest litter (Giltrap 1982). The aim of this chapter is to review works assessing the genetic diversity of ECM fungi associated with the main forest species. The goal is to identify and select the most efficient isolates to be used in mycorrhizal inoculation programs in forest nurseries. We also relate the ECM fungi diversity according to the tree-host species, to their ecology (soil abiotic characteristics) and probably their biology (ectomycorrhizal vs saprophytic).

2. Host Specificity for Moroccan Plant Ectotrophic Communities Given the ecological importance of host specificity for ectotrophic plant communities and the associated mycota, studies describing the specificity patterns occurring in selected ecosystems are of premium significance, as they can contribute to a better definition of the environmental biotic and abiotic factors that affect specificity phenomena, and how the specialization of ectomycorrhizal fungi and plant hosts originated and evolved (Molina et al. 1992; Erland and Taylor 2002; Van der Heijden and Sanders 2002).

2.1. Phylogenetic Diversity of Cork Oak Ectomycorrhizal Fungi Cork oak (Quercus suber) extending an area of 377 482 ha, with 51% and 49% in the plains and the mountains, respectively. These formations are located in humid bioclimatic zones sub-humid, semi-arid but also in case of sufficient humidity. Some lowland forests, like

The Mycorrhizal Symbiosis in Mediterranean Environment: Importance in Ecosystem Stability and in Soil Rehabilitation Strategies : Importance in

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

6

Bakkali Yakhlef Salah Eddine and Duponnois Robin

those of Maâmora and the region of Larache, are sufficiently dense and extensive. The floristic of this national treasure is about a thousand of vascular species and subspecies, including a fifty endemic. This diversity represents 21% of the Moroccan flora and more than 900 taxa with a dominance of Ericaceae or Cistaceae. The cork forests are often training in three strata - trees, shrub and grass - and the level of biodiversity in areas well conserved is estimated at 135 species per 1000 m2. Inter-specific and intra-specific fungi diversity is very important and most genres inventoried in Morocco are well represented. In terms of symbiotic associations, cork oak and some species associated with it, oak deciduous in cistaceae also form ectomycorrhizae or external symbiotic associations of many types. Community studies of ectomycorrhizal fungi are based either on identification of mycorrhizas (the socalled below ground view), or on monitoring of fruit body production (above-ground view) (Richard et al. 2005). It is clear that fruit body surveys reveal the presence of ECM taxa in a fast and inexpensive way (Richard et al. 2004). Interspecific variation among sporocarps of fungi collected in Moroccan cork oak woodlands were evaluated by phenotypic and molecular analysis (Bakkali et al. 2009a, 2010). The amplification products for the ITS region of 39 species (100%) of fungi ranging from 500 to 950 bp, coincided with the sizes obtained for the other fungi (Gardes et al. 1991a; Karen et al. 1997; Martin et al. 1998). Despite the length polymorphism observed for many of the species, ITS analysis alone was not able to separate all the genotypes. RFLP analysis of the ITS region has been suggested by several authors as a means for discriminating between fungi at the inter-specific and intra-specific level (Gardes et al. 1990, 1991b; Bruns et al. 1991; Manassila et al. 2005; Guerin-Laguette 1998). Cleavage of the ITS region with AluI allowed differentiation of 40 out of the 39 identified species according to macroscopic and microscopic characteristics (Table 1). In fact, two ITS-RFLP types of Pisolithus are identified and ITS sequence analysis indicated that the two ITS-RFLP types correspond to two distinct Pisolithus species, species 6 and species 4, recognized by Martin et al. (2002). Furthermore, none of the restriction enzymes produced a distinct pattern for the 2 Russula species (R. decipiens and R. straminea). Henrion et al. (1992) considered those types of species, which do not differ enough from the ITS region to be distinguished with this marker (e.g. Laccaria bicolor and L. laccata) as closely related species. Due to this high degree of interspecific variation of the ITS, a matching of RFLP types of mycorrhizas and identified sporocarps (reference) RFLP types within the same geographical area will be likely to indicate identical species. Basing on PCR/RFLP of the ITS of the ectomycorrhizal fungi, a RFLP database has been setting up in a World Wide Web Internet server (Martin et al. 1998). However fruit body studies often underestimate the presence of numerous taxa, like resupinate and hypogeous fungi and taxa lacking an apparent sexual stage (Horton et al. 2001). In Morocco, studies on forest tree ectomycorrhizas are poorly documented. Abourouh (2000) analyzed for the first time the morphological diversity of cork oak ectomycorrhizas. Morphotypes of Cenococcum, Pisolithus, Russula, Amanita, Boletus, etc. have been identified. Recently, as part of the agreement between the Forest Research Center (CRFMorocco) and the National Research Council (CNR-Italy), a phenotypic and molecular survey of ectomycorrhizas diversity is conducted jointly in the cork oak forests in Morocco and in Italy.

The Mycorrhizal Symbiosis in Mediterranean Environment: Importance in Ecosystem Stability and in Soil Rehabilitation Strategies : Importance in

Natural Diversity of Ectomycorrhizal Fungi in Moroccan Forest Woodlands

7

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

Table 1. Restriction fragments sizes of ITS regions of the studied fungi digested with AluI, EcoRI and HinfI

2.2. Diversity of Ectomycorrhizal Fungi Associated to Quercus ilex With an estimated area of about 1.34 million ha approximately 29% of the total Moroccan forest area, Green oak (Quercus ilex) is the most important tree species (Boudy 1958; Seigue 1985). Green oak, characterized by a remarkable ecological plasticity, is common on all types of substrates, in bioclimates wet sub-humid and semi-arid, temperate, cool and cold, and its altitudinal range is between 300 and 2700 m. However, despite their valuable social, economic and ecological functions, no information was available about the diversity of ECM fungi in Q. ilex stands. It should therefore value this diversity and make the service of the forest ecosystem. Phenotypic analysis of ectomycorrhizas showed the existence of 8 morphotypes (Table 2). Among those, 2 morphotypes were phenotypically determined as Pisolithus sp. and Cenococcum geophilum.

The Mycorrhizal Symbiosis in Mediterranean Environment: Importance in Ecosystem Stability and in Soil Rehabilitation Strategies : Importance in

8

Bakkali Yakhlef Salah Eddine and Duponnois Robin Table 2. Phenotypic description of the different morphotypes

Morphological description Simple ectomycorrhiza with a irregularly shaped, dark color brown at the apex, with white mycelium 2 Ectomycorrhiza simple dichotomy. Light brown with cream apices. Surface cottony coat, with some emerging hyphae. Presence of white mycelial strands 3 Ectomycorrhiza yellow, smooth surface and presence of mycelial cords 4 Ectomycorrhiza simple, sometimes dichotomous, shaggy black, with stiff black hyphae, radiant 5 Ectomycorrhiza simple dichotomous. Coat surface cottony, pink 6 ectomycorrhiza grayish brown, cottony mycelium with brown wrapping apex 7 Ectomycorrhiza brown to dark-green top with white dichotomy. Presence of hyphae at the base 8 Ectomycorrhiza clear dark brown with apex white dichotomy (1) ND. Not Determined.

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

Morphotype 1

Consensus taxon ND (1) ND

Pisolithus spp. Cenococcum geophylum ND ND ND ND

Figure 1. Electrophoresis of the ITS of different morphotypes of ectomycorrhizae associated with holm oak (M: molecular weight marker (smart leader)).

The identification of some fungal species through the investigation of morphological characteristics of mycorrhizal anatomy is possible, but it presents some difficulties, particularly in relation to the age of mycorrhizae, the type of host plant and the environmental conditions. The recent application of the phylogenetic species concept, which describes the species as a monophyletic group within which the rate of homology between sequences is high, has been fostered by advances in molecular analysis techniques, and tends to be heavily used in mycology (Mischler and Brandon 1987). The PCR / RFLP of the ITS of 8

The Mycorrhizal Symbiosis in Mediterranean Environment: Importance in Ecosystem Stability and in Soil Rehabilitation Strategies : Importance in

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

Natural Diversity of Ectomycorrhizal Fungi in Moroccan Forest Woodlands

9

morphotypes, using the endonucleases used, has discriminated eight ITS-RFLP types (Figure 1, Table 3). The interpretation of ITS sequences identified fungi belonging to two phyla, Ascomycota and Basidiomycota. Also, phylogenetic analysis has identified three morphotypes at a species level (C. geophilum, P. arhizus and S. collinitus) and five at the genus level (Phaengium, Rhizopogon, Tomentella, Sebacina and Tuber) (Figure 2). C. geophilum was by far the most abundant morphotype with a relative abundance of 28.5%. This can be explained by the arid conditions of the study site and the drought tolerance of this fungal species (Molina et al. 1992; LoBuglio 1999). It is well known that this fungal species shows a better adaptation to Mediterranean ecosystems. Most studies of ECM communities published to date report that this species still has the highest relative abundance in forest ecosystems, ranging from 11 to 29% (Abourouh and Najim 1995; Al Sayegh-Petkovsek and Kraigher 2000; Dahlberg et al. 1997). The Thelephoraceae (Tomentella spp.) is also well present with a relative abundance of 17%. In Spain, species belonging to the Thelephora genus represent 25% of the total ectomycorrhizal morphotype recorded in a Q. ilex forest (De Román and De Miguel 2002). The Thelephoraceae also dominated the ectomycorrhizal community in two Mediterranean ecosystems in California (Gardes and Bruns 1996). The fungal species, Rhizopogon sp., with a relative abundance of 14%, ranked third. This genus is mainly detected under Pinaceae tree species (Molina et al. 1999). Phaeangium sp., currently considered as a fungal symbiont belonging to the Picoa genus (Moreno et al. 2000 a, b), is fourth with a relative abundance of 11%. The species of this genus, belonging to the desert truffles have been reported in ecosystems ranging from Mediterranean areas to arid lands of the Middle East (Moreno et al. 2000 a,b; Ammarellou and Trappe 2007). They are mainly associated with the roots of the genus Helianthemum, family Cistaceae (Gutierrez et al. 2003; Slama et al. 2006). The ECM of Tuber sp. were also reported, with a relative abundance of 10%. As for Suillus ectomycorrhizas, basidiomycetes temperate and Mediterranean forests, they occupy the sixth place with a relative abundance of 8%. These fungi form ectomycorrhizas with Pinaceae tree species primarily, but also with hardwood species (Dahlberg and Finlay 1999; Wu et al. 2000). Table 3. Size of restriction fragments of the ITS region, obtained with restriction enzymes EcoRI and HinfI Morphotypes MP1 MP2 MP3 MP4 MP5 MP6 MP7 MP8

Morphotype size bands ITS (bp) ITS EcoRI 750 290, 190, 65 750 290 650 190, 120 604 150, 65 760 290 770 300, 325 770 440, 230 780 300, 290

HinfI 340, 240 340, 240 240, 170, 130, 110 290, 200, 110 310 340, 290 340, 290 240, 170

The Mycorrhizal Symbiosis in Mediterranean Environment: Importance in Ecosystem Stability and in Soil Rehabilitation Strategies : Importance in

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

10

Bakkali Yakhlef Salah Eddine and Duponnois Robin

Figure 2. Maximum likelihood phylogenetic tree of the different morphotypes based on internal transcribed spacer (ITS) sequences. Bootstrap values calculated from 500 replicates (using phyML) are shown at tree nodes, only for values over 70%.

In the Mediterranean forest ecosystems, S. collinutus is used as mycorrhizal inoculant in nurseries and experimental plantations, to enhance growth, mineral nutrition and survival of pine (El Karkouri et al. 2002, 2004; Gonzalez-Ochoa et al. 2003). The Sebacinaceae, showed a relative abundance of 7.5%. Taxa of this family have been considered among the most frequently encountered in a Mediterranean forest of aged Q. ilex (Richard et al. 2005) and sclerophyll eucalypt forests in Australia (Glen et al. 2002). Finally, morphotype Pisolithus, with the lowest relative abundance (4%) belong to P. arhizus, versatile fungus adapted to marginal conditions. Bakkali et al. (2009b) reported for the first time in Morocco, the presence of fruiting bodies of P. arhizus under Q. ilex.

2.3. Diversity of Cistus Ectomycorrhizal Fungi Some genera of shrubs and a very small number of herbaceous species of angiosperm are routinely found to be ECM. Of these, the shrub Cistus (Cistaceae) is of particular ecological significance associated with numerous tree species. It is also of interest because this genus comprises a group of about 20 shrub species found in wide areas throughout the whole

The Mycorrhizal Symbiosis in Mediterranean Environment: Importance in Ecosystem Stability and in Soil Rehabilitation Strategies : Importance in

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

Natural Diversity of Ectomycorrhizal Fungi in Moroccan Forest Woodlands

11

Mediterranean region (Arrington and Kubitzki 2003). Being one of the main constituents of the Mediterranean-type maquis, this plant genus is peculiar in that it has developed a range of specific adaptations to resist summer drought and frequent disturbance events, such as fire and grazing. Cistus may form both ectomycorrhizas and vesicular arbuscular mycorrhizas, the other widespread type of mycorrhizal association (Smith and Read 1997). Knowledge of the mycorrhizal ecology and biology of Cistus and its fungal associates, focusing on topics such as mycobiont diversity, host specificity, and impact of disturbances are needed. Current knowledge about Cistus ectomycorrhizal fungal diversity is based mostly on above-ground observations of fungal fruitbodies (Malloch and Thorn 1985; Lavorato 1991; Ballero et al. 1992; Vila and Llimona 1999, 2002). About 230 fungal species belonging to 40 genera are identified, belonging to both Ascomycota and Basidiomycota. Early studies reviewing ectomycorrhizal fungi and relevant plant hosts overlooked Cistus and its mycoflora (e.g., Trappe 1962), partly due to a focus on forests rather than shrublands. Comandini et al. (2006) reported that Cortinarius and Russula are the best represented ECM genus, followed by Inocybe, Amanita, Hygrophorus, Lactarius, Hebeloma, Boletus, Tuber, Tricholoma. At the family level, Cortinariaceae and Russulaceae, clearly form the prevalent groups. The association of Cistus with numerous hypogeous ascomycetes seems to be a common feature of the Cistaceae as a whole, as other genera, such as Helianthemum, also show similar mycorrhizal preferences (e.g., Malloch and Thorn 1985). The largest number of records was originally collected in Spain, followed by peninsular and insular Italy and southern France, and a few in Morocco. In Morocco, Bakkali et al. (2008) have shown that the different species of the genus Cistus -C. salviifolius, C. crispus and C. monspeliensis- have a double mycorrhizal status endo- and ectomycorrhizal. Ectomycorrhizas phenotypic analysis of the three Cistus species showed the existence of 15 morphotypes (MT). The interpretation of ITS sequences identified fungi belonging to two phyla, Ascomycota and Basidiomycota. Also, phylogenetic analysis has identified eight ectomycorrhizal genera: Russula, Peziza, Tomentella, Thelephora, Pisolithus (Pisolithus sp. 3 Cistus specific, see below), Scleroderma, Hebeloma and Chamonixia (Bakkali et al., unpublished observations). In this study, Chamonixia sp. (hypogeous Boletaceae) has been identified for the first time associated with Cistus in Maâmora forest (Bakkali and Duponnois, unpublished observations). Considering the ecological niches occupied by Cistus and the intermediate position of this host genus in the vegetation series leading to evergreen Quercus or Pinus climax forests on one side, and to impoverished pastures and/or desertified lands on the other, a deeper knowledge of Cistus ectomycorrhizal fungal communities may well prove to be of wider significance and to contribute to understand the role and dynamics of mycorrhizas in inherently unstable ecosystems, especially if integrated into broader ecological investigations.

3. Spatial Distribution of Pisolithus sp. Ectomycorrhizal Fungi Recent studies reported that Pisolithus species can be structured not only according to the tree-host species but also to their ecology (soil abiotic characteristics) and probably their biology (ectomycorrhizal vs saprophytic).

The Mycorrhizal Symbiosis in Mediterranean Environment: Importance in Ecosystem Stability and in Soil Rehabilitation Strategies : Importance in

12

Bakkali Yakhlef Salah Eddine and Duponnois Robin

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

3.1. Tree-Host Specificity While many of the fungal partners of ECM symbioses also lack specificity, it is becoming increasingly clear that there are greater levels of specialization among the fungi than was hitherto believed. The application of molecular tools to enable secure identification of fungi forming mycorrhizas on individual root tips has confirmed that, while generalist fungi can be present on a high proportion of the roots of co-associated tree species (Horton and Bruns 1998), there are a number of ecologically important fungal taxa which may be present on smaller numbers of roots and which show specialization towards particular species of autotroph. Considerable attention has been devoted to the Pisolithus, an Agaricomycete (family of Sclerodermataceae) genus, has a worldwide distribution and forms ectomycorrhizal associations with a wide range of woody plants (Marx 1977), including members of the Pinaceae, Myrtaceae, Fagaceae, Mimosaceae, Dipterocarpaceae and Cistaceae. It has been recorded as occurring in a range of habitats including forests, orchard, urban sites and eroded and mine site soils (Marx 1977; Malloch and Kuja 1979; Castellano and Trapp 1991). This ectomycorrhizal genus is commercially important since its basidiospore inoculum may be used to enhance tree establishment and growth of several species of pine, eucalypt and acacia (Marx 1977; Garbaye et al. 1988; Duponnois and Ba 1999). The host range of Pisolithus fungi has been generally considered to be relatively wide, because the genus develops their basidiomata in association with many tree species and has also been confirmed as forming ectomycorrhizas in laboratory synthesis experiments with 20 host genera (Marx 1977; Martin et al. 1998; Chambers and Cairney 1999). The survey results for the fruitbodies in Morocco showed that Pisolithus is a common ectomycorrhizal fungus in native Quercus forests, Pinus and Eucalyptus woodlands and Cistus scrubs. These results compound well with those of Calonge and Demoulin (1975), Diez et al. (2001) and Martin et al. (2002). In natural environments, the ecology and geographical distribution of ectomycorrhizal species are similar to these of the host-species with which they are associated (Martin et al. 2002; Moyersoen et al. 2003). In Morocco, Bakkali et al. (2009b) studied the phylogenetic relationships among 200 Pisolithus basidiomata collected from pine, oak, and eucalypt forests and rockrose scrubs in Morocco were investigated (Table 4). Maximum likelihood phylogenetic analysis based on ITS sequences clearly separated Pisolithus samples in Morocco into five groups (Figure 3). It showed that all samples from oaks and pines forests were classified into Pisolithus arhizus (sp. 6) and Pisolithus sp. 4 of Martin et al. (2002). This suggests that pine and oak can be associated with the same Pisolithus isolates as reported by Anderson et al. (2001). P. arhizus (Scop. Pers.) Rauschert is distributed widely in the Northern Hemisphere, where it associates in natural ecosystems with pines and oaks (Marx 1977; Martin et al. 2002). Pisolithus sp. 4 is a basophilous species and has been identified only in Spain (Diez et al. 2001). In eucalypt forests, Pisolithus samples were identified as P. albus and P. microcarpus, respectively designated as sp. 7 and sp. 9 by Martin et al. (2002). Species 7 contains two Moroccan isolates, mar 01 and mar 02, while species 9 contains only one isolate, mam17, originated from eucalypt forests. The present result confirms those obtained by Martin et al. (2002), suggesting that these isolates were introduced with eucalypt seedlings from Australia. Furthermore, none of the species 2, species 5, species 8 and species 10 of Martin et al. (2002) have been found in Moroccan pine and eucalypt forests. Until now, one Pisolithus, species 3, has been identified with Cistaceae. Diez et al. (2001) considered

The Mycorrhizal Symbiosis in Mediterranean Environment: Importance in Ecosystem Stability and in Soil Rehabilitation Strategies : Importance in

Natural Diversity of Ectomycorrhizal Fungi in Moroccan Forest Woodlands

13

this species as Cistus-specific. It is interesting to note that this species has been found only in the northern part of the country.

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

Table 4. List of locations and tree plantations under which Pisolithus were collected Host

Geographic location N34°01.935‘ W06°30.617‘

Number of samples 97

Year collected period 2006-2008

Quercus suber, Pinus pinaster Q. ilex Cistus crispus, C. salviifolius, C. monspeliensis Eucalyptus camaldulensis, E. gomphocephala E. camaldulensis

N34°00.406‘ W03°57.006‘ N35°06.168‘ W05°18.074‘

3 15

2007-2008 2008-2009

N34°13.561‘ W06°39.202‘

50

2006-2008

N34°17.814‘ W06°15.578‘

35

2006-2008

Figure 3. Maximum likelihood phylogenetic tree of Pisolithus species based on internal transcribed spacer (ITS) sequences. Paxillus involutus and Suillus luteus were used as an outgroup. Bootstrap values calculated from 500 replicates (using phyML) are shown at tree nodes, only for values over 70%.

The Mycorrhizal Symbiosis in Mediterranean Environment: Importance in Ecosystem Stability and in Soil Rehabilitation Strategies : Importance in

14

Bakkali Yakhlef Salah Eddine and Duponnois Robin

3.2. Ecology and Biology of Pisolithus sp.

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

Soil physical and chemical characteristics, especially carbon and nitrogen sources, greatly influence the survival and growth of ectomycorrhizal fungi in the field as well as in controlled conditions (Lilleskov et al. 2002). In Morocco, the above- and below-ground distribution of the ectomycorrhizal fungi (EMF) Pisolithus spp. was studied according to changes in soil chemistry in a Quercus suber plantation located in the Bir Chleuh region of the Maâmora forest (Bakkali et al. 2011). A multivariate analysis PCA suggested a strong influence of the soil characteristics upon the distribution of the two Pisolithus species (Figure 4). The restricted distribution of P. arhizus to the low-nutrient niche agrees with other studies which reported that this fungus is well adapted to disturbed sites (McAfee and Fortin 1988). However, Pisolithus sp. 4 is likely to be adapted to acidified and high-nutrient niche. In the Bir Chleuh plantation, host range of P. arhizus includes Q. suber, whereas the ectomycorrhizal status of Pisolithus sp. 4 remains doubtful since no ectomycorrhizas of this species have been observed on the roots of Q. suber. The same result have been obtained by "cross inoculations" of cork oak and holm oak from various phylogeographical origins with Pisolithus spp. (including strains of species 6 and 4 and of a species associated with Eucalyptus sp.): the 2 last species did not generate mycorrhizas with the Mediterranean oaks, contrary to the strains of species 6 (Mousain et al. unpublished data). On another hand, ―species 4‖ is known to form ectomycorrhizas with pines and oaks in basic soils (Martin et al. 2002). Even if the genus Pisolithus, as several other ectomycorrhizal species, is able to grow saprophytically (Maijala et al. 1991; Bending and Read 1995), it is probable that Pisolithus sp. (species 4) is an inconsistent ectomycorrhizal partner of Q. suber in acidic soils, permitting formation of erratic fruit bodies but not typical Pisolithus-like ectomycorrhizas.

Figure 4. Plot of factors scores for Pisolithus spp. fruit bodies along principal components axes 1 and 2.Vectors indicate quantitative soil parameters (pH, Clay, N, P, M). The Mycorrhizal Symbiosis in Mediterranean Environment: Importance in Ecosystem Stability and in Soil Rehabilitation Strategies : Importance in

Natural Diversity of Ectomycorrhizal Fungi in Moroccan Forest Woodlands

15

Table 5. Soil analysis data in Pisolithus arhizus and Pisolithus sp. niches

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

Species Pisolithus arhizus Pisolithus sp. 4

pH 6.1  0.01 5.8  0.03

Clay type molasic red

Clay percent 11.5  0.06 18.5  0.53

N (mg/kg) 48.8  1.24 134  2.13

P2 O5 (mg/kg) 75.8  1.14 194.8  6.59

Such situation has already been recognized for Paxillus involutus that can grow saprophytically, but needs connections with a plant host to produce fruit bodies (Laiho 1970). A saprophytic growth mode was also evident for individual genets of Suillus pungens enabling its survival for short periods in the absence of plant host, for example after wildfires, by persisting on the dead host root systems or other dead organic matter (Pierluigi et al. 1998). Finally, as various levels of saprophytic abilities were demonstrated for several ectomycorrhizal fungi (Laiho 1970; Pierluigi et al. 1998; Maijala et al. 1991; Bending and Read 1995), species 4 of Pisolithus could be of this type in the present case. Contrary to Moyersoen and Beever (2004) who reported the co-existence of three species of Pisolithus associated with one single host, Kunzea ericoides, in similar ecological conditions without mutual competitive exclusion, in the Bir Chleuh plantation, fruit bodies of Pisolithus spp., ―species 4‖ and ―species 6‖, co-exist in different soil conditions (Table 5). Furthermore, Pisolithus species 4, considered as ―basophilous‖ by Diez et al. (2001), is found in acid soils (pH # 5.5-5.8) in Maâmora and in a cork oak plantation at Montesquieu-desAlbères (Eastern Pyrenees, France). In addition, Bowen (1994) reported that different ecological and physiological factors affect the growth of mycorrhizal fungi and also mycorrhizal formation and functioning in nature. Increased N leads to a decrease in total production and diversity of ectomycorrhizal fruit bodies above-ground and to shifts in the composition of ectomycorrhizal communities present on roots, in conifer forests experimentally fertilized (Jonsson et al. 2000; Peter et al. 2001). Indeed, N supply level is a critical factor in fruit bodies production and the colonization of roots by ectomycorrhizal fungi in a range of terrestrial ecosystems (Peter et al. 2001; Lilleskov et al. 2001, 2002). Increasing nutrient status (phosphorus, in particular) could reduce infection levels by Pisolithus spp. and induce growth responses in Eucalyptus diversicolor (Marx et al. 1982; Beckjord et al. 1985; Bougher and Malajczuk 1990), as in some N and P richer parts of the Bir Chleuh plantation.

4. Characterisation of the ECM Communities of the Quercus Suber – Cistus Association The degradation of forest cover has profound physical characteristics, chemical and biological soil (erosion, declining fertility, etc.) which significantly limits the process of natural renewal of forest formations (Garcia et al. 1997; Caravaca et al. 2003). Among the biological dysfunctions recorded in these ecosystems, structure and function of soil microbial communities are generally deeply weathered. Communities of ectomycorrhizal fungi, essential to the development of species of the ectotrophic Quercus genus (Smith and Read 1997; Dickie et al. 2002, 2004) are heavily modified both in their species richness than abundance (Dickie and Reich 2005).

The Mycorrhizal Symbiosis in Mediterranean Environment: Importance in Ecosystem Stability and in Soil Rehabilitation Strategies : Importance in

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

16

Bakkali Yakhlef Salah Eddine and Duponnois Robin

In these degraded areas where forest cover is very diffuse, ectomycorrhizal fungal microflora showed a heterogeneous distribution in the form of islands (or "patches") that will be there by the ectotrophic remaining trees (Dickie et al. 2002 ; Dickie and Reich 2005; Jones et al. 2003). These islands facilitate the development of young ectotrophic tree seedlings and consequently the natural regeneration of the species (Dickie et al. 2002, 2004). Recent studies have shown that the sustainability of these pockets rich in ectomycorrhizal symbionts could also be provided by herbaceous or pioneer shrubs (Dickie et al. 2004; Hogetsu and Nara 2004). As the shrub layer of cork oak forests frequently hosts the pioneer shrubs of the Cistus genus with a complex mycorrhizal status ectomycorrhizal and endomycorrhizal type and the cork oak is mainly associated with the same ectomycorrhizal symbionts (Smith and Read 1997), these shrub species could act as a vector for the spread of fungal symbionts likely to promote the potential of soil ectomycorrhizal propagules and facilitate the processes of natural regeneration of ectotrophic trees in these regions. In this context and to rehabilitate these ecosystems, it is necessary to promote the formation of this shrub layer and ensure its sustainability through the management of biological vectors compatible with the environmental and socio-economic encountered in these regions. A recent research program (PRAD 04-10) focusing on the characterization of the ectomycorrhizas formed by the associated Cistus spp. - Quercus suber in natural and disturbed situation was implemented in 2010. The main objective of this project is that the management of the mycorrhizal potential "in situ" of the vegetation in this ecosystem, namely species of the genus Cistus, could promote mycorrhizal seedlings of cork oak and consequently improve their establishment after outplanting. This research program is part of a microbial ecology problem to describe and understand certain biological mechanisms governing the spatiotemporal evolution of the ectomycorrhizal cover associated with cork oak and its impact on the conservation and productivity of cork oak in Morocco. The expected results will highlight the links between the density of tree and shrub cover on abundance, diversity and distribution of ectomycorrhizal fungi communities in cork forests more or less degraded. In describing the possible links between the state of the cork oak forests and the parameters measured on populations of ectomycorrhizal symbionts, it will be possible to determine the basis for a predictive model to assess the impact of symbiotic microflora in the soil changes in forest cover.

CONCLUSION AND PERSPECTIVES The results outlined in the present chapter show a relatively high interspecific variation of the ectomycorrhizal fungi in Moroccan Forest ecosystems. It appears that molecular analysis of the ITS of the rDNA is a potent tool for studying fungi population heterogeneity in the field and the identification and monitoring of specific indigenous strains (Bakkali et al. 2008, 2009a, 2009b, 2010, 2011). Our results also show significant diversity of Pisolithus species in Morocco, and confirm the host specificity grouping of Pisolithus taxa suggested by several others studies (Diez et al. 2001; Martin et al. 2002). Also, two Pisolithus species can be structured not only according to the tree-host species but also to their ecology (soil abiotic characteristics) and probably their biology (ectomycorrhizal vs saprophytic). We suggest

The Mycorrhizal Symbiosis in Mediterranean Environment: Importance in Ecosystem Stability and in Soil Rehabilitation Strategies : Importance in

Natural Diversity of Ectomycorrhizal Fungi in Moroccan Forest Woodlands

17

future studies on the description of ectomycorrhizal fungal communities and the use of more efficient fungal symbionts are able to enhance the performances rehabilitation programs of degraded forest formations using advanced techniques such as controlled mycorrhization. Furthermore, considering the ecological niches occupied by some shrub species and the intermediate position of these hosts in the Moroccan forest ecosystems on one side, and to impoverished pastures and/or desertified lands on the other, a deeper knowledge of shrubs ectomycorrhizal fungal communities may contribute to help shape future programs of protection and management of natural resources in those ecosystems.

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

References Abourouh M (2000) Ectomycorhizes et mycorhization des principales essences forestières du Maroc. Thèse d'Etat, Facultés des Sciences de Rabat : 170 p. Abourouh M, Najim L (1995) Les différents types d‘ectomycorhizes naturelles de Cedrus atlantica Manetti au Maroc. Cryptogam. Bot. 5:332-340. AEFCS (1996) Actes du colloque national sur la forêt, du 21 au 23 mars 1996 à Ifrane. AEFCS, Rabat. 120 p. Agerer R (1991) Characterization of ectomycorrhiza. In: Norris J.R., Read D.J. and Varma A.K., eds. Methods in microbiology. London: Academic Press, pp. 25-73. Al Sayegh-Petkovsek S, Kraigher H (2000) Types of ectomycorrhizae from Kocevska Reka. Phyton-Annales Rei Botanicae 40 (4): 37-42. Amicucci A, Rossi I, Potenza L, Zambonelli A, Agostini D, Palma F, Stocchi V (1996) Identification of ectomycorrhizae from Tuber species by RFLP analysis of the ITS region. Biotechnol. Lett. 18: 821-826. Ammarellou A, Trappe JM (2007) A first Ascomycete genus (Picoa sp.) record for the fungi flora of Iran. Pak. J. Biol. Sci. 10:1772. Anderson IC, Chambers SM and Cairney JWG (2001) ITS-RFLP and ITS sequence diversity in Pisolithus from central and eastern Australia sclerophyll forests. Mycological Research 105: 1304-1312. Arrington JM, Kubitzki K (2003) Cistaceae. In: Kubitzki K, Bayer C (eds) The families and genera of vascular plants. V. Flowering plants. Dicotyledons. Malvales, Capparales and Non-betalain Caryophyllales. Springer, Berlin Heidelberg New York, pp 62-70. Azcón-Aguilar C, Palenzuela J, Roldán A, Bautista S, Vallejo R, Barea JM (2003) Analysis of the mycorrhizal potential in the rhizosphere of representative plant species from desertification-threatened Mediterranean shrublands, Appl. Soil. Ecol. 22: 29-37. Bakkali Yakhlef S, Chakir S, Kerdouh B, Mousain D, Duponnois R et Abourouh M (2008) Analyse phénotypique et moléculaire du potentiel mycorhizogène in situ des cistes associés au Chêne-liège au Maroc. Journées Francophones Mycorhizes. 04 et 05 septembre 2008, Dijon, France. Bakkali Yakhlef S, Kerdouh B, Mousain D, Ducousso M, Duponnois R and Abourouh M (2009a) Phylogenetic diversity of Moroccan Cork oak woodlands fungi. Biotechnol. Agron. Soc. Environ. 13(4): 521-528.

The Mycorrhizal Symbiosis in Mediterranean Environment: Importance in Ecosystem Stability and in Soil Rehabilitation Strategies : Importance in

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

18

Bakkali Yakhlef Salah Eddine and Duponnois Robin

Bakkali Yakhlef S, Mousain D, Duponnois R, Ducousso M, Belkouri A, Kerdouh B, Perrineau M and Abourouh M (2009b) Molecular phylogeny of Pisolithus species from Moroccan forests woodlands. Symbiosis 49 (3):157-162. Bakkali Yakhlef S, Abourouh M, Moreau PA, Richard F et Mousain D (2010) Diversité des champignons basidiomycètes dans les subéraies du Rif occidental. Société d‘horticulture et d‘histoire naturelle de l‘Herault. Les Rencontres du Cent cinquantenaire Colloque Forestiers / Naturalistes. 21 octobre 2010, Montpellier, France. Bakkali Yakhlef S, Abourouh M, Ducousso M, Duponnois R, Delaruelle C and Mousain D (2011) Intraspecific variability of Pisolithus spp. as a response of soil characteristic change in a Moroccan cork oak plantation. Mycology: An International Journal on Fungal Biology, 2 (4): 283-290. Ballero M, Contu M, Fogu MC (1992) Contributo alla conoscenza dei macromiceti presenti nei cisteti della Sardegna. Rev. Iberoamericana Micol. 9:58-60. Beckjord PR, Melhuish JH, Mc lntosh MS (1985) Effects of nitrogen and phosphorus fertilization on growth and formation of ectomycorrhizae of Quercus alba and Q. rubra seedlings by Pisolithus tinctorius and Scleroderma aurantium. Canadian Journal of Botany 63: 677-1680. Bending GD, Read J (1995) The structure and function of the vegetative mycelium of ectomycorrhizal plants. V. Foraging behaviour and translocation of nutrients from exploited litter. New Phytologist 130 : 401-409. Birot Y (1991) Boisement et reboisement. In "Actes du Dixième Congrès Forestier Mondial 1991". R. F. F., hors série, 5: 9-19. Boudy P (1958) Économie Forestière nord-africaine. Description forestière du Maroc. Bougher NL, Malajczuk N (1990) Effects of high soil moisture on formation of ectomycorrhizas and growth of karri (Eucalyptus diversicolor) seedlings inoculated with Descolea maculate, Pisolithus tinctorius and Laccaria laccata. New Phytologist 114: 8791. Bruns TD, White TJ and Taylor JW (1991) Fungal molecular systematics. Ann. Rev. Ecol. Syst. 22 : 525-564. Calonge FD, Demoulin V (1975) Les Gastéromycètes d‘Espagne. Bulletin Trimestriel de la Société Mycologique de France 91: 247-292. Caravaca F, Alguacil MM, Figueroa D, Barea JM, Roldán A (2003a) Re-establishment of Retama sphaerocarpa as a target species for reclamation of soil physical and biological properties in a semiarid Mediterranean land. For. Ecol. Manage 182: 49-58. Caravaca F, Barea JM, Palenzuela J, Figueroa D, Alguacil MM, Roldán A (2003b) Establishment of shrub species in a degraded semiarid site after inoculation with native or allochthonous arbuscular mycorrhizal fungi. Appl. Soil. Ecol. 22:103-111. Castellano MA and Trappe JM (1991) Pisolithus tinctorius fails to improve plantation performance of inoculated conifers in south-western Oregon. New Forests 5: 349-358. Chambers SM and Cairney JWG (1999) Pisolithus. In: Ectomycorrhizal Fungi. Key Genera in Profile. Cairney, J.W.G. and Chambers, S.M., eds. Springer-Verlag, Berlin, Germany, pp. 1-31. Dahlberg A, Jonsson L, Nylund LE (1997) Species diversity and distribution of biomass above and belowground among ectomycorrhizal fungi in an old-growth Norway spruce forest in south Sweden. Canadian Journal of Botany 75:1323-1335.

The Mycorrhizal Symbiosis in Mediterranean Environment: Importance in Ecosystem Stability and in Soil Rehabilitation Strategies : Importance in

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

Natural Diversity of Ectomycorrhizal Fungi in Moroccan Forest Woodlands

19

Dahlberg A, Finlay RD (1999) Suillus. In: Cairney JEGC, Chambers SM (eds) Ectomycorrhizal fungi, key genera in profile. Springer, Berlin Heidelberg New York, pp 33-64. De Román M, Agerer R, De Miguel AM (2002) ―Quercirhiza cumulosa‖ + Quercus ilex L. subsp. ballota (Desf.) Samp. Descr. Ectomycorrhizae 6:13-18. Di Battista C (1997) Comportement en pépinière et en plantation d‘un champignon ectomycorhizien Laccaria bicolor S238N inoculé sur Épicéa (Picea abies) et Douglas (Pseudotsuga menziesii). Typage moléculaire de la souche introduite. Thèse de doctorat : Université Henri Poincarré de Nancy (France). Dickie IA and Reich PB (2005) Ectomycorrhizal fungal communities at forest edges. Journal of Ecology 93: 244-255. Dickie IA, Roger TK, Kim CS. (2002) Influences of established trees on mycorrhizas, nutrition, and growth of Quercus rubra seedlings. Ecological Monographs 72: 505-521. Dickie IA, Rebecca CG, Sarah EK, Peter BR (2004) Shared ectomycorrhizal fungi between a herbaceous perennial (Helianthenum bicknellii) and oak (Quercus) seedlings. New Phytologist 164: 375-382. Diez J, Anta B, Manjon JL, Honrubia M (2001) Genetic variability of Pisolithus isolates associated with native hosts and exotic eucalyptus in the western Mediterranean region. New Phytologist 149:577-587. Duponnois R and Ba AM (1999) Growth stimulation of Acacia mangium Willd. by Pisolithus sp. in some Senegalese soils. Forest Ecology and Management 119: 209-215. El Karkouri KE, Martin F, Mousain D (2002) Dominance of the mycorrhizal fungus Rhizopogon rubescens in a plantation of Pinus pinea seedlings inoculated with Suillus collinitus. Ann. For. Sci. 59: 197-204. El Karkouri KE, Martin F, Mousain D (2004) Diversity of ectomycorrhizal symbionts in a disturbed Pinus halepensis plantation in the Mediterranean region. Ann. For. Sci. 61: 705710. Erland S, Taylor AFS (2002) Diversity of ectomycorrhizal fungal communities in relation to the abiotic environment. In: van der Heijden MGA, Sanders IR (eds) Mycorrhizal ecology. Ecological studies analysis and synthesis, vol 157. Springer, Berlin Heidelberg New York, pp 163–200. Garbaye J, Delwaulle JC and Diangana D (1988) Growth response of eucalyptus in the Congo to ectomycorrhizal inoculation. Forest Ecology and Management 24: 151-157. Garcia C, Rodan A, Hernandez T (1997) Changes in microbial activity after abandonment of cultivation in a semi-arid Mediterranean environment. Journal of Environmental Quality 26: 285-291. Gardes M, Fortin JA, Mueller GM and Kropp BR (1990) Restriction fragment length polymorphisms in the nuclear ribosomal DANN of four Laccaria spp.: L. bicolour, L. laccata, L. proxima and L. amethystina. Phytopathology 80: 1312-1317. Gardes M, Mueller GM, Fortin JA and Kropp BR (1991a) Mitochondrial DNA polymorphisms in Laccaria bicolor, L. Laccat, L. Proxima and L. amethystina. Mycological Res. 95: 206-216. Gardes M, White TJ, Fortin JA, Bruns TD, and Taylor JW (1991b) Identification of indigenous and introduced symbiotic fungi in ectomycorrhizae by amplification of nuclear and mitochondrial ribosomal DNA. Can. J. Bot. 69: 180-190.

The Mycorrhizal Symbiosis in Mediterranean Environment: Importance in Ecosystem Stability and in Soil Rehabilitation Strategies : Importance in

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

20

Bakkali Yakhlef Salah Eddine and Duponnois Robin

Gardes M and Bruns TD (1996) Community structure of ectomycorrhizal fungi in a Pinus muricata forest: above- and below-ground views. Can. J. Bot. 74:1572-1583. Giltrap NJ (1982) Production of polyphenol oxidases by ecto-mycorrhizal fungi with special reference to Lactarius spp. Trans Brit. Mycol. Soc. 78: 75-81. Glen M, Tommerup IC, Bougher NL, O‘Brien PA (2002) Are Sebacinaceae common and widespread ectomycorrhizal associates of Eucalyptus species in Australian forests? Mycorrhiza 12: 243-247. González-Ochoa AI, De las Heras J, Torres P, Sánchez-Gómez E (2003) Mycorrhization of Pinus halepensis Mill. and Pinus pinaster seedlings in two commercial nurseries. Ann. For. Sci. 60: 43-48. Grove TS and Le Tacon F (1993) Mycorrhiza in plantation forestry. Advances in Plant Pathology 23:191-227. Guerin-Laguette L (1998) Les lactaires à lait rouge : mycorrhization contrôlée des pins et characterisation moléculaire. Application à l‘étude de la compétence écologique et de la compétitivité d‘isolats de Lactarius delicious. Thèse de doctorat : École Nationale Supérieure Agronomique de Montpellier (France). Gutierrez A, Morte A, Honrubia M (2003) Morphological characterization of the mycorrhiza formed by Helianthemum almeriense Pau with Terfezia claveryi Chatin and Picoa lefebvrei (Pat) Maire. Mycorrhiza 13: 299-307 Henrion B, Le Tacon F and Martin F (1992) Rapid identification of genetic variation of ectomycorrhizal fungi by amplification of ribosomal RNA genes. New Phytol. 122: 289298. Herrera MA, Salamanca CP and Barea JM (1993) Inoculation of woody legumes with selected arbuscular mycorrhizal fungi and rhizobia to recover desertified Mediterranean ecosystems. Appl. Environ. Microb. 59: 129-133. Hogetsu T and Nara K (2004) Ectomycorrhizal fungi on established shrubs facilitate subsequent seedling establishment of successional plant species. Ecology 85: 1700-1707. Horton TR and Bruns TD (1998) Multiple-host fungi are the most frequent and abundant ectomycorrhizal types in a mixed stand of Douglas fir (Pseudotsuga menziesii) and bishop pine (Pinus muricata). New Phytologist 139: 331-339. Horton JS, Palmer GE and Shah A (2001) Mushroom development and cAMP signalling in Schizophyllum commune. The Genetics and Cellular Biology of Basidiomycetes V, Abstract, p. 11.Mississauga, ON, Canada. Jones MD, Durall DM, Cairney JWG (2003) Ectomycorrhizal fungal communities in young forest stands regenerating after clearcut logging. New Phytologist 157: 399-422. Jonsson L, Dahlberg A and Brandrud TE (2000) Spatiotemporal distribution of an ectomycorrhizal community in an oligotrophic Swedish Picea abies forest subjected to experimental nitrogen addition: above and below ground views. Forest Ecol. Manage 132: 143-156. Karén O, Högberg N, Dahlberg A, Jonsson L and Nylund J-E (1997) Inter- and intraspecific variation in the ITS region of rDNA of ectomycorrhizal fungi in Fennoscandia as detected by endonuclease analysis. New Phytol. 136: 313-325. Kropp BP and Langlois CF (1990) Ectomycorhizae in reforestation. Can. J. Forest. Researh 20: 438-451. Laiho O (1970) Paxillus involutus as a mycorrhizal symbiont of forest trees. Acta Forestalia Fennica 106:1-73.

The Mycorrhizal Symbiosis in Mediterranean Environment: Importance in Ecosystem Stability and in Soil Rehabilitation Strategies : Importance in

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

Natural Diversity of Ectomycorrhizal Fungi in Moroccan Forest Woodlands

21

Lavorato C (1991) Chiave analitica e note bibliografiche della micoflora del cisto. Boll. AMER 24:16-45. Le Tacon F, Alvarez IF, Bouchard D, Henrion B, Jackson RM, Luff S, Parlade JI, Pera J, Stenström E, Villeneuve N, Walker C (1992) Variations in field response of forest trees to nursery ectomycorrhizal inoculation in Europe, in: Read DJ, Lewis D, Fitter A, Alexander I, (Eds), Mycorrhizas in Ecosystems. CAB International, Wallington, UK. p 119-134. Lilleskov EA, Fahey TJ, Lovett GM (2001) Ectomycorrhizal fungal aboveground community change over an atmospheric nitrogen deposition gradient. Ecological Applications 11: 397-410. Lilleskov EA, Hobbie EA, Fahey TJ (2002) Ectomycorrhizal fungal taxa differing in response to nitrogen deposition also differ in pure culture organic nitrogen use and natural abundance of nitrogen isotopes. New Phytologist 159: 219-231. LoBuglio KF (1999) Cenococcum. In: Cairney JWG, Chambers SM (eds) Ectomycorrhizal fungi. Key genera in profile. Springer, Berlin Heidelberg, New York, pp 287-310. Maijala P, Fagerstedt KV, Raudaskoski M (1991) Detection of extracellular cellulolytic and proteolytic activity in ectomycorrhizal fungi and Heterobasidion annosum (Fr.) Bref. New Phytologist 117:643-648. Malloch D and Kuja AL (1979) Occurrence of the ectomycorrhizal fungus Pisolithus tinctorius in Ontario. Canadian Journal of Botany 57: 1848-1849. Malloch D, Thorn RG (1985) The occurrence of ectomycorrhizae in some species of Cistaceae in North America. Can. J. Bot. 63:872–875. Manassila M, Sooksa-nguan T, Boonkerd N, Rodtong S, and Teaumroong N (2005) Phylogenetic diversity of wild edible Russula from north-eastern Thailand on the basis of internal transcribed spacer sequence. Science Asia 31: 323-328. Martin F, Delaruelle C, Ivory M (1998) Genetic variability in intergenic spacers of ribosomal DNA in Pisolithus isolates associated with pine, eucalyptus and Afzelia in lowland Kenyan forests. New Phytologist. 139: 341-352. Martin F, Diez J, Dell B, Delaruelle C (2002) Phylogeography of the ectomycorrhizal Pisolithus species as infered from nuclear ribosomal DNA ITS sequences. New Phytologist. 153:345-357. Marx DH, Hatchell GE (1986) Root stripping of ectomycorrhizae decreases field performance of loblolly and longleaf pine seedlings. South J. Appl. For. 10: 17-179. Marx DH (1977) Tree host range and world distribution of the ectomycorrhizal fungus Pisolithus tinctorius. Canadian Journal of Microbiology 23:217-233. Marx DH, Ruehle JL, Kenney DS, Cordell CE, Riffle JW, Molina RJ, Pawuk WH, Navratil S, Tinus RW, Goodwin OC (1982) Commercial vegetative inoculum of Pisolithus tinctorius and inoculation techniques for development of ectomycorrhizae on container-grown tree seedlings. Forest Science 28: 373-400. Mishler BD and Brandon RN (1987) Individuality, pluralism, and the phylogenetic species concept. Biology and Philosophy 2:397-414. Molina R, Trappe JM, Grubisha LC, Spatafora JW (1999) Rhizopogon. In: Cairney JWG, Chambers SM, eds. Ectomycorrhizal fungi: key genera in profile. Heidelberg: SpringerVerlag. pp 129-161. Molina R, Massicotte HB, Trappe JM (1992) Ecological role of specificity phenomena in ectomycorrhizal plant communities: potentials for interplant linkages and guild

The Mycorrhizal Symbiosis in Mediterranean Environment: Importance in Ecosystem Stability and in Soil Rehabilitation Strategies : Importance in

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

22

Bakkali Yakhlef Salah Eddine and Duponnois Robin

development. In: Mycorrhizas in Ecosystems / D.J. Read, D.H. Lewis, A.H. Fitter, I.J. Alexander Eds. Oxon (UK): CAB International, pp 106-112. Moreno G, Diez GJ, Manjon JL (2000a) Picoa melospora sp nov (Pezizales) from the Iberian Peninsula. Bull. Fed. Assoc. Mycol. Medit. 18: 87-92. Moreno G, Diez J, Manjon JL (2000b) Picoa lefebvrei and Tirmania nivea, two rare hypogeous fungi from Spain. Mycol. Res. 104: 378-381. Mousain D, Plassard C, Argillier C, Sardin T, Leprince F, El Kerkouri K, Arvieu JC, CleyetMarel JC (1994) Stratégie d'amélioration de la qualité des plants forestiers et des reboisements méditerranéens par utilisation de la mycorhization contrôlée en pépinière. Acta Bot. Gallica 141: 571-580. Moyersoen B, Beever RE, and Martin F (2003) Genetic diversity of Pisolithus in New Zealand indicates multiple longdistance dispersal from Australia. New Phytologist 160: 569-579. Moyersoen B, Beever RE (2004) Abundance and characteristics of Pisolithus ectomycorrhizas in New Zealand geothermal areas. Mycologia 96: 1225-1232. Ouahmane (2007) Rôle des mycorhizes et des plantes compagnes (lavande et thym) sur la croissance du cyprès de l‘Atlas (Cupressus atlantica G.) : conséquences sur la biodiversité rhizosphérique et la réhabilitation des sites dégradés. Thèse de Doctorat National, Uni. Cadi Ayyad, Fac Sc Sémlalia Marrakech, pp227. Peter M, Ayer F, Egli S (2001) Fire and vegetation effects on productivity and nitrogen cycling across a forest-grassland continuum. Ecology 82: 1703-1719. Petkovsek Al Sayegh S and Kraigher H (2000) Types of ectomycorrhizae from Kocevska Reka. - Phyton (Horn, Austria) 40 (4): 37-42. Pierluigi B, Bruns TD, Gardes M (1998) Genetic structure of a natural population of the ectomycorrhizal fungus Suillus pungens. New Phytologist 138: 533-542. Pritsch K, Boyle H, Munch JC and Buscot F (1997) Characterization and identification of black alder ectomycorrhizas by PCR/RFLP analyses of the rDNA internal transcribed spacer (ITS). New Phytol. 137: 357-369. Requena N, Jeffries P, Barea JM (1996) Assessment of natural mycorrhizal potential in a desertified semiarid ecosystem. Appl. Environ. Microbiol. 62: 842-847. Requena N, Jimenez I, Toro M and Barea JM (1997) Interactions between plant-growthpromoting rhizobacteria (PGPR), arbuscular mycorrhizal fungi and Rhizobium spp. in the rhizosphere of Anthyllis cytisoides, a model legume for revegetation in Mediterranean semiarid ecosystems. New Phytol. 136: 667-677. Richard F, Moreau PA, Selosse MA and Gardes M (2004) Diversity and fruiting patterns of ectomycorrhizal and litter saprobic fungi in an old-growth Mediterranean forest dominated by Quercus ilex L. Can. J. Bot. 82: 1711-1729. Richard F, Millot S, Gardes M and Selosse MA (2005) Diversity and specificity of ectomycorrhizal fungi retrieved from an old-growth Mediterranean forest dominated by Quercus ilex. New Phytol. 166 : 1011-1023. Seigue A (1985) La forêt circumméditerranéenne et ses problèmes. Paris: Éditions Maisonneuve et Larose pp502p. Sen R (1990) Intraspecific variation in two species of Suillus from scots pine (Pinus sylvestris L.) forests based on somatic incompatibility and isozyme analyses. New Phytol. 114: 607-616.

The Mycorrhizal Symbiosis in Mediterranean Environment: Importance in Ecosystem Stability and in Soil Rehabilitation Strategies : Importance in

Natural Diversity of Ectomycorrhizal Fungi in Moroccan Forest Woodlands

23

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

Shemakhonova NM (1962) Mycotrophy of woody plants. Academiya Naur SSSR Institut Microbiologi. pp330. Slama A, Fortas Z, Neffati M, Khabar L, Boudabbous A (2006) Etude taxonomique de quelques Ascomycota hypogéss (Terfeziaceae) de la Tunisie méridionale. Bull. Soc. Mycol. Fr. 122: 187-195. Smith FA, Smith SE (1997) Structural diversity in (vesicular) – arbuscular mycorrhizal symbiosis. New Phytol. 137:373-388. Trappe JM (1962) Fungus associates of ectotrophic mycorrhizae. Bot. Rev. 28: 538-606. Van der Heijden MGA, Sanders IR (2002) (eds) Mycorrhizal ecology. Ecological studies analysis and synthesis, vol 157. Springer, Berlin Heidelberg New York. Vila J, Llimona X (1999) Els fongs del Parc Natural del Cap de Creus i Serra de Verdura (Girona). II. Aproximaciò al component fùngic del Cistion. Rev. Catalana Micol. 22: 95114. Vila J, Llimona X (2002) Noves dades sobre el component fúngic de les comunitats de Cistus de Catalunya. Rev. Catalana Micol. 24:75-121. Wu QX, Mueller GM, Lutzoni FM, Huang YQ, Guo SY (2000) Phylogenetic and biogeographic relationship of eastern Asian and eastern north American disjunct Suillus species (Fungi) as inferred from nuclear ribosomal DNA ITS sequences. Mol. Phylogenet. Evol. 17: 37-47.

The Mycorrhizal Symbiosis in Mediterranean Environment: Importance in Ecosystem Stability and in Soil Rehabilitation Strategies : Importance in

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved. The Mycorrhizal Symbiosis in Mediterranean Environment: Importance in Ecosystem Stability and in Soil Rehabilitation Strategies : Importance in

In: The Mycorrhizal Symbiosis in Mediterranean Environment ISBN: 978-1- 62081-278-5 Editors: Mohamed Hafidi and Robin Duponnois © 2012 Nova Science Publishers, Inc.

Chapter II

Ectotrophic Mycorrhizal Symbioses Are Dominant in Natural Ultramafic Forest Ecosystems of New Caledonia Y. Prin,1 M. Ducousso,1,2 J. Tassin,2,3 G. Béna,4 P. Jourand,5 V. Dumontet,6 L. Moulin,4 C. Contesto,4 J. P. Ambrosi,5, 7 C. Chaintreuil,4 B. Dreyfus 4 and M. Lebrun4 1

CIRAD, UMR LSTM, France IAC, Nouméa, Nouvelle Calédonie 3 CIRAD, UPR Dynamique des forêts naturelles Montpellier Cedex, France 4 IRD, UMR LSTM, F-Montpellier Cedex, France 5 IRD, Nouméa Cedex, Nouvelle Calédonie 6 CNRS, Laboratoire des plantes Médicinales, Nouvelle Calédonie 7 CNRS UMR CEREGE, Provence, France

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

2

Dedicated to the memory of our friend Nicolas Perrier

Abstract Insularity, geological history and biogeography have made from New-Caledonia a hot spot of biodiversity where extremely diversified ecosystems occupies ultramafic terrains with drastic edaphic conditions in terms of fertility and metallic toxicity. In the framework of the mine project of the Koniambo Massif, a large nickel deposit, we tried to explore the diversity of ectomycorrhizal symbioses within these poorly explored natural ultramafic ecosystems. Floristic inventories along an altitudinal gradient ranging from 700 to 900 m evidenced 4 different plant communities. The 2 lower plant communities, 3 and 4, were dominated by 2 endemic tree genera, Tristaniopsis (Leptospermoideae) and Nothofagus

The Mycorrhizal Symbiosis in Mediterranean Environment: Importance in Ecosystem Stability and in Soil Rehabilitation Strategies : Importance in

26

Y. Prin, M. Ducousso, J. Tassin et al. (Nothofagaceae) respectively, whose ectomycorrhizal (ECM) status was shown and explored through molecular methods on sporocarps, mycorrhizae and soil mycelium. We evidenced a diversified fungal community in the basal plant community dominated by two tree species of the genus Nothofagus. The molecular characterization of these ECM fungi was established on the total ribosomal inter transcribed spacer (ITS) by PCR-sequencing and BLASTn analysis, revealing the relative abundance of the Cortinariaceae among our samples. Samples belonging to this fungal family were phylogenetically analyzed on the same ITS, in reference to sequences of samples with geographically different origins, including countries derived from the Gondwanaland fragmentation. If no clear phylogenetical relationships were evidenced, our study confirmed the same relative dominance of ECM Nothofagaceae, as well as the relative abundance of associated Cortinariaceae, in New Caledonia as in several of the Gondwanaland-originating countries.

Keywords: Ectomycorrhiza, New Caledonia, Cortinarius, Nothofagus, Metal toxicity

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

Introduction New Caledonia is a relatively small group of islands (20,000 Km2) located in the South Pacific Ocean. Its geological history is directly related to that of the Gondwana super continent until 80 million years ago when the Eastern margin of the Gondwana started to break up (Veevers 1986, Veevers et al. 1991). A complex geological history has led to the presence of large nickel ore deposits located within the lateritic regolith, making, according to the International Nickel Study Group, this island the 4th world producer of nickel ore for the period 1993-2001. The Koniambo Massif is one of these ore deposits, relatively isolated from each other‘s, in the Northern Province. It is in exploration by the mining group KNS (Xtrata and SMSP consortium) to establish an open pit nickel mine and smelter a high grade nickel ore located in the thick regolith. The soils present on the Koniambo massif can be classified as highly weathered oxisols formed of a majority (50 to 85%) of nodular iron oxides. These soils are characterised by a deficiency in the main plant nutrients (N, P, K), high concentrations of toxic heavy metals: Fe, Ni, Cr, Co, Mn (Perrier et al. 2006a) and an unbalanced Ca/Mg ratio. Specific vegetation conditions have risen as a result of the specific geological history of New Caledonia. The local flora comprises approximately 3,300 species of which 74.5 % are endemic to the island (Jaffré et al. 2001). This endemism increases to 90 % on the ultramafic terrains, which count approximately 1,840 species (Jaffré 1974). In these particularly drastic edaphic conditions, mining companies have the constraint to proceed to the ecological restoration of mine sites after exploitation, which is a big challenge considering the lack of knowledge on these ecosystems, their flora and associated symbioses. Symbioses, be they ectomycorrhizal (ECM) or arbuscular (AM) are known to be essential in plant adaptation to soil conditions (Jentschke and Godbold 2000, Stahl et al. 1988). Concerning AM symbioses, Perrier et al. (2006b) described the status (mycorrhizal colonization frequency and intensity) of 10 different plant species in the Koniambo Massif. For ECM in these ecosystems, only partial information were available either for some of the plant species studied by Perrier et al. (2006b) or through the description of a new Cantharellus species by Ducousso et al. (2004). However, the diversity and the distribution of

The Mycorrhizal Symbiosis in Mediterranean Environment: Importance in Ecosystem Stability and in Soil Rehabilitation Strategies : Importance in

Ectotrophic Mycorrhizal Symbioses Are Dominant …

27

ECM symbioses in the different endemic vegetation ecosystems using molecular typing were not known. The aim of this study is (i) to explore the floristic composition of the ecosystems in a zone representative of those targeted by the mining group as prioritized mining sites (ie over 700 m) (ii) to characterize the diversity of the ectomycorrhizal (ECM) fungi naturally associated to ECM plants within these inventories and (iii) to replace these data in the evolutionary and bio-geographical context of New Caledonia. Additionally, these data should later allow giving to the mining consortium recommendations on the use of ECM plants and management of topsoils in future revegetation processes.

Materials and Methods

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

 Study Site The Koniambo massif is one of the isolated ultramafic massifs of the West coast (Figure 1 A and B). It has a total area of 381 km2 and peaks at 930 m. The climate on the Koniambo Massif can be defined by two different seasons, a humid season ranging from January to April (an average of 250 mm monthly precipitation) and a drier season from May to December (an average of 100 mm monthly precipitation with a 50 mm low peak in September). The wind regime is governed generally by the trade winds blowing from an ESE direction.

Figure 1. (A) Map of the ultramafic bodies of New Caledonia (in grey), (B) Shaded relief map of the Koniambo Massif with study site location, (C) Partial shaded relief map of the Pandanus river watershed zone of the Koniambo Massif with location of plant communities and sampling sites.

The Mycorrhizal Symbiosis in Mediterranean Environment: Importance in Ecosystem Stability and in Soil Rehabilitation Strategies : Importance in

28

Y. Prin, M. Ducousso, J. Tassin et al.

The average daily temperature on Koniambo ranges from 22°C (at 900 m altitude) in the summer months (January-March) to 14°C in the winter months (July to September). This massif is mainly composed of a harzburgite substrate (a type of peridotite formed of the minerals olivine and orthopyroxene) with some dunite and gabbro inclusions, the lateritic weathering of this body has led to the presence of a thick nickel rich regolith. The sampling site was located in the watershed of the Pandanus river on a slope ranging from 883 to 700 m (21°00‘32‖S; 164°50‘17‖E and 21°00‘25‖S; 164°49‘45‖E). In a floristic analysis of the whole massif, Jaffré (1974) identified 650 plant species and described, according to plant abundance, 12 different plant communities. The vegetation is an assemblage of maquis and rainforests. The maquis can be of different types, dominated, below 500 m, by the shrubby/arborescent angiosperm species (height of 6-10 m) Gymnostoma chamaecyparis Poiss. L.A.S. Johnson or, from 400 to 900 m, by species of the more shrubby genera Tristaniopsis. In the valleys above 600 m, rainforests are dominated by Nothofagus balansae (Baill.) Steenis and Nothofagus codonandra (Baill.) Steenis. Botanical nomenclature follows The International Plant Index (2008).

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

 Floristic analyses Floristic data were collected from 80 randomly located 100 m2 plots placed along a topological sequence ranging from the plateau at 882 m to the talweg at 700 m and spanning different vegetation types. Sixty plots were located in the maquis and 20 in the forest. The number of plots in each vegetation type was approximately proportional to the area represented in the landscape (Figure 1C). The Braun-Blanquet cover-abundance scale was used to describe the species abundance in each plot. Frequencies percentages were inferred from each Braun-Blanquet class (Braun-Blanquet et al. 1932). Data were analyzed using correspondence analysis (Greenacre 1984). They were processed using ADE4 software (Thioulouse et al. 1997).  Ectomycorrhizas surveys Sporocarps of ectomycorrhizal (ECM) fungi were collected at 4 different dates (June and July 2002, July and December 2003) under identified tree species, all located in plant communities 3 and 4 in the same site of the Pandanus river watershed. A photograph of each sampled sporocarp was taken and a sample number was given. A small portion of the flesh (≈ 0.5 cm3) of each sample was placed on a cotton layer in 10 ml plastic tubes half filled with Silicagel (Prolabo, France) for rapid drying and kept at room temperature for subsequent DNA extraction. The sporocarps were air-dried at 40°C, and they were deposited in Paris Cryptogamie (PC) herbarium (Paris, France). Identification was based on macroscopic and microscopic characters of basidiomata. For most specimens, identification was not possible at the species level (restricted to the genus level) because the study was conducted in ecosystems that have been very poorly studied by taxonomists (Horak and Mouchacca 1998) and where many species remain to be described. Complementarily to sporocarps, some morphotypically different (color, shape) ECM apices from each of the four ECM plant species were randomly collected from superficial roots excavated all the way from the trunk to the ultimate fine ECM roots to determine their host-plant without any error, ECM. For DNA extraction, ECM samples were rapidly dried in Silicagel and kept at room temperature. Other samples of the same origin and morphotype were fixed in glycerol, ethanol and water (1:1:1, v/v/v) for morphological and microscopic observations. Fine roots with

The Mycorrhizal Symbiosis in Mediterranean Environment: Importance in Ecosystem Stability and in Soil Rehabilitation Strategies : Importance in

Ectotrophic Mycorrhizal Symbioses Are Dominant …

29

ectomycorrhizas were gently washed under tap water. Freehand sections were cleared with a 20% sodium hypochlorite solution, rinsed in water, stained with Congo red and observed under the microscope. Roots were considered to be ECM when they showed a distinctive mantle and a Hartig net. Soil samples aggregated by visible mycelium were also collected and kept fresh at 4°C in the soil until DNA extraction. Other biological material included nodular iron oxide soil particles with their colonizing hyphae, and soil mycelial fragments.

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

 Molecular characterizations DNA was extracted from the dried sporocarp flesh, from a single ECM tip, from soil mycelium or from a single iron oxide particle (or bead), using a Dneasy Plant Mini kit following the manufacturer‘s recommendations (Qiagen, Courtabœuf, France). An approximately 600-bp fragment of the nuclear ribosomal internal transcribed spacer (ITS) rDNA containing the 5.8S region was amplified using the specific primers ITS1 (5‘TCCGTAGGTGAA CCTGCGG-3‘) and ITS4 (5‘-TCCTCCGCTTATTGATATGC-3‘ (White et al. 1990). PCR reaction was made in total volume of 25 µl, containing aliquots of 1 l of genomic DNA, 1 µM of each primer, 1.5 units of Taq DNA Polymerase (Amersham Pharmacia Biotech), 200 µM of each dNTP, 10 mM Tris-HCl, 50 mM KCl, and 1.5 mM MgCl2. Amplification was performed with a DNA thermal-cycler (GenAmp PCR System 2400, Perkin Elmer) programmed as follows: 1 cycle for 5 min at 95°C followed by 35 cycles at 94°C for 30 s, 50°C for 30 s, 72°C for 1 min 30 s, and a final extension at 72°C for 7 min. PCR products were separated by electrophoresis in 1 % (wt/vol) agarose gels (Sigma) in 1x TAE with ethidium bromide at 10 µg/ml in the running buffer. DNA bands were visualised by fluorescence under UV light and photographed.  Sequencing and phylogeny Sequencing was performed with each ITS1/ITS4 primers. Each PCR product was extracted from agarose gel and purified using a QIA (Quick Gel Extraction) kit following the manufacturer‘s recommendations. Sequencing was performed with the ABI Prism BigDye Terminator Cycle sequence kit (Applied Biosystems, Foster City, California) and analysed on an Applied Biosystems model 310 DNA sequencer (Applied Biosystems, Foster City, California). DNA sequences were submitted to the NCBI database (http://www.ncbi.nlm. nih.gov/) under the accession numbers FJ 656000 to FJ 656047. Table 1. List of reference material and GenBank accession numbers included in this study. Names and subgenus classification after Peintner et al. (2001, 2004) Genus / Species Cortinarius acutus Cortinarius albocanus Cortinarius alboviolaceus 1 Cortinarius alboviolaceus 2 Cortinarius allutus Cortinarius amoenus Cortinarius anomalus 1 Cortinarius anomalus 2 Cortinarius armeniacus 1 Cortinarius armeniacus 2

Geogr. origin Europe Chile USA Europe Europe Chile Europe Europe Europe Europe

Subgenus Acutus Myxotelamonia Telamonia Telamonia Allutus Icterinula Anomali Anomali Telamonia Telamonia

GenBank access. AF325578 AF325599 AF325597 AF325596 AF325585 AF539721 AF325581 AJ236071 AF325595 AJ236074

The Mycorrhizal Symbiosis in Mediterranean Environment: Importance in Ecosystem Stability and in Soil Rehabilitation Strategies : Importance in

30

Y. Prin, M. Ducousso, J. Tassin et al.

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

Table 1. (Continued) Genus / Species Cortinarius armillatus Cortinarius austrovenetus Cortinarius bigelowii Cortinarius brunneus 1 Cortinarius brunneus 2 Cortinarius caelicolor Cortinarius callochrous Cortinarius campbellae 1 Cortinarius campbellae 2 Cortinarius caninus Cortinarius caperatus 1 Cortinarius caperatus 2 Cortinarius carneolus Cortinarius cinereobrunneus Cortinarius citriolens Cortinarius collinitus Cortinarius corrosus Cortinarius corrugatus Cortinarius cupreorufus Cortinarius delibutus 1 Cortinarius delibutus 2 Cortinarius elaiochrous Cortinarius elaphinus Cortinarius elegantior Cortinarius evernius Cortinarius favrei Cortinarius flavaurora Cortinarius fragilis Cortinarius fraudulosus Cortinarius gentilis 1 Cortinarius gentilis 2 Cortinarius glaucopus Cortinarius globuliformis Cortinarius hercynicus Cortinarius humicola Cortinarius laniger 1 Cortinarius laniger 2 Cortinarius leucopus Cortinarius limonius 1 Cortinarius limonius 2 Cortinarius lividoochrascens Cortinarius luteistriatulus Cortinarius magnivelatus Cortinarius mucifluus Cortinarius mucosus Cortinarius muscigenus Cortinarius obscurooliveus Cortinarius obtusus Cortinarius odorifer Cortinarius olivaceobubalinus Cortinarius olivaceopictus 1 Cortinarius olivaceopictus 2

Geogr. origin Europe Europe USA Europe Europe Chile Europe Australia Australia USA Europe Europe Chile Argentina USA Europe Europe USA Europe Europe Europe New Zealand Chile Europe Europe Europe USA Australia Europe Europe USA Europe Australia Europe Europe Europe Europe Europe Europe Europe Europe Chile USA USA USA USA Chile Europe Europe Chile USA USA

Subgenus Telamonia Dermocybe Phlegmacium Telamonia Telamonia Phlegmacium Calochroi Purpurascentes Phlegmacium Anomali Rozites Rozites Telamonia Myxotelamonia Phlegmacium Myxacium Phlegmacium Corrugatus Phlegmacium Delibuti Delibuti Cuphocybe Telamonia Calochroi Telamonia Myxacium Calochroi Purpurascentes Phlegmacium Telamonia Telamonia Phlegmacium Dermocybe Cortinarius Telamonia Telamonia Telamonia Telamonia Limonius Limonius Myxacium Dermocybe Calochroi Myxacium Myxacium Myxacium Dermocybe Acutus Calochroi Dermocybe Dermocybe Dermocybe

GenBank access. AJ236075 AF112147h AF325617 AF325590 AJ236076 AF539715 AF325619 AF325558 Specimen ref Tr 18323 U56024 AJ238033 AF325614 AF539712 AF325600 AF325607 AF325565 AF325618 AF325611 AY174831 AJ236065 AF325580 AY033100 AF539725 AF325622 AJ236077 AF325575 AF325621 AF325559 AF325605 AF325589 U56026 AF325604 AF325582 AF062631 AF325594 AF325592 AF325591 AF325593 U56028 AF325588 AF325565 AF539707 AF325615 AF182795 AF325574 AF182800 AF539708 AJ238035 AF325620 AF539736 U56049 U56050

The Mycorrhizal Symbiosis in Mediterranean Environment: Importance in Ecosystem Stability and in Soil Rehabilitation Strategies : Importance in

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

Ectotrophic Mycorrhizal Symbioses Are Dominant … Genus / Species Cortinarius paragaudis Cortinarius parahumilis Cortinarius pavelekii Cortinarius pholideus Cortinarius pingue 1 Cortinarius pingue 2 Cortinarius piriforme Cortinarius porphyroides 1 Cortinarius porphyroides 2 Cortinarius porphyropus 1 Cortinarius porphyropus 2 Cortinarius pseudosalor Cortinarius pugionipes Cortinarius rapaceus 1 Cortinarius rapaceus 2 Cortinarius saginus Cortinarius salor Cortinarius scaurus 1 Cortinarius scaurus 2 Cortinarius scaurus 3 Cortinarius sp. Cortinarius squamiger Cortinarius subfoetidus Cortinarius talus Cortinarius tenellus Cortinarius traganus 1 Cortinarius traganus 2 Cortinarius trivialis Cortinarius umbilicatus Cortinarius vanduzerensis Cortinarius variecolor Cortinarius vernicosus Cortinarius verrucisporus Cortinarius vibratilis 1 Cortinarius vibratilis 2 Cortinarius violaceus 1 Cortinarius violaceus 2 Cortinarius violaceus 3 Cortinarius viridibasalis Cuphocybe melliolens Dermocybe cinnamomea Dermocybe crocea Dermocybe malicoria Dermocybe phoenicea Dermocybe splendid Hebeloma circinans Hebeloma crustuliniforme Hebeloma fastibile Hymenogaster remyi Hymenogaster sublilacinus Protoglossum luteum Protoglossum sp. 1 Protoglossum sp. 2 Quadrispora oblongispora Quadrispora sp.

Geogr. origin USA Chile USA Europe USA USA Australia New Zealand New Zealand Europe Europe USA Chile Chile Chile Europe Europe Europe Europe Europe USA Chile USA Europe Chile Europe Europe Europe USA USA Europe USA USA Europe USA Europe Europe USA Chile New Zealand Europe Europe USA Europe New Zealand

Subgenus Telamonia Telamonia Myxacium Sericeocybe Myxacium Myxacium Myxacium Thaxterogaster Myxacium Purpurascentes Purpurascentes Myxacium Phlegmacium Phlegmacium Phlegmacium Phlegmacium Delibuti Purpurascentes Purpurascentes Purpurascentes Phlegmacium Telamonia Phlegmacium Allutus Telamonia Telamonia Telamonia Myxacium Telamonia Myxacium Phlegmacium Myxacium Phlegmacium Ochroleuci Ochroleuci Cortinarius Cortinarius Cortinarius Telamonia Cuphocybe Dermocybe Dermocybe Dermocybe Dermocybe Dermocybe

Europe USA Australia New Zealand Australia Australia Australia

Phlegmacium Phlegmacium Corrugatus Acutus Myxacium Myxacium

GenBank access. U56030 AF539731 AF325564 AJ236072 AF325570 AF325571 AF325569 AF325576 AF325577 AF325560 AJ236069 AF182792 AF539713 AF539723 AF539724 AF325608 AF325579 AJ236070 AF325562 AF325563 AF325606 AF539729 AF325609 AF325586 AF539728 AF037224 AF325598 AJ236066 U56032 AF182793 AJ238082 AF182799 AF325616 AJ238032 AF325584 AJ236059 AF325601 AF389130 AF539717 AF325610 AJ238030 AJ238031 U56045 U56055 AF325583 AF124699 AF124716 AF325643 AF325602 AF325603 AF325612 AF325613 AF325561 AF325566 AF325567

The Mycorrhizal Symbiosis in Mediterranean Environment: Importance in Ecosystem Stability and in Soil Rehabilitation Strategies : Importance in

31

32

Y. Prin, M. Ducousso, J. Tassin et al. Table 1. (Continued)

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

Genus / Species Thaxterogaster redactus Thaxterogaster sp. Thaxterogaster violaceum 1 Thaxterogaster violaceum 2

Geogr. origin Australia USA Argentina Argentina

Subgenus Myxacium Myxacium Thaxterogaster Thaxterogaster

GenBank access. AF325568 AF325572 AF325556 AF325557

 Phylogenetic analyses New ITS sequences were compared to the Genbank database using the BLASTn algorithm (http://www.ncbi.nlm.nih.gov/blast). Multiple alignments were performed with Clustal X (Thompson et al. 1997) or Muscle 3.6 (Edgar 2004), and manually cured under Genedoc software (Nicholas and Nicholas 1997). The phylogenetic analysis was focused on Cortinarius species (as it was the most represented genus) by using the full spacer sequences (ITS1-5.8S-ITS2) hand-aligned with the Peintner et al. (2001) alignment (Treebase, accession number S636: M988-990), also enriched with some Cortinarius sequences from the southern hemisphere (Table 1). A Maximum Likelihood (ML) phylogeny was established using the best-fit model detected by Model test (Posada and Crandall 1998) under the Akaike information criterion (TrN+I+G: transition only, estimation of invariant sites, gamma distribution of substitution). Internal gaps were treated as effective data (fifth base). Maximum-likelihood (ML) analyses were carried out with the computer program PAUP 4.0b5 under heuristic searches with ―asis‖ addition sequence and TBR branch swapping. Bootstrapping replicates were performed using PHYML on-line using 100 replicates under a GTR+I+G model (Nst=6, Invariant sites, Gamma distribution) (20). The Cortinariaceae phylogeny was also inferred by a Bayesian approach (Huelsenbeck et al. 2001), using MrBayes 3.1.2 (Ronquiste and Huelsenbeck 2003) with four Monte Carlo Markov Chains (MCMC) run on 3 millions generations, with print frequency and chain temperature set to 50,000 and 0.2, respectively, and saving branch lengths. A 50% majority rule consensus analysis was performed on trees sampled after the chains had reached stationarity. Priors used to run the chains were the parameters estimated by Modeltest (TrN+I+G). Trees topology congruence between ML and Bayesian inferences of Cortinariaceae ITS sequences was assessed using the Shimodaira-Hasegawa Likelihood based test (S-H test using 1000 RELL bootstrap replicates) (Shimodaira and Hasegawa 1999), implemented in PAUP4. Congruence was found between trees obtained by both methods (-ln L = 12183 by ML; -ln L=12200 by MrBayes; S-H test P value=0.254)

Results  Vegetation distribution along the topographic gradient From the plateau located at 882 m to the talweg at 700 m, the site presented four different plant communities among the twelve groups defined by Jaffré (1974). Statistical analyses confirmed the drastic changes in floristic composition between the different plant communities, excepted between plant communities 2 and 3 where the discontinuity was less strongly marked.

The Mycorrhizal Symbiosis in Mediterranean Environment: Importance in Ecosystem Stability and in Soil Rehabilitation Strategies : Importance in

Ectotrophic Mycorrhizal Symbioses Are Dominant …

33

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

The Araucaria montana Brongn. and Gris and Nothofagus plant communities are strongly isolated on the ordination graph (Figure 2). Floristic biodiversity increases downwards the toposequence. Plant community 1: On the plateau, the dominant plant species of the plant community is Araucaria montana, and 53 other plant species were numbered. The ligneous stratum is the most abundant in terms of cover abundance (33.9 %), with Codia montana J.R. Forst. and G. Forst. (15.7 %), Tristaniopsis guillainii Viell. ex Brongn. and Gris (5.7 %) Araucaria montana (4.3 %) and Dracophyllum verticillatum Labill. (3.5 %). The herbaceous stratum is less abundant (27.3 %), with the pteridophytes Dicranopteris linearis J. Underw. (15.0 %) and Pteridium esculentum (G. Forster) Cockayne (13.9 %). Plant community 2: On the slope, the dominant plant community is a ligno-herbaceous maquis where 58 plant species were numbered. The herbaceous stratum is the most abundant (85.0 %) with Costularia nervosa Raynal (60.1 %), Dicranopteris linearis (24.8 %), Pteridium esculentum (15.0 %), Schoenus neocaledonicus C.B. Clarke (11.8 %) and Lepidosperma perteres C.B. Clarke (9.8 %).

Figure 2. Ordination of records along the two main factors of correspondence analysis, showing drastic changes in plant communities within the toposequence, from the plateau (plant community 1) to the thalweg (plant community 4).

The ligneous stratum is less abundant (55.1 %), and it is mainly formed of Codia montana (35.9 %), Dracophyllum ramosum Pancher ex Brongn. and Gris (9.7 %) and Myodocarpus crassifolius Dubard and R. Viguier (7.0 %). Plant community 3: On the lower part of the slope, the dominant plant community is a maquis, with 72 plant species. The ligneous stratum is the most abundant (80.0 %), and is mainly formed of Tristaniopsis guillainii (50.5 %), Tristaniopsis callobuxus Brongn. and Gris (27.0 %) and

The Mycorrhizal Symbiosis in Mediterranean Environment: Importance in Ecosystem Stability and in Soil Rehabilitation Strategies : Importance in

34

Y. Prin, M. Ducousso, J. Tassin et al.

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

Codia montana (23.5 %). The herbaceous stratum is less abundant (34.5%) with Costularia nervosa (22.2 %) and Lepidosperma perteres (12.13%). Plant community 4: In the talweg, the plant community is a rainforest dominated by 2 Nothofagus species and 81 other species were numbered. The dominant plant species is Nothofagus balansae (62.5%), which represents the tallest stratum, Callophylum caledonicum Vieill. (8.9 %) is another member of the tallest stratum. The species Nothofagus codonandra is also present in this forest but forms monodominant patches, with only a small overlap between the two Nothofagus species, not illustrated in this paper. The medium stratum is heterogeneous; the most abundant species are Styphelia pancheri (Brongn. and Gris) F. Muell. (11.7 %), Basselinia gracilis (Brongn. and Gris) Vieill. (6.45 %) Pancheria ferruginea Brongn. (3.3 %) and Rapanea assymetrica Mez. The herbaceous stratum is represented essentially by Lepidosperma perteres (7.23 %) and Costularia arundinacea (Soland. ex Vahl) Kük. (1.5 %).

Figure 3. A to D: aspect of some ECM sporocarps from plant communities 3 and 4. A: Pisolithus albus K66C, associated to Tristaniopsis guillainii, in plant community 3. B: Cortinarius sp nov. K12C, associated to Nothofagus balansae in plant community 4. C: Multifurca auranthiophylla K18C, associated to Nothofagus balansae in plant community 4. D: Tricholoma sp nov. aff ustale K10C, associated to Nothofagus balansae in plant community 4. E: Cross section through an ECM apex of Tristaniopsis guillainii (plant community 3), after staining with Trypan Blue, showing the fungal mantle and the Hartig net.

The Mycorrhizal Symbiosis in Mediterranean Environment: Importance in Ecosystem Stability and in Soil Rehabilitation Strategies : Importance in

Y. Prin, M. Ducousso, J. Tassin et al.

35

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

 Ectomycorrhizal tree species Roots of the plant species of each plant community were observed under the microscope in order to characterize the symbiotic associations. In the Araucaria montana group on the plateau (plant community 1) and the ligno-herbaceous maquis on the slope (plant community 2), all plants were endomycorrhizal confirming observations by Perrier et al. (2006b) and no ECM symbiosis was observed. Ectomycorrhization in the Tristaniopsis maquis (plant community 3) and Nothofagus forest (plant community 4) was limited to the dominant plant species (Tristaniopsis guillainii, T. calobuxus, Nothofagus balansae and N. codonandra, respectively) presenting typical fungal mantle around the roots and a Hartig net under the microscope.

Figure 4. A: Aspect of a soil aggregate with yellow gold mycelium and rhizomorphs, harvested in plant community 3 under Pisolithus albus K66C sporocarp. The spherical aspect of iron oxide beads that compose the soil is clearly visible. B and C: High magnification of iron oxide beads under the stereomicroscope, with numerous non-identified hyphal links between beads.

The Mycorrhizal Symbiosis in Mediterranean Environment: Importance in Ecosystem Stability and in Soil Rehabilitation Strategies : Importance in

36

Y. Prin, M. Ducousso, J. Tassin et al.

 Fungal diversity A total of 48 samples were analyzed, 11 from plant community 3 and 37 from plant community 4. Twenty-eight samples were sporocarps among which only 2 from plant community 3. Figure 3A to D illustrates some of the sporocarps harvested in plant communities 3 and 4. Twelve samples were mycorrhizal apices with 3 and 9 collected from plant communities 3 and 4, respectively. The last type of samples was from soil aggregates as illustrated in Figure 4A with yellow-gold mycelium harvested in plant community 3 under Pisolithus albus K66C sporocarp. The spherical aspect of iron oxide beads that compose the soil is clearly visible. A closer examination of these iron oxide beads under the stereomicroscope is presented Figure 4B and C, with unidentified hyphal links between beads. Other soil samples, with different mycelial colors, were also harvested in plant communities 3 and 4. Six samples were analyzed from plant community 3 and 2 from plant community 4. The material used for extraction was a fragment of mycelium or rhizomorph, and in plant community 3, 4 samples were individual iron oxide beads, with their colonizing hyphae as illustrated by Figure 4B and C. Table 2 lists the different ECM samples collected in the two plant communities, indicating the morphological determination of sporocarps, ITS sequence length and BLASTn identity. Table 2. Ectomycorrhizal samples collected in the different plant communities, submitted ITS sequence length, closest match with GenBank genera, query coverage, maximum identity and e-value (www.ncbi.nlm.nih.gov/blast/) Ref*

Plant community

Host plant

Macroscopy

ITS1/ITS4 submitted length

BLAST closest genera

Query Coverage

Max identity

K01C

4

Nothofagus balansae

Tricholoma sp nov

701

Tricholoma

98%

88%

4

Nothofagus balansae

Boletus sp

609

Boletus

91%

76%

4

Nothofagus balansae

Cortinarius sp nov

551

Cortinarius

99%

83%

K06 C

4

Nothofagus balansae

Phellodon sp

605

Phellodon

81%

85%

K09 C

4

Nothofagus balansae

nd**

547

Clavariadelphus

70%

90%

4

Nothofagus balansae

Tricholoma sp nov aff ustale

670

Tricholoma

98%

93%

4

Nothofagus balansae

Cortinarius sp. nov

572

Cortinarius

99%

92%

4

Nothofagus balansae

Cortinarius sp.

506

Cortinarius

98%

95%

4

Nothofagus balansae

Lactarius sp

706

Lactarius

99%

91%

4

Nothofagus balansae

Multifurca auranthiophylla

598

Lactarius

98%

87%

K22 C

4

Nothofagus balansae

Inocybe sp

582

Inocybe

99%

83%

K66 C

3

Tristaniopsis guillainii

Pisolithus albus

527

Pisolithus

99%

98%

436

Cortinarius

97%

89%

3e-154

612

Russula

100%

93%

0.0

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

K02 C K05 C

K10 C K12 C K14 C K16 C K18 C

KC02 C

4

KC03 C

4

Nothofagus balansae

Cortinarius sp nov aff lividoochrascens

Nothofagus balansae

Russula sp

The Mycorrhizal Symbiosis in Mediterranean Environment: Importance in Ecosystem Stability and in Soil Rehabilitation Strategies : Importance in

evalue

0,0 2e-120 3e-155 4e-155 8e-87 0.0 0.0 0.0 0.0 0.0 2e-159 0.0

Ectotrophic Mycorrhizal Symbioses Are Dominant …

Ref*

Plant community

Host plant

Macroscopy

ITS1/ITS4 submitted length

BLAST closest genera

Query Coverage

Max identity

KC05 C

4

Nothofagus balansae

Inocybe sp

580

Inocybe

94%

84%

4

Nothofagus balansae

nd

436

Phaeocollybia

94%

95%

4

Nothofagus balansae

Lactarius sp nov

670

Lactarius

98%

91%

KC10 C

4

Nothofagus balansae

447

Thaxterogaster

99%

93%

KC11 C

4

Nothofagus balansae

609

Russula

99%

93%

4

Nothofagus balansae

Cortinarius sp

518

Cortinarius

100%

84%

4

Nothofagus balansae

Cortinarius sp

574

Cortinarius

100%

86%

KC17 C

4

Nothofagus balansae

Cortinarius sp.

574

Cortinarius

99%

83%

KC19 C

4

Nothofagus balansae

Cortinarius np perrierii

583

Cortinarius

99%

86%

4

Nothofagus balansae

nd

528

Clavariadelphus

74%

94%

3

Tristaniopsis guillainii

nd

414

Cortinarius

71%

88%

KD10 M

4

Nothofagus balansae

-

552

Oidiodendron

91%

92%

KD18 M

4

Nothofagus codonandra

-

474

Cortinarius

98%

89%

3

Tristaniopsis guillainii

-

698

Lycoperdon

94%

93%

3

Tristaniopsis guillainii

-

592

Cortinarius

99%

92%

3

Tristaniopsis guillainii

-

410

Cortinarius

97%

85%

3

Tristaniopsis guillainii

-

616

Cortinarius

100%

85%

3

Tristaniopsis guillainii

-

585

Cortinarius

100%

85%

3

Tristaniopsis guillainii

-

615

Cortinarius

100%

86%

4

Nothofagus codonandra

-

608

Cortinarius

99%

91%

4

Nothofagus codonandra

-

570

Cortinarius

99%

85%

4

Nothofagus codonandra

-

682

Tomentella

98%

93%

4

Nothofagus codonandra

-

584

Cortinarius

99%

86%

4

Nothofagus codonandra

Cortinarius sp nov

585

Cortinarius

99%

93%

4

Nothofagus codonandra

Cortinarius sp nov

582

Cortinarius

98%

94%

4

Nothofagus codonandra

Tricholoma sp nov

458

Tricholoma

94%

88%

3

Tristaniopsis guillainii

-

457

Piloderma

98%

88%

3

Tristaniopsis guillainii

-

511

Cortinarius

98%

79%

3

Tristaniopsis guillainii

-

438

Piloderma

97%

88%

4

Nothofagus balansae

-

584

Cortinarius

100%

91%

KC06 C KC08 C

KC12 C KC16 C

KC22 C KC23 C

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

37

KD19-1 Sn KD19-2 Sm KD19-9 Sm KD20-2 Sn KD20-5 Sn KD20-6 Sn KD29-2 M KD31''-2 M KD31' M KD36-2 M KD36 C KD37 C KD42 C KE01-2 M KE02 M KE04 M KE06 M

Cortinarius sp nov aff teraturgus Russula sp nov aff occidentalis

The Mycorrhizal Symbiosis in Mediterranean Environment: Importance in Ecosystem Stability and in Soil Rehabilitation Strategies : Importance in

evalue

4e-161 6e-176 0.0 0.0 0.0 9e-143 0.0 1e-147 0.0 2e-93 6e-100 0.0 3e-155 0.0 0.0 6e-106 0.0 0.0 0.0 0.0 1e-172 0.0 0.0 0.0 0.0 3e-154 2e-150 2e-126 2e-143 0.0

38

Y. Prin, M. Ducousso, J. Tassin et al. Table 2. (Continued)

Ref*

Plant community

Host plant

Macroscopy

ITS1/ITS4 submitted length

BLAST closest genera

Query Coverage

Max identity

KE10 M

4

Nothofagus balansae

-

572

Thaxterogaster

100%

86%

4

Nothofagus balansae

-

620

Tricholoma

99%

86%

4

Nothofagus balansae

-

608

Tricholoma

99%

87%

4

Nothofagus codonandra

-

540

Cortinarius

98%

83%

KE12-1 M KE12-2 Sm KE18-2 Sm

evalue

0.0 0.0 0.0 1.9e150

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

*Suffixes refer to the type of material: C: sporocarp, M: mycorrhizal apex, S: soil aggregate with hyphal mats, with two different types: Sn: one single iron oxyde bead, Sm: one mycelial or rhizomorph fragment. **: nd: not determined.

Most sporocarps (suffix ―C‖) were identified on their macroscopic and microscopic characteristics at least at the genus level, only four of them (―nd‖, not determined) were not attributed any taxonomic name. Mycorrhizal root apices (suffix ―M‖) and soil samples (suffix ―S‖) were obviously left without taxonomic name. None of the sequences compared to the Genbank database gave 100 % similarities with previously described species. On the number of samples analyzed and considering the relatively small area of the prospected site (roughly estimated to represent 5 ha, in a massif of more than 38,000 ha) a large diversity of fungal taxons was found. Among sporocarps, there was generally a good correspondence between macroscopic and molecular determinations. In plant community 3, 4 different genera were detected among 11 different samples. Seven of these samples belonged to the genus Cortinarius, 2 to Piloderma, 1 to Pisolithus, sampled as a sporocarp, and 1 to Lycoperdon, detected in one soil iron oxide bead and generally considered as non-mycorrhizal. The genus Cortinarius was detected in one sporocarp, in ECM, mycelium and soil beads. In plant community 4, the diversity is much greater with 12 different genera recorded from ITS sequences, corresponding to 37 samples. The dominant genus is Cortinarius with a total of 18 samples (including 2 species formerly identified as Thaxterogaster) thus reaching almost 50 % of the samples for Cortinariaceae. Genera Tricholoma and Lactarius-Russula followed with 5 samples each. Dominance of the Cortinariaceae is observed for the different forms of fungal samples. Out of 26 collected sporocarps, 11 belonged to this group. Among ECM, 9 different samples were successfully analyzed, belonging to 5 different genera. The dominant genus also is Cortinarius with 5 different samples. In the soil, only 2 samples could be analyzed and they belonged to genera Cortinarius and Tricholoma.  Phylogenetic grouping The analysis (Figure 5) of the Cortinariaceae family was focused on the whole 18S-28S portion (ie including both ITS and 5.8S sequences) and referred to 124 database sequences (121 from Cortinariaceae and 3 from Hebeloma) as listed in Table 1. On the 18 different samples analyzed, 5 were from plant community 3 and 13 from plant community 4. The different forms of the 18 samples were sporocarps (8), mycorrhizal tips (5) and soil mycelium (5). On this limited number of samples, the diversity appears to be high, all samples being distinct from each other and from the database sequences. New Caledonian Cortinariaceae are

The Mycorrhizal Symbiosis in Mediterranean Environment: Importance in Ecosystem Stability and in Soil Rehabilitation Strategies : Importance in

Ectotrophic Mycorrhizal Symbioses Are Dominant …

39

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

distributed in 6 different clades. A few clades mix sequences from plant community 3 and 4, but there obviously is a need for more samples analyses to evaluate a putative link between fungal communities, as for example there is no grouping between ECM apices from both plant communities and no total sequence identity between any sample. Cortinarius armeniacus 2 Cortinarius armeniacus 1 Cortinarius alboviolaceus 1 Cortinarius alboviolaceus 2 0.809 Cortinarius evernius Cortinarius leucopus Cortinarius laniger 1 Cortinarius laniger 2 Cortinarius humicola 0.958 Cortinarius paragaudis Cortinarius armillatus Cortinarius pholideus Cortinarius traganus 2 0.983 Cortinarius traganus 1 Cortinarius umbilicatus Cortinarius gentilis 2 Cortinarius gentilis 1 0.838 Cortinarius brunneus 1 Cortinarius brunneus 2 Cortinarius squamiger Cortinarius tenellus Cortinarius obtusus 0.918 Cortinarius acutus Protoglossum sp. Dermocybe crocea 0.844 Dermocybe cinnamomea 0.934 Dermocybe phoenicea Dermocybe malicoria 0.721 Cortinarius olivaceopictus 1 Cortinarius olivaceopictus 2 K12C Dermocybe splendida 0.957 Cortinarius globuliformis 0.755 Cortinarius luteistriatulus Cortinarius obscurooliveus Cortinarius amoenus Cortinarius carneolus Cortinarius elaphinus Cortinarius austrovenetus Cortinarius violaceus 2 Cortinarius violaceus 1 1.000 Cortinarius hercynicus Cortinarius violaceus 3 Cortinarius delibutus 1 Cortinarius delibutus 2 Cortinarius salor Cortinarius anomalus 1 0.764 Cortinarius anomalus 2 Cortinarius caninus KD31-2M Cortinarius saginus Cortinarius caelicolor Cortinarius viridibasalis KD20 5Sn 0.990 KD20 6Sn 0.731 KD20 2Sn 0.961 KE06M KD36C 0.895 KD29 2M Cortinarius elaiochrous 0.832 Cortinarius parahumilis Cortinarius variecolor Cortinarius citriolens Cortinarius sp. Cortinarius fraudulosus 0.978 Hymenogaster remyi Hymenogaster sublilacinus Cortinarius glaucopus Cortinarius subfoetidus 1.000 Cortinarius limonius 1 Cortinarius limonius 2

The Mycorrhizal Symbiosis in Mediterranean Environment: Importance in Ecosystem Stability and in Soil Rehabilitation Strategies : Importance in

40

Y. Prin, M. Ducousso, J. Tassin et al. Cortinarius trivialis Cortinarius vernicosus Cortinarius favrei Cortinarius muscigenus Cortinarius pingue 1 Cortinarius pingue 2 Thaxterogaster sp. Cortinarius collinitus Cortinarius mucosus Cortinarius piriforme Thaxterogaster redactus Quadrispora sp. nov. Quadrispora oblongispora Cortinarius pseudosalor Cortinarius mucifluus Cortinarius vanduzerensis 0.843 Cortinarius paveleckii Cortinarius lividoochrascens Cortinarius porphyroides 1 Cortinarius porphyroides 2 KD36 2M KC19C 1.000 KC17C 0.990 KC12C 0.721 KC16C K05C Protoglossum luteum Cortinarius corrugatus 0.913 Cuphocybe melliolens Protoglossum sp. 0.877 KE18-2Sm 0.892 KE02M Cortinarius cinereobrunneus Cortinarius albocanus Cortinarius olivaceobubalinus Cortinarius porphyropus 1 Cortinarius porphyropus 2 Cortinarius fragilis Cortinarius campbellae 1 0.748 Cortinarius campbellae 2 0.770

0.715

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

0.979

Cortinarius scaurus 2 Cortinarius scaurus 3 Cortinarius scaurus 1 KD37C 1.000 KD19-2Sm Cortinarius pugionioides Cortinarius vibratilis 2 Cortinarius vibratilis 1 Cortinarius caperatus 2 Cortinarius caperatus 1 Cortinarius rapaceus 2 Cortinarius rapaceus 1 Thaxterogaster violaceum 1 Thaxterogaster violaceum 2 Cortinarius magnivelatus Cortinarius bigelowii Cortinarius callochrous Cortinarius corrosus Cortinarius verrucisporus 0.841 Cortinarius flavaurora Cortinarius cupreorufus 0.998 Cortinarius odorifer Cortinarius elegantior Cortinarius talus Cortinarius allutus

Hebeloma circinans Hebeloma fastibile Hebeloma mesophaeum 0.1

Figure 5. Maximum likelihood phylogram (congruent with Bayesian tree, see Material & Methods) of 136 Cortinariaceae rDNA ITS sequences, including 18 New Caledonian samples harvested in the Pandanus river watershed, in the Koniambo massif. Outgroup was made with 3 Hebeloma spp sequences. Only bootstrap values superior to 70% and also supported by Bayesian Posterior Probabilities superior to 70% are indicated. The ML model used is described in Material and Methods.

The Mycorrhizal Symbiosis in Mediterranean Environment: Importance in Ecosystem Stability and in Soil Rehabilitation Strategies : Importance in

Ectotrophic Mycorrhizal Symbioses Are Dominant …

41

Telamonia Eur, Rus, USA

Acutus

Dermocybe NCal

NCal Aust, N Zel S Am

Icterinula Telamonia

Cortinarius Eur, Rus, USA

Delibuti Anomali

NCal

Ncal Phlegmacium

S Am

Telamonia

NCal

NCal

Aust, N Zel S Am

Cuphocybe Telamonia

Aust, N Zel

Eur, Rus, USA

Phlegmacium

Limonius

Eur, Rus, USA

Myxacium Aust, N Zel Eur, Rus, USA

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

Aust, N Zel NCal

NCal

Eur, Rus, USA

Aust, N Zel

Corrugatus

NCal

NCal

S Am

Myxotelamonia

Eur, Rus, USA

Aust, N Zel Purpurascentes Eur, Rus, USA

NCal

NCal

S Am Eur, Rus, USA

S Am

Ochroleuci Rozites Phlegmacium Thaxterogaster

Eur, Rus, USA Phlegmacium (incl. Calochroi)

Hebeloma (outgroup)

Figure 6. Schematic representation of the phylogenetic tree presented in Fig. 5A and B, but converted as a cladogram, and illustrated with a colour code for the geographical origin of the Cortinariaceae (left side tree) or the taxonomic grouping (genera and subgenera) according to Peintner et al. (2004) (right side tree). European, Russian and North American Cortinariaceae were arbitrarily considered as fitting in the same clusters.

The Mycorrhizal Symbiosis in Mediterranean Environment: Importance in Ecosystem Stability and in Soil Rehabilitation Strategies : Importance in

42

Y. Prin, M. Ducousso, J. Tassin et al.

New Caledonian samples fit sometime in distinctive clusters, among the 118 reference sequences from the Cortinariaceae, whatever their origin, including geographically linked countries like Australia, New Zealand, Chile or Papua New Guinea, indicating the presence of several new taxons. Reporting on the tree (drawn as a cladogram) the phyto-geographical origin of the samples from sequences taken as references (Figure 6) the existence of several New Caledonian clades, but not always positioned closer to clades from Gondwanaland fragments than to clades from Northern hemisphere. This study did not aim at giving a taxonomical description of New Caledonian Cortinariaceae due to the heterogeneity of our material (not only sporocarps). However, after reporting on the cladogram (Figure 6) most of the different genera and subgenera described by Peintner et al. (2004) with a colour code, the New Caledonian clades seemed to either fit within existing clades like Dermocybe, Purpurascentes or constituted separated and potentially new taxons, one of them being possibly related to Myxacium. Descolea sequences that we first included in our phylogeny were finally removed as these sequences fitted in a separated cluster (Moncalvo et al. 2002) with no affinity with any of our New Caledonian species.

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

Discussion  Floristic diversity, dominance and symbioses As stated by Jaffré (1974), the floristic diversity of the Koniambo massif is quite high as referred to those of New Caledonia: the 453 species inventoried by this author represented 15% of the diversity of the island, for a geographic surface ratio of less than 1%. Our floristic inventories, limited to 4 of the 12 phyto-ecological plant communities listed by Jaffré (1974) on the massif, are consistent in terms of species number with those given by this author. Dominant plant species of plant communities 3 and 4 were found ECM. Such a situation has often been described in tropical ecosystems (ie Dipterocarps in Asian forests), although it is far from being systematic (Torti and Coley 1999). In New Caledonia, it has been hypothesized that this dominance could be only a transient stage linked to exogenous disturbance, like fire or cyclones (Read et al. 1995). This study confirmed the description (Perrier et al. 2006b) of ectomycorrhizas on the roots of Nothofagus balansae (Nothofagaceae) and Tristaniopsis, from the Myrtaceae (Leptospermoideae) family. The genus Tristania, close to Tristaniopsis, has already been described as ECM in Brunei (Moyersoen et al. 2001). Nothofagus is the well-known Southern beech, reported as naturally ECM in Australia, New Zealand and South America (Halling 2001, Tedersoo et al. 2008, McKenzie et al. 2000, Valenzuela et al. 1999). Mycorrhizal species were present as ECM roots, soil mycelia and sporocarps enabling us to get a picture of the general diversity since several studies have shown that there was little correlation between diversity of sporocarps and ectomycorrhizal roots (Buscot et al. 2000, Dahlberg et al. 1996, Gardes and Bruns 1996, Horton and Bruns 2001). Furthermore, Landeweert et al. (2003) showed that using internal transcript spacers on mycelium from soils gave an extra insight on the diversity of the fungal communities. Only 1 sporocarp was collected in plant community 3, which again shows the divergence between the above- and below-ground fungal communities. This probably resulted from the

The Mycorrhizal Symbiosis in Mediterranean Environment: Importance in Ecosystem Stability and in Soil Rehabilitation Strategies : Importance in

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

Ectotrophic Mycorrhizal Symbioses Are Dominant …

43

environmental conditions of the maquis, which could be not optimal for fungal fruiting as very dry conditions prevail and as the soil contains little organic matter (Brundrett 1991, Slankis 1974, Smith and Read 1997). Furthermore, the presence of typical sporocarps of Pisolithus albus (Figure S3A), which presented identical morphological and anatomical features to those described by Anderson et al. (1998, 2001) in Australia and Aggangan et al. (1998) in New Caledonia, is not surprising in the maquis-type vegetation dominated by sclerophyllous plants, since this genus is known to be well-adapted to growth in mineral soils as an ―early stage‖ mycorrhizal fungus, until forest soil conditions develop (Brundrett 1991, Gardner and Malajczuk 1988, Stahl et al. 1988). The presence of Cortinarius in this ecosystem is also of prime importance: it is one of the taxonomically most complex genera in the Basidiomycetes (with ca. 2000 species described so far), being the most frequently recorded ectomycorrhizal fungal genus in many European and North American conifer forests (Alexander and Watling 1987, Hoiland and Holst-Jensen 2000, Villeneuve et al. 1989). Its occurrence in the Southern hemisphere is still poorly documented (Garnica et al. 2005, Tedersoo et al 2008). In the tropics, the genus was reported in tropical North America (Murrill 1912), South America (Singer et al. 1983, Garnica et al. 2003), India (Peintner et al. 2003, Natarajan et al. 2005) along with Australian eucalypt forests (Chambers et al. 1999, Malajczuk et al. 1987, Sawyer et al. 1999, 60). Cortinariaceae seem to be poorly represented in African rainforests as stated by Rivière et al. (2007) in Southern Guinea who did not identify any Cortinariaceae within sequences of 119 sporocarps and 55 ECM and Onguene and Kuyper (2001) in Cameroon. In plant community 4, ECM symbioses were found with a much greater diversity of genera, confirmed by molecular methods. This diversity mostly included ECM taxons, like Russula, Lactarius, Tricholoma, Boletus, Inocybe, etc, and was already reported in other tropical forest ecosystems like India, Guinea (Rivière et al. 2007) or Cameroon (Onguéné and Kuyper 20001). However, in each type of sample, the presence of the genus Cortinarius and other genera of the Cortinariaceae is noted and this taxon is dominant among the sporocarps and ectomycorrhizas. If the dominance of the genus Cortinarius and family Cortinariaceae in Nothofagus forests is well documented in South America (Garnica et al. 2003, Garnica et al. 2005, Valenzuela et al. 1999), Australia (Bougher et al. 1994, Halling 2001) and New Zealand (McKenzie et al. 2002), this study represents the first record its dominance in New Caledonia.

Biogeography and Evolution The dominance of Cortinariaceae in the Nothofagus forest of the Koniambo massif gives a new insight into the phylogeography of this fungal family in the Southern hemisphere. Nothofagus incorporates 4 subgenera and 35 species in 6 isolated landmasses: Australia, New Caledonia, New Guinea, New Zealand and South America (Hill and Dettman 1996, Swenson et al. 2000). Swenson et al. (2001b) showed that the biogeography of Nothofagus supports the Gondwanaland break-up sequence and that the four major lineages were already present 80 My ago prior to the break up of the Gondwanaland eastern margin. The Nothofagus members of New Caledonia all belong to Brassospora subgenus, which also encompasses species from New Guinea and fossil species from New Zealand, Antarctica, South America and Australia. The presence of species belonging to Brassospora subgenus in Papua New Guinea and New

The Mycorrhizal Symbiosis in Mediterranean Environment: Importance in Ecosystem Stability and in Soil Rehabilitation Strategies : Importance in

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

44

Y. Prin, M. Ducousso, J. Tassin et al.

Caledonia can be explained by dramatic tectonic events since the mid-Cenozoic (Hallam 1994; Veevers et al. 1991), which provided habitable niches in tropical latitudes (Swenson et al. 2001a). Strong evidence exists to support the possible co-evolution of Nothofagus and ECM fungi from the Cortinariaceae family (Bougher et al. 1994, Halling 2001, Halling and Mueller 2002) in the Southern Hemisphere. The data accumulated in the Nothofagus forest of the Koniambo massif identifies several fungal clades specific to New Caledonia, inferring that both plant and fungal ancestors already co-existed at the time they colonized the island. Along with Nothofagaceae, Leptospermoideae (dry-fruit Myrtaceae) like Tristaniopsis have their geographical distribution closely linked to the fragmentation of Gondwanaland. The presence of Cortinarius species in the Tristaniopsis plant community concern 6 among 10 samples, among those only one was an ECM apex, the other being soil samples, i.e. either mycelium or iron oxyde beads from soil mycelia aggregates, whose actual symbiosis with Tristaniopsis was not attested. Sawyer et al. (1999) characterized genets of Cortinarius rotundisporus reaching 9 to 30 m in diameter in Australian schlerophyll forests. It cannot be rejected that mycelium from Nothofagus-associated Cortinariaceae from plant community 4 explore soil from under Tristianopsis in plant community 3. The ability of Cortinariaceae to switch hosts was demonstrated in the laboratory by pure culture synthesis of ECM with Nothofagus and Eucalyptus (Bougher 1987, Bougher et al. 1994) opening the door to putative exchange of ECM fungi between plant communities 3 and 4. Phylogenetic analyses showed that New Caledonian Cortinariaceae did not constitute a unique separated clade but were rather dispersed among or close to several different taxonomical groups. They neither constituted a systematic sister clade of other Cortinariaceae from countries derived from the Gondwanaland break up, although the size of our sampling, and its relatively confined area (only a few hundred square meters, on one watershed, in one isolated ultramafic massif in New Caledonia) might clearly not represent the whole diversity of New Caledonian Cortinariaceae. It is remarkable that all the New Caledonian Cortinariaceae were different from molecular databases existing taxons, and from each other, as if this study only scratched the surface of the diversity present. More systematic sampling of Cortinariaceae and ECM root apices within Nothofagus forests in all regional Gondwanaland countries would be necessary to further analyse the possible phylogeography and co-evolution of both plant and fungal partners. The dominance of the ECM symbioses in two plant communities shows the necessity to consider these partners in the early stages of restoration processes, e.g. when choosing plant species to be planted or when optimizing the management of topsoils. We also showed the existence of soil fungal mats and hyphal networks, with several ECM taxons, that could be of key interest to stabilize the iron oxide spherical beads that almost exclusively compose the soil, in often steep slopes and under cyclonic rains.

Acknowledgments Nicolas Perrier received a PhD grant from KNS Consortium and a financial support from CIRAD. This study was partially funded by the French Embassy in Australia. Thanks are expressed to S. Nourissier for technical help with VA symbioses and Abdala Diédhiou with ECM. Vegetation data were collected with the assistance of Alexandre Lagrange.

The Mycorrhizal Symbiosis in Mediterranean Environment: Importance in Ecosystem Stability and in Soil Rehabilitation Strategies : Importance in

Ectotrophic Mycorrhizal Symbioses Are Dominant …

45

References Aggangan NS, Dell B, Malajczuk N (1998) Effects of chromium and nickel on growth of the ectomycorrhizal fungus Pisolithus and formation of ectomycorrhizas on Eucalyptus urophylla S.T. Blake. Geoderma, 84,15-27. Alexander IJ, Watling R (1987) Macrofungi of Sitka spruce in Scotland. Proceedings of the Royal Society Edinburgh, 93B,107-115. Anderson IC, Chambers SM, Cairney JWG (2001) Distribution and persistence of Australian Pisolithus species genets at native sclerophyll forest field sites. Mycological Research, 105, 971-976. Anderson IC, Chambers SM, Cairney JWG (1998) Molecular determination of genetic variation in Pisolithus isolates from a defined region in New South Wales, Australia. New Phytologist, 138,151-162. Bougher NL (1987) The systematic position and ectomycorrhizal status of the fungal genus Descolea. Ph.D. thesis. University of Western Australia, Perth, WA. Bougher NL, Fuhrer BA, Horak E (1994) Taxonomy and biogeography of Australian Rozites species mycorrhizal with Nothofagus and Myrtaceae. Australian Systematic Botany, 7, 353-375. Braun-Blanquet G, Fuller GD, Conard HS (1932) Plant sociology: the study of plant communities. McGraw-Hill book, New York, USA. Brundrett M (1991) Mycorrhizas in natural ecosystems. Advances in Ecological Research, 21, 171-313. Buscot F, Munch JC, Charcosset JY, Gardes M, Nehls U, Hampp R (2000) Recent advances in exploring physiology and biodiversity of ectomycorrhizas highlight the functioning of these symbioses in ecosystems. FEMS Microbiology Reviews, 24, 601-614. Chambers SM, Sawyer NA, Cairney JWG (1999) Molecular identification of co-occurring Cortinarius and Dermocybe species from Southeastern Australian sclerophyll forests. Mycorrhiza, 9, 85-90. Dahlberg A, Jonsson L, Nylund JE (1996) Species diversity and distribution of biomass above and below ground among ectomycorrhizal fungi in an old-growth Norway spruce forest in South Sweden. Canadian Journal of Botany, 75, 1323-1335. Ducousso M, Contesto C, Cossegal M, Prin Y, Rigault F, Eyssartier G (2004) Cantharellus garnierii sp. nov., une nouvelle chanterelle de Nouvelle-Calédonie. Cryptogamie Mycologie, 25, 115-125. Edgar RC (2004) MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Research, 32, 1792-1797. Gardes M, Bruns TD (1996) Community structure of ectomycorrhizal fungi in a Pinus muricata forest: above and below ground views. Canadian Journal of Botany, 74, 15721583. Gardner JH, Malajczuk N (1988) Recolonisation of rehabilitated bauxite mine sites in Western Australia by mycorrhizal fungi. Forest Ecology and Management, 24, 27-42. Garnica S, Weiss M, Oberwinkler F (2002) New Cortinarius species from Nothofagus forests in South Chile. Mycologia, 94, 136-145. Garnica S, Weiss M, Oberwinkler F (2003) Morphological and molecular phylogenetic studies in South American Cortinarius species. Mycological Research, 107, 1143-1156.

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

.

The Mycorrhizal Symbiosis in Mediterranean Environment: Importance in Ecosystem Stability and in Soil Rehabilitation Strategies : Importance in

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

46

Y. Prin, M. Ducousso, J. Tassin et al.

Garnica S, Weiss M, Oertel B, Oberwinkler F (2005) A framework for a phylogenetic classification in the genus Cortinarius (Basidiomycota, Agaricales) derived from morphological and molecular data. Canadian Journal of Botany, 83, 1457-1477. Greenacre MJ (1984) Theory and applications of correspondence analysis, Academic Press, London. Guindon S, Lethiec F, Duroux P, Gascuel O (2005) PHYML Online--a web server for fast maximum likelihood-based phylogenetic inference. Nucleic Acids Research, 33, W557-9. Hallam A (1994) An outline of Phanerozoic biogeography. Oxford University Press, Oxford. Halling RE (2001) Ectomycorrhizae: co-evolution, significance, and biogeography. Annals of the Missoury Botanical Garden, 88, 5-13. Halling RE, Mueller GM (2002) Agarics and Boletes of neotropical oakwoods. In: Tropical Mycology, Vol 1, Macromycetes (ed. Watling R, Frankland JC, Ainsworth AM, Isaac S, Robinson CH). CAB International, New York, USA. Hill RS, Dettmann ME (1996) Origin and diversification of the genus Nothofagus. In: The Ecology and Biogeography of Nothofagus forest (ed. Veblen TT, Hill RS, Read J) pp. 1124. Yale University Press, New Haven, CT. Hoiland K, Holst-Jensen A (2000) Cortinarius phylogeny and possible taxonomic implications of ITS rDNA sequences. Mycologia, 92, 694-710. Horak E, Mouchacca J (1998) Annotated checklist of New Caledonian Basidiomycota. I . Holobasidiomycetes. Mycotaxon, 68, 75-129. Horton R, Bruns TD (2001) The molecular revolution in ectomycorrhizal ecology: peeking into the black-box. Molecular Ecology, 10, 1855-1871. Huelsenbeck JP, Ronquist F, Nielsen R, Bollback JP (2001) Bayesian inference of phylogeny and its impact on evolutionary biology. Science, 294, 2310-4. Jaffré T (1992) Floristic and ecological diversity of the vegetation on ultramafic rocks in New Caledonia. In: The vegetation of ultramafic (serpentine) soils (ed. Baker AJM, Proctor J, Reeves RD) pp. 101-107. Intercept Ltd, Andover, NL. Jaffré T (1974) La végétation d'un massif de roches ultrabasiques de Nouvelle-Calédonie: le Koniambo. Candollea, 29, 427-456. Jaffré T, Morat P, Veillon JM, Rigault F, Dagostini G (2001) Composition et caractérisation de la flore indigène de Nouvelle-Calédonie. (ed. IRD Centre de Noumea) Documents Scientifiques et Techniques vol. II 4. IRD Nouméa. Jentschke G, Godbold DL (2000) Metal toxicity and ectomycorrhiza. Physiologia Plantarum, 109, 107-116. Landeweert R, Leeflang P, Kuyper TW, Hoffland E, Rosling A, Wernars K, Smit E (2003) Molecular identification of ectomycorrhizal mycelium in soil horizons. Applied Environmental Microbiology, 69, 327-333. Malajczuk N, Dell B, Bougher NL (1987) Ectomycorrhiza formation in Eucalyptus IIIsuperficial ectomycorrhizas initiated by Hysterangium and Cortinarius species. New Phytologist, 105, 421-428. McKenzie EHC, Buchanan PK, Johnston PR (2000) Checklist of fungi on Nothofagus species in New Zealand. New Zealand Journal of Botany, 38, 635-720. Moncalvo JM, Vilgalys R, Redhead SA, et al. (2002) One hundred and seventeen clades of euagarics. Molecular Phylogenetics and Evolution, 23, 357-400. Moyersoen B, Becker P, Alexander IJ. 2001. Are ectomycorrhizas more abundant than arbuscular mycorrhizas in tropical heath forests? New Phytologist, 150, 591-599.

The Mycorrhizal Symbiosis in Mediterranean Environment: Importance in Ecosystem Stability and in Soil Rehabilitation Strategies : Importance in

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

Ectotrophic Mycorrhizal Symbioses Are Dominant …

47

Murrill WA (1912) Agaricaceae of tropical North America. Mycologia, 4, 72-83. Natarajan K, Senthilarasu G, Kumaseran V, Rivière T (2005) Diversity in ectomycorrhizal fungi of a dipterocarp forest in Western Ghats. Current Science, 88, 1893-1895. Nicholas KB, Nicholas BJ (1997) Genedoc: a tool for editing and annotating multiple sequence alignments. Distributed by the author. Onguéné NA, Kuyper TW (2001) Mycorrhizal associations in the rainforest of South Cameroon. Forest Ecology and Management, 40, 277-287. Peintner U, Bougher N, Castellano M et al. (2001) Multiple origins of sequestrate fungi related to Cortinarius (Cortinariaceae). American Journal of Botany, 88, 177-184. Peintner U, Moser MM, Thomas KA, Manimohan P (2003) First record of ectomycorrhizal Cortinarius species (Agaricales, Basidiomycetes) from tropical India and their phylogenetic position based on rDNA ITS sequences. Mycological Research, 107, 485494. Peintner U, Moncalvo JM, Vilgalys R (2004) Toward a better understanding of the infrageneric relationships in Cortinarius (Agaricales, Basidiomycota). Mycologia, 96, 1042-1058. Perrier N, Ambrosi JP, Colin F, Gilkes RJ (2006a) Biogeochemistry of a regolith: The New Caledonian Koniambo ultramafic massif. Journal of Geochemical Exploration, 88, 5458. Perrier N, Amir H, Colin F (2006b) Occurrence of mycorrhizal symbioses in the metal rich lateritic soils of the Koniambo Massif, New Caledonia. Mycorrhiza, 16, 449-458. Posada D, Crandall KA (1998) Modeltest: testing the model of DNA substitution. Bioinformatics, 14, 817-818. Read J, Hallam P, Cherrier JF (1995) The anomaly of mono-dominant tropical rainforests: some preliminary observations in the Nothofagus-dominated rainforests of New Caledonia. Journal of Tropical Ecology, 11, 359-389. Rivière T, Diédhiou AG, Diabaté M et al. (2007) Genetic diversity of ectomycorrhizal Basidiomycetes from African and Indian tropical rainforests. Mycorrhiza, 17, 415-428. Ronquist F, Huelsenbeck JP (2003) MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics, 19, 1572-4. Sawyer NA, Chambers SM, Cairney JWG (1999) Molecular investigation of genet distribution and genetic variation of Cortinarius rotundisporus in eastern Australian sclerophyll forests. New Phytologist, 142, 561-568. Shimodaira H, Hasegawa M (1999) Multiple Comparisons of Log-Likelihoods with Applications to Phylogenetic Inference. Molecular Biology and Evolution, 16, 11141116. Singer R, Araujo I, Ivory MH (1983) The ectotrophically mycorrhizal fungi of the neotropical lowlands, especially Central Amazonia. Beiheft zur Nova Hedwigia, 77, 1-339. Slankis V (1974) Soil factors influencing formation of mycorrhizae. Annual Review of Phytopathology, 12, 437-457. Smith SE, Read DJ (1997) Mycorrhizal Symbiosis, 2nd Edition. Academic Press, London. Stahl PD, Williams SE, Christensen M (1988) Efficacy of native vesicular-arbuscular mycorrhizal fungi after severe soil disturbance. New Phytologist, 110, 347-354. Swenson U, Backlund A, McLoughlin S, Hill RS (2001a) Nothofagus biogeography revisited with special emphasis on the enigmatic distribution of subgenus Brassospora in New Caledonia. Cladistics, 17, 28-47.

The Mycorrhizal Symbiosis in Mediterranean Environment: Importance in Ecosystem Stability and in Soil Rehabilitation Strategies : Importance in

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

48

Y. Prin, M. Ducousso, J. Tassin et al.

Swenson U, Hill RS, McLoughlin S (2000) Ancestral area analysis of Nothofagus (Nothofagaceae) and its congruence with fossil record. Australian Systematic Botany, 13, 469-478. Swenson U, Hill RS, McLoughlin S (2001b) Biogeography of Nothofagus supports the sequence of Gondwana break-up. Taxon, 50, 1025-1041. Tedersoo L, Jairus T, Horton BM et al. (2008) Strong host preference of ectomycorrhizal fungi in a Tasmanian wet schlerophyll forest as revealed by DNA barcoding and taxonspecific primers. New Phytologist, 180, 479-490. The International Plant Index (2008) Published on the Internet http://www.ipni.org [accessed 20 October 2008]. Thioulouse J, Chessel D, Dolédec S, Olivier JM (1997) ADE4: a multivariate analysis and graphical display software. Statistics and Computing, 7, 75-83. Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG (1997) The clustal-X windows interface: flexible strategies for multiple sequence alignment aided by quality tools. Nucleic Acids Research, 25, 4876-4882. Torti SD, Coley PD (1999) Tropical monodominance: A preliminary test of the ectomycorrhizal hypothesis. Biotropica, 31, 220-228. Valenzuela E, Moreno G, Garnica S, Godoy R, Ramirez C (1999) Mycosociology in native forests of Nothofagus of the X region of Chile, diversity and ecological role. Mycotaxon, 72, 217-226. Veevers JJ (1986) Breakup of Australia and Antarctica estimated as Mid-Cretaceous (95±5 Ma) from magnetic and seismic data at the continental margin. Earth and Planetary Science Letters, 77, 91-99. Veevers JJ, Powell CM, Roots SR (1991) Review of seafloor spreading around Australia. 1. Synthesis of the patterns of spreading. Australian Journal of Earth Science, 38, 373-389. Villeneuve NM, Grandtner M, Fortin JA (1989) Frequency and diversity of ectomycorrhizal and saprophytic macrofungi in the Laurentide Moutains of Quebec. Canadian Journal of Botany, 67, 2616-2629. White TJ, Bruns T, Lee S, Taylor J (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: PCR protocols: A guide to methods and applications (ed. Innis MA, Gelfand H, Sninsky JS, White TJ) pp.185-189. Academic Press, New York.

The Mycorrhizal Symbiosis in Mediterranean Environment: Importance in Ecosystem Stability and in Soil Rehabilitation Strategies : Importance in

In: The Mycorrhizal Symbiosis in Mediterranean Environment ISBN: 978-1- 62081-278-5 Editors: Mohamed Hafidi and Robin Duponnois © 2012 Nova Science Publishers, Inc.

Chapter III

Mycorrhizal Status and Arbuscular Mycorrizal Fungal Diversity of Endangered Plant Species in the Sierra Nevada National Park Concepción Azcón-Aguilar,1 Javier Palenzuela,1 Nuria Ferrol,1 Fritz Oehl2 and José Miguel Barea1

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

1

Departamento de Microbiología del Suelo y Sistemas Simbióticos Estación Experimental del Zaidín, CSIC, Granada, Spain 2 Agroscope Reckenholz-Tänikon Research Station ART Ecological Farming Systems, Zürich, Switzerland

1. Abstract Mycorrhizas have played a key role in plant evolution on Earth as well as on the development of the structure and diversity of terrestrial ecosystems. Most plants depend on mycorrhizas to thrive, particularly in fragile and stressed environments, as those in certain areas of the high Mediterranean mountains of the Sierra Nevada National Park (Granada, Spain). Sierra Nevada constitutes an exceptional refuge for the flora and one of the enclaves with higher biodiversity levels of the European continent. It presents about 2100 plant species and 80 exclusive endemisms, some of them threatened with extinction. With the objective of ascertaining the impact of mycorrhizal associations at facilitating the conservation of species from the threatened flora of Sierra Nevada a research programme was initiated aiming at (i) determining the mycorrhizal status of selected species of the endangered flora of Sierra Nevada, (ii) analysing the diversity of the mycorrhizal fungi associated with the selected species, and (iii) establishing a mycorrhizal fungi germplasm bank. Thirty four plant species belonging to 22 different botanical families were selected. All of them belong to the endangered and/or endemic flora of Sierra Nevada. The results showed that six out of the 34 selected species had no mycorrhizal colonization. All the other 28 species (about 80 % of the studied plant species) showed arbuscular mycorrhizal (AM) colonization and one of them (Salix hastata) also ectomycorrhizas. In most mycorrhizal plants, the typical structures

The Mycorrhizal Symbiosis in Mediterranean Environment: Importance in Ecosystem Stability and in Soil Rehabilitation Strategies : Importance in

50

Concepción Azcón-Aguilar, Javier Palenzuela, Nuria Ferrol et al. characteristic of the AM symbiosis, mainly arbuscules and vesicles, could be observed. Arum, Paris and intermediate type AM were detected, produced by the colonization with coarse endophytes in most cases, although fine endophytes were also evident in some plant species. Approximately one third of the studied plants were colonised by dark septate endophytes. The density of AM fungal spores in the soil around the selected plants was relatively low, except for certain plant species, such as the fern Ophioglossum vulgatum. By using morphological and molecular criteria most of these spores were identified up to species level. AM fungal species richness was, however, quite high. More than 60 different AM fungal species were detected, belonging to the genera Glomus, Acaulospora, Entrophospora, Gigaspora, Scutellospora, Pacispora, Diversispora, Ambispora and Paraglomus. The most frequent genera in Sierra Nevada are Glomus and Acaulospora. Acaulospora species are mainly found at the highest altitudes and in acidic soils, being A. laevis the most common species. Glomus species predominate at lower altitudes and in neutral and alkaline soils, with G. constrictum and G. etunicatum as the most frequent species. A new AM fungal species (Entrophospora nevadensis) was described recently. Some other spore types do not correspond to any of the species described so far. A germplasm collection of autochthonous AM fungi from Sierra Nevada has been established with the isolated fungi that could be grown as monospecific cultures. This collection is being used to ascertain the effect of a tailored mycorrhizal inoculation, with autochthonous mycorrhizal fungi, on the nursery production of target plants to be reintroduced in their natural habitats according to the conservation initiatives in the National Park.

Keywords: Arbuscular mycorrhiza (AM), dark septate endophytes (DSE), endangered plant species, endemisms, Glomeromycota, high Mediterranean mountains, mycorrhizal fungi

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

2. Introduction The Mediterranean Basin has been declared one of the 25 biodiversity hot-spots in the world. In these 25 diversity hotspots live a 44 % of the plant species of the Planet when the area is 1.4 % de total Earth surface (Myers et al. 2000). Sierra Nevada (the Spanish name for snowy range) is a rugged and extensive mountain range, included in the Baetic Cordillera and located in south-western Europe (Andalucía, provinces of Granada and Almería, Spain). It constitutes the most southern mountain range of the European continent, having not only the highest points of continental Spain (more than 20 peaks above 3000 m), but also the highest of continental Europe excluding the Alps. It was designated a UNESCO Biosphere Reserve in 1986 and a National Park in 1999 in recognition of its exceptionally high plant and animal diversity. High mountains often bear plant diversities richer than those in their surrounding lowlands, including large numbers of threatened species (Körner 2003; Grabherr et al. 2003). This is especially true in the case of the Sierra Nevada. Sierra Nevada constitutes an exceptional refuge for the flora and one of the enclaves with higher biodiversity levels of the European continent. It hosts about 2100 plant species (7 % of the Mediterranean region flora, with an area lower to the 0.01%) and about 80 exclusive endemisms, many of them in serious extinction danger (Blanca et al. 1998, 2002). In the highest peaks, 30-40 % of the flora is exclusive to Sierra Nevada, while in some ecological niches, such as on rocky ground or screes, the proportion of endemic species can be as high as 80% (Blanca et al. 1998).

The Mycorrhizal Symbiosis in Mediterranean Environment: Importance in Ecosystem Stability and in Soil Rehabilitation Strategies : Importance in

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

Mycorrhizal Status and Arbuscular Mycorrizal Fungal Diversity ...

51

This high plant diversity is due to its geographic isolation and abruptness of their ecological gradients with large altitudinal ranks thus creating a diversity of ecological niches (Blanca et al. 2002). It contains five of the six bioclimatic belts identified for the Mediterranean region (according to the classification of Rivas Martínez 1990), making it the only mountain range in the southern half of the Iberian Peninsula to contain the upper bioclimatic level (cryoro-Mediterranean) or the nival zone, according to the definitions adopted for alpine areas in Europe (Grabherr et al. 2003), where temperatures of -20°C or even lower are commonly recorded. This is in sharp contrast with the mild climate which characterises the rest of southern Spain. Thus, the highest areas of the Sierra Nevada currently behave as a biological island (Blanca et al. 1998). The high altitudes together with its southern situation mean that plants from a number of different origins (from central and northern Europe to Africa and south-western Asia) are currently found in Sierra Nevada (Blanca et al. 1998). Additionally Sierra Nevada was identified as the enclave with the largest number of threatened taxa in Peninsular and Balearic Spain (Domínguez Lozano et al. 1996). In high mountain habitats, plants have developed numerous adaptations imposed by the harsh conditions derived from the increase in altitude (low temperatures, intense winds, short growing season, nutrient-poor soils, etc.). These adaptations are manifested in different life strategies and physiological processes. Adaptive strategies can also involve the establishment of symbiotic association with mycorrhizal fungi, and several types of mycorrhizas have been described in high altitude plant communities (Körner 2003). Mycorrhizal simbioses have played a key role in plant evolution on Earth (Simon et al. 1993; Redecker et al. 2000) as well as in the development and maintenance of the structure and diversity of terrestrial ecosystems (van der Heijden et al. 1998). This is mainly due to the ability of mycorrhizal fungi to increase plant fitness and soil quality (Smith and Read 2008). Actually, mycorrhiza formation provides the plant with an increased ability for nutrient capture and cycling in soils with low nutrient availability. In addition, mycorrhizal symbioses improve plant protection against environmental stresses, either biotic or abiotic, and enhance soil structure through the formation of hydro-stable aggregates necessary for good soil tilth (Barea et al. 2011). Most plants depend on mycorrhizas to thrive, particularly in fragile and stressed environments as those in certain areas of the high Mediterranean mountains of Sierra Nevada National Park (Smith et al. 2010). Little information is available on the mycorrhizal status of the dominant plant species in alpine plant communities. Pioneer studies were carried out in the Austrian Alps by Haselwandter (Haselwandter and Read 1980; Read and Haselwandter 1981; Haselwandter 1987) and afterwards by other authors in Mt. Paras (northern Norway) (Ruotsalainen et al. 2004), the Rocky Mountains (USA) (Cripps and Eddington 2005; Schmidt et al. 2008), the Andes (Schmidt et al. 2008), the Pennsylvania Mountains (Becklin and Galen 2009), the Tatra Mountains (Western Carpathians) (Zubek et al. 2009a) and the Swiss Alps (Oehl et al. 2011). The general conclusion of these studies is that although mycorrhizal colonization overall decreases with increasing altitude, arbuscular mycorrhizal (AM) associations and dark septate endophytes (DSE) are present even in the highest habitats (Haselwandter and Read 1980; Read and Haselwandter 1981; Ruotsalainen et al. 2004; Zubek et al. 2009a). Endangered plant species are threatened by human exploitations, alterations in their natural habitats or environmental changes. Especially global warming may result in the loss of species, particularly the high mountain species. It is well known that the in situ and ex situ conservation of endangered plant species has ecophysiological constraints which difficult

The Mycorrhizal Symbiosis in Mediterranean Environment: Importance in Ecosystem Stability and in Soil Rehabilitation Strategies : Importance in

52

Concepción Azcón-Aguilar, Javier Palenzuela, Nuria Ferrol et al.

regeneration in their natural habitats. One of these constraints can be mycorrhiza formation. In fact, AM colonization can affect vegetative and sexual reproduction of plants by impacting on the number of inflorescences, fruit and seed production, and offspring vigour (Koide and Dickie 2002; Bothe et al. 2010). Several research programs are being developed to conserve threatened plant species in Sierra Nevada, but little effort has been directed so far to document and preserve mycorrhizal symbionts that co-exist with plant communities in the area. Due to the importance of mycorrhizal symbioses for plant establishment and development in stress conditions, and since information concerning the mycorrhizal status of endangered plants is of major importance for their potential re-establishment (Fuchs and Haselwandter 2004; Zubek et al. 2008; 2009b; Bothe et al. 2010), a research project aiming at (i) determining the mycorrhizal status of selected species of the endangered and/or endemic flora of Sierra Nevada, (ii) analysing the diversity of mycorrhizal fungi associated to the selected species, and (iii) establishing a germplasm bank of autochthonous AM fungi from Sierra Nevada was initiated. The final aim of the study is to check the effect of a tailored mycorrhizal inoculation with specific mixes of suitable, autochthonous AM fungi, on the nursery production of target plant species to facilitate the successful re-establishment of the endangered plants in their natural habitats in the context of the corresponding conservation programs.

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

3. Selection and Sampling of the Target Plant Species The mycorrhizal status and the AM fungi present as spores in the soil around their roots were investigated for 34 plant species belonging to 22 different botanical families, either endemic to the Sierra Nevada or threatened with extinction according to the Red List of Endangered Plant Species of Andalucía (Blanca et al. 1999, 2000) and the compilation of Threatened and Endemic Flora of Sierra Nevada (Blanca et al. 2002). The selected plant species, their geographical distribution and the conservation status in Sierra Nevada are recorded in Table 1. Twenty-one of the selected species are endemic to the Sierra Nevada and all of them, with the exception of Plantago nivalis, present a certain degree of extinction threat in Sierra Nevada according to the criteria established by the UICN (1994). For each plant species five individual plants from the same location were sampled. Each sample consisted of root fragments and the surrounding soil from a depth of 5–25 cm. The root samples were used to determine the mycorrhizal status of the plant and the soil samples to isolate AM fungal spores and to determine soil characteristics. Soil pH, organic matter and essential macronutrient content are shown in Table 2. In accordance with the high diversity of ecological niches in Sierra Nevada there is a high variability in soil types. With some exceptions corresponding to soils around plants growing close to streams or in wet meadows (mainly those of O. vulgatum, N. nevadensis, S. hybrida and S. elodes), most of them are stony soils and screes, poor in organic matter and with very low fertility level, as it is usual at high mountain ecosystems (Billings 1979). In Sierra Nevada there are two well differentiated soil environments according to the nature of the parent rock. The central core of the massif, which includes the highest peaks, is formed by siliceous materials composed mainly of mica-schists. This central core is partially surrounded

The Mycorrhizal Symbiosis in Mediterranean Environment: Importance in Ecosystem Stability and in Soil Rehabilitation Strategies : Importance in

Mycorrhizal Status and Arbuscular Mycorrizal Fungal Diversity ...

53

by a calcareous belt mainly composed of limestones and dolomites. Soils corresponding to plants growing at higher altitudes belong to the central siliceous nucleus of Sierra Nevada and had usually acidic or neutral pH, while those at lower altitudes were alkaline soils. Consequently, according to the ecology of the different target species there were also important differences in pH and Ca and Mg content of the soils around them. Table 1. Target plant species, geographical distribution and their conservation status in Sierra Nevada Family

Species

Ophioglossaceae Aspidiaceae Ranunculaceae Papaveraceae Fumariaceae Caryophyllaceae Plumbaginaceae

Ophioglossum vulgatum Dryopteris tyrrhena Delphinium nevadense Papaver lapeyrousianum Sarcocapnos speciosa Arenaria nevadensis Armeria filicaulis subsp. trevenqueana Salix hastata subsp. sierrae-nevadae Sempervivum tectorum Sibbaldia procumbens Sorbus hybrida Sorbus torminalis Alchemilla fontqueri Chamaespartium undulatum Hippocrepis nevadensis Hippocrepis prostrata Ilex aquifolium Erodium astragaloides Erodium boissieri Erodium daucoides Laserpitium longiradium Gentiana sierrae Plantago nivalis Odontites granatensis Pinguicola grandiflora Pinguicola nevadensis Scabiosa pulsatilloides Artemisia alba subsp. nevadensis Artemisia granatensis Artemisia umbelliformis Erigeron frigidus Senecio elodes Avenula laevis Narcissus nevadensis

Salicaceae Crassulaceae Rosaceae

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

Leguminosae

Aquifoliaceae Geraniaceae

Umbelliferae Gentianaceae Plantaginaceae Scrophulariaceae Lentibulariaceae Dipsacaceae Compositae

Graminae Amaryllidaceae

Geographical distribution1

An-endemic SN-endemic SN-endemic SN-endemic SN-endemic

SN-endemic SN-endemic SN-endemic SN-endemic SN-endemic SN-endemic SN-endemic SN-endemic SN-endemic SN-endemic SN-endemic SN-endemic SN-endemic An-endemic SN-endemic SN-endemic SN-endemic SN-endemic An-endemic

IUCN category in SN2 EN CR VU EN VU CR EN CR VU VU CR EN CR VU VU VU EN VU VU VU CR VU CR VU VU VU VU CR EN VU CR VU CR

1

Geographical distribution: SN-endemic = endemism exclusive from Sierra Nevada; An-endemic = exclusive endemism from Andalucía. 2 IUCN categories of threats in Sierra Nevada: CR: Critically Endangered; EN: Endangered; VU: Vulnerable.

The Mycorrhizal Symbiosis in Mediterranean Environment: Importance in Ecosystem Stability and in Soil Rehabilitation Strategies : Importance in

54

Concepción Azcón-Aguilar, Javier Palenzuela, Nuria Ferrol et al.

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

Table 2. Characteristics of the soils where the target plant species develop Plant species

pH (H2O)

Ophioglossum vulgatum Dryopteris tyrrhena Delphinium nevadense Papaver lapeyrousianum Sarcocapnos speciosa Arenaria nevadensis Armeria filicaulis ssp. trevenqueana Salix hastata subsp. sierrae-nevadae Sempervivum tectorum Sibbaldia procumbens Sorbus hybrida Sorbus torminalis Alchemilla fontqueri Chamaespartium undulatum Hippocrepis nevadensis Hippocrepis prostrata Ilex aquifolium Erodium astragaloides Erodium boissieri Erodium daucoides Laserpitium longiradium Gentiana sierrae Plantago nivalis Odontites granatensis Pinguicola grandiflora Pinguicola nevadensis Scabiosa pulsatilloides Artemisia alba subsp. nevadensis Artemisia granatensis Artemisia umbelliformis Erigeron frigidus Senecio elodes Avenula laevis Narcissus nevadensis

6.5 5.6 6.8 6.6 6.9 6.5 7.8 6.7 5.7 6.4 6.0 7.1 6.5 7.6 7.8 6.1 6.2 7.9 7.9 7.3 7.2 6.9 5.1 7.7 6.7 5.9 7.3 7.9 6.0 6.2 6.8 5.8 5.6 6.4

Organic matter (%) 52.05 10.95 6.62 0.84 8.29 0.93 6.28 10.84 6.91 1.56 22.71 4.50 15.18 10.88 4.40 5.08 5.75 10.11 5.96 8.33 3.25 10.56 6.59 1.83 12.29 3.28 5.02 2.54 0.81 1.89 1.51 19.96 7.72 19.96

N (%)

P (%)

K (%)

Ca (%)

Mg (%)

1.56 0.44 0.36 0.03 0.49 0.04 0.06 0.47 0.51 0.05 0.91 0.27 0.71 0.12 0.18 0.28 0.30 0.04 0.09 0.37 0.20 0.55 0.36 0.13 0.60 0.16 0.07 0.19 0.11 0.13 0.07 0.92 0.38 1.34

0.04 0.04 0.06 0.06 0.13 0.03 0.02 0.03 0.05 0.03 0.02 0.07 0.06 0.02 0.02 0.03 0.04 0.01 0.02 0.06 0.02 0.06 0.07 0.04 0.07 0.04 0.01 0.02 0.05 0.09 0.06 0.07 0.06 0.04

0.21 0.40 0.88 0.51 0.80 0.30 0.05 0.42 0.71 0.42 0.64 0.48 0.46 0.09 0.41 0.78 0.42 0.02 0.04 1.13 1.21 1.06 0.55 1.22 0.58 0.77 0.06 0.49 0.49 0.37 0.69 0.43 0.96 0.46

0.61 0.44 0.93 0.69 0.80 0.11 15.72 0.38 0.50 0.16 0.35 0.22 1.07 20.37 20.62 0.24 0.21 22.03 15.70 16.19 1.45 1.02 0.30 5.88 0.83 0.27 23.94 18.86 0.12 0.34 0.21 0.35 0.24 1.18

0.35 0.58 0.62 1.06 0.85 0.55 12.75 0.55 0.36 0.61 0.52 0.09 0.69 10.95 9.50 0.37 0.36 11.74 4.99 5.48 1.29 0.62 0.70 1.75 0.53 0.65 13.18 1.42 0.32 0.70 0.63 0.40 0.45 0.43

It is noteworthy the high Ca content of soils around species characteristics of calcareous substrates, such as Artemisia alba subsp. nevadensis, or the high Ca and Mg content of those around plants from dolomitic lands, such as Scabiosa pulsatilloides, Armeria filicaulis subsp. trevenqueana, Erodium astragaloides or Chamaespartium undulatum. To multiply the AM fungi trap cultures were established using the soil collected around the plants grown in their natural environment. Those AM fungi that proliferate in the trap cultures were multiplied in monospecific cultures. Monospecific cultures were established in pots containing a mixture of steam-sterilized soil, vermiculite and sand (2:1:1, v:v) by adding 20 apparently healthy spores isolated from the trap cultures. The isolated AM fungi were characterized by using a combination of morphological and molecular methods. The morphological characterization was mainly based on the observation of spore formation and the morphology of spores. The molecular methods were based on the

The Mycorrhizal Symbiosis in Mediterranean Environment: Importance in Ecosystem Stability and in Soil Rehabilitation Strategies : Importance in

55

Mycorrhizal Status and Arbuscular Mycorrizal Fungal Diversity ...

amplification by nested PCR of a portion of the 18S small subunit ribosomal gene (Palenzuela et al. 2010). Table 3. Mycorrhizal status and colonization by dark septate endophytes (DSE) of the target plant species in the Sierra Nevada National Park

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

Plant species Ophioglossum vulgatum Dryopteris tyrrhena Delphinium nevadense Papaver lapeyrousianum Sarcocapnos speciosa Arenaria nevadensis Armeria filicaulis subsp. trevenqueana Salix hastata subsp. sierrae-nevadae Sempervivum tectorum Sibbaldia procumbens Sorbus hybrida Sorbus torminalis Alchemilla fontqueri Chamaespartium undulatum Hippocrepis nevadensis Hippocrepis prostrata Ilex aquifolium Erodium astragaloides Erodium boissieri Erodium daucoides Laserpitium longiradium Gentiana sierrae Plantago nivalis Odontites granatensis Pinguicola grandiflora Pinguicola nevadensis Scabiosa pulsatilloides Artemisia alba subsp. nevadensis Artemisia granatensis Artemisia umbelliformis Erigeron frigidus Senecio elodes Avenula laevis Narcissus nevadensis 1

Mycorrhizal status NM AM1 ++ +++ +    + +  + + + +++ + +++ +++ +++ + +++ +++ +++ + +++ +++   +++ +++ ++ + + +++ +++ +++

DSE1 ECM

1

+

+

+++

++ + + + +

+ +

+

+

From (+) some colonization (1-10%) to (+++) heavily colonized (> 50%).

4. Mycorrhizal Status of the Target Plant Species The results showed that six out of the 34 selected species had no mycorrhizal colonization: Papaver lapeyrousianum, Sarcocapnos speciosa, Arenaria nevadensis, Sempervivum tectorum, Pinguicola grandiflora and Pinguicola nevadensis (Table 3). Although there are no previous records on the mycorrhizal status of most of these plant

The Mycorrhizal Symbiosis in Mediterranean Environment: Importance in Ecosystem Stability and in Soil Rehabilitation Strategies : Importance in

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

56

Concepción Azcón-Aguilar, Javier Palenzuela, Nuria Ferrol et al.

species, their non-mycorrhizal status was expected since they belong to botanical families composed of non-mycotrophic species. This is the case of Arenaria nevadensis (Caryophyllaceae), Sempervivum tectorum (Crassulaceae) and the two Pinguicola species which are carnivorous bog plants belonging to the Lentibulariaceae (Harley and Harley 1987; Cripps and Eddington 2005; Brundrett 2009). Papaver lapeyrousianum and Sarcocapnos speciosa belong to families (Papaveraceae and Fumariaceae) reported to have a majority of non-mycorrhizal species, although some mycotrophic species have also been described (Harley and Harley 1987; Wijesinghe et al. 2001; Brundrett 2009). One of the studied species (Salix hastata subsp. sierrae-nevadae) showed simultaneously ecto- and AM colonization (Table 3, Figure 1). The level of root colonization by AM fungi was low and it was limited to some coils, intercellular hyphae and vesicles. Even though most plants form only one type of mycorrhiza, the ability of the Salix species to establish both ecto- and arbuscular mycorrhizas has widely been reported (Dhillion 1994; van der Heijden 2001; Trowbridge and Jumpponen 2004; Becerra et al. 2009). All the other 27 species (approximately 80% of the studied plant species) showed AM colonization (Table 3). Our results agree with those of Read and Haselwandter (1981). These authors studied mycorrhizal associations in some Austrian alpine plant communities. They concluded that more than half of the observed plant species had typical AM colonization. In most AM plants, the typical structures characteristic of the AM symbiosis, mainly arbuscules, vesicles and coils, could be observed (Figure 1). Arum, Paris and intermediate AM structures were detected, produced by the colonization with coarse endophytes in most cases, although fine endophytes were also evident in one-third of the AM plant species, in some cases co-existing with coarse endophytes. Fine endophytes seem to be well adapted to cold and harsh climatic conditions. An increase in their relative presence colonizing mycorrhizal plants as altitude increases has been described in the Austrian Alps (Read and Haselwandter 1981), the Tatra mountains (Zubek et al. 2009), and at high latitudes in polar regions (Olsson et al. 2004; Ruotsalainen et al. 2004; Newsham et al. 2009). All these results suggest that fine endophytes play a key role, though still largely unknown, in the fitness of artic and alpine plants. As reported for most ferns in other parts of the world (Iqbal et al. 1981; Zhang et al. 2004), the studied pteridophytes from Sierra Nevada were colonized by AM fungi but fungal morphology in these plant species differed from the common pattern found in most AM associations. Ophioglossum vulgatum sporophytes exhibited Paris-type AM colonization, as it has been previously reported for this species in other environments (Harley and Harley 1987; Zhang et al. 2004). However, arbuscules had a coralloid shape with large vesicular swellings, similar to those reported for other ophioglossaceous species (Schmid and Oberwinkler 1996). Dryopteris tyrrhena sporophytes were also colonized by AM fungi. As far as we know, this is the first description of mycorrhizal colonization in this fern. Other Dryopteris species and members of the Aspidiaceae family have been reported to form arbuscular mycorrizas (Harley and Harley 1987; Turnau et al. 1999; Reyes-Jaramillo et al. 2008). Dryopteris tyrrhena showed an unusual Paris-type mycorrhizal colonization, with cells colonized simultaneously by coils and vesicles, vesicles and arbuscules or several vesicles (Figure 1).

The Mycorrhizal Symbiosis in Mediterranean Environment: Importance in Ecosystem Stability and in Soil Rehabilitation Strategies : Importance in

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

Mycorrhizal Status and Arbuscular Mycorrizal Fungal Diversity ...

57

Figure 1. Mycorrhizal structures of the target plant species. a: Arbuscules of coralloid shape in roots of Ophioglossum vulgatum. b: Intracellular coils and vesicles of Dryopteris tyrrhena. c: Coils of AM fungi in roots of Salix hastata. d: Ectomycorrhiza of S. hastata. e: Arbuscules in roots of Armeria filicaulis. f: Arbuscule-containing cell of Alchemilla fontqueri. g: Intracellular vesicles of Chamaespartium undulatum. h: Arum-type colonization of Hippocrepis prostrata. i: Paris-type colonization of Ilex aquifolium, with hyphae spreading from cell-to-cell. j: Coexistence of fine and coarse endophytes in roots of Erodium boissieri. k) Colonization of Erodium daucoides with arbuscules and lipid droplets filled vesicles. l: Arbuscules-containing cells in roots of Odontites granatensis. m: Coils of Scabiosa pulsatilloides. n: Appressorium on roots of Artemisia alba. o: Round-shaped vesicle of Artemisia granatensis. p: Ovoid and irregularly shaped vesicles of Artemisia umbelliformis. q: Colonization by AM fungi and DSE of Erigerum frigidus. r: Intensive root colonization by fine endophytes of Senecio elodes. s: Avenula laevis root cells showing coils and arbuscules. t: Coiled hyphae in root epidermal cells of Narcissus nevadensis. Note the cell membrane surrounding the coiled hyphae in b, s and t.

The Mycorrhizal Symbiosis in Mediterranean Environment: Importance in Ecosystem Stability and in Soil Rehabilitation Strategies : Importance in

58

Concepción Azcón-Aguilar, Javier Palenzuela, Nuria Ferrol et al.

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

Figure 2. Dark septate endophyte (DSE) colonization of some of the target plant species. Melanized septate hyphae (a, c, d, e and f) and microsclerotia (b, c and e) are observed.

As shown in previous studies on other alpine plant communities (Read and Haselwandter 1981), nearly one third of the plants were colonised by DSE. These fungi, which are not considered as true mycorrhizal symbionts (Smith and Read 2008), were indistinctly detected in roots of non-mycorrhizal plants or in those of plants colonized by ecto- and/or AM fungi. However, root colonization by this group of endophytes was generally low (Table 3, Figure 2). DSE have been repeatedly described in alpine plants (Read and Haselwandter 1981; Gardes and Dahlberg 1996; Ruotsalainen et al. 2002, 2004; Schmidt et al. 2008; Zubek et al. 2009a), but little is known about their ecological significance and phylogenetic identities (Rodriguez et al. 2009). Several studies suggest beneficial effects of DSE on nutrient uptake (N and P, especially from recalcitrant or complex organic sources), growth and survival of their host plants in natural habitats (Mandyam and Jumpponen 2005; Newsham et al. 2009; Newsham 2011) and mainly in cold- and water-stressed environments as those in certain areas of in the high mountains of Sierra Nevada.

5. AMF Diversity in the Rhizosphere of the Target Plant Species The density of AM fungal (AMF) spores in the soil around the selected plants was low, usually less than 200 spore per 100 g of soil (Table 4). However, the AMF spore densities generally increased in soils with high organic matter content, as it was the case of those supporting the growth of O. vulgatum. There are evidences supporting a positive effect of organic matter on AMF spore production (Gould et al. 1996; Quilliam et al. 2010).

The Mycorrhizal Symbiosis in Mediterranean Environment: Importance in Ecosystem Stability and in Soil Rehabilitation Strategies : Importance in

Mycorrhizal Status and Arbuscular Mycorrizal Fungal Diversity ...

59

Table 4. Spore density and species richness of arbuscular mycorrhizal fungi in the soil around the target plants

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

Plant species Ophioglossum vulgatum Dryopteris tyrrhena Delphinium nevadense Papaver lapeyrousianum Sarcocapnos speciosa Arenaria nevadensis Armeria filicaulis subsp. trevenqueana Salix hastata subsp. sierrae-nevadae Sempervivum tectorum Sibbaldia procumbens Sorbus hybrida Sorbus torminalis Alchemilla fontqueri Chamaespartium undulatum Hippocrepis nevadensis Hippocrepis prostrata Ilex aquifolium Erodium astragaloides Erodium boissieri Erodium daucoides Laserpitium longiradium Gentiana sierrae Plantago nivalis Odontites granatensis Pinguicola grandiflora Pinguicola nevadensis Scabiosa pulsatilloides Artemisia alba subsp. nevadensis Artemisia granatensis Artemisia umbelliformi Erigeron frigidus Senecio elodes Avenula laevis Narcissus nevadensis

AMF spore density (/100 g dry soil) 1188.8  305.6 54.4  23.4 217.2  40.5 86.0  11.4 70.0  18.0 4.4  1.6 72.8  18.0 82.8  22.3 63.2  25.1 52.8  35.1 27.2  10.1 54.0  12.4 173.6  71.3 56.0  13.3 64.0  17.1 90.4  8.0 142.4  34.8 64.8  11.1 46.0  7.6 53.6  12.6 30.8  6.6 183.3  47.8 482.7  38.1 131.6  44.2 429.0  145.1 115.2  38.3 8.0  1.7 187.2  81.2 19.6  8.4 50.8  49.3 465.2  240.7 352.0  102.8 92.4  12.2 275.2  177.9

AMF species richness 4.0  0.8 2.6  0.5 5.8  0.4 5.2  0.6 3.0  1.0 1.2  0.4 4.0  1.0 5.6  0.8 4.0  0.3 1.6  0.6 3.8  0.4 5.0  0.3 7.2  0.9 4.6  0.4 4.0  0.7 4.6  0.4 4.0  0.6 4.2  0.4 3.6  0.6 4.6  0.7 3.0  0.3 6.0  1.2 3.0  0.3 4.6  0.4 5.8  0.6 6.8  0.4 2.6  0.5 5.4  0.4 2.0  0.3 1.4  1.0 2.8  0.6 6.4  0.2 2.4  0.4 6.6  0.8

The low AMF spore densities found in most of the studied soils are similar to those described in the soil near the roots of representative plant species from desertificationthreatened Mediterranean shrublands (Azcón-Aguilar et al. 2003; Barea et al. 2011), but lower than those reported for other Mediterranean environments (Ouahmane et al. 2008; Rodríguez-Echevarría et al. 2008; Curaqueo et al. 2011). Especially low was the spore density found in the soil around A. nevadensis, a non-mycorrhizal plant growing on stony soils and screes at 3000 m asl, and S. pulsatilloides, a plant heavily colonized by mycorrhizal fungi but growing in dolomite sandy places, with very high levels of Ca and Mg. The low AMF spore

The Mycorrhizal Symbiosis in Mediterranean Environment: Importance in Ecosystem Stability and in Soil Rehabilitation Strategies : Importance in

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

60

Concepción Azcón-Aguilar, Javier Palenzuela, Nuria Ferrol et al.

density can probably be explained by the severe conditions (poor soils and harsh climate) prevailing in the habitats of both species. No significant differences were detected among the AMF spore densities in the soils around mycorrhizal and non-mycorrhizal plants. Probably, AMF in the soils near the nonmycorrhizal plants were symbiotically associated with plants accompanying the target species. Species richness ranged from a minimum of 1.2 AMF species for A. nevadensis and a maximum value of 7.2 for Alchemilla fontqueri (Table 4). In general, no correlation could be detected between spore density and species richness, and those plant species with a higher number of AMF spores in the soil around their roots were not the ones showing a higher AMF diversity. More than 60 different AMF morphotypes were detected, belonging to the genera Acaulospora, Ambispora, Diversispora, Entrophospora, Gigaspora, Glomus, Pacispora, Paraglomus and Scutellospora (Table 5). Most of these morphotypes have been identified up to species level by using morphological and molecular tools. It has been verified that some of the isolated morphotypes do not correspond to any of the species described so far. The most frequent genus in Sierra Nevada is Glomus, with approximately 30 different species isolated from the soil around the target plants, being G. constrictum and G. etunicatum the most common species (Table 5). These two species have been described among the most frequent in Mediterranean environments (Requena et al. 1996; AzcónAguilar et al. 2003; Ferrol et al. 2004; Alguacil et al. 2009) and could be considered as ubiquitous in Sierra Nevada since they were found in soils with different physico-chemical characteristics, at different altitudes and in the soil around diverse plant species. The next best represented AMF genus in Sierra Nevada is Acaulospora, with more than 10 species detected, being the most frequent A. laevis (Table 5). Acaulospora species are usually found in acid soils (Morton 1986; Sieverding 1991; Moutoglis and Widden 1996; Clark 1997; Castillo et al. 2006). This fact has been corroborated in the present study since, with the exception of A. thomii, present in the rhizosphere of plants grown in alkaline soils, all other Acaulospora species were detected in acid soils, where they usually constituted the best represented genus. According to this, Acaulospora is the dominant genus in the soils around plants living in the nival zone (cryoro-Mediterranean bioclimatic level), as these are growing in base-poor soils, on mica-schist litological substrates. As altitude decreases, what usually implies an increase in soil pH in Sierra Nevada, Acaulospora species decrease their relative frequency and increase the presence of Glomus species and, to a lower extent, those of Ambispora, Pacispora and of the only species detected of Scutellospora, S. calospora. Acaulospora alpina, a species that up to know has only be found in the high mountains of the Swiss Alps (Oehl et al. 2006) and Chilean Andes (Castillo et al. 2006), has also been found in Sierra Nevada soils. It is noteworthy that it is in the Acaulospora genus where there are apparently more new species, whose characteristics do not correspond to any of the species previously recorded in the scientific literature. As it happens to the flora, it seems that also at the AMF level it is in the highest peaks where there can be higher rates of endemic species. However, this may also reflect the fact that in higher altitudes, where Acaulospora species have increasingly been found, hitherto AMF diversity studies have rarely been performed (Oehl et al. 2011a).

The Mycorrhizal Symbiosis in Mediterranean Environment: Importance in Ecosystem Stability and in Soil Rehabilitation Strategies : Importance in

61

Mycorrhizal Status and Arbuscular Mycorrizal Fungal Diversity ... Table 5. Arbuscular mycorrhizal fungi found in Sierra Nevada and frequency of detection Genus

Species

Acaulospora

alpina elegans laevis longula mellea morrowiae paulinae scrobiculata spinosa thomii sp. nov.1 sp. nov.2 sp1 sp2 gerdemanii fennica sp1 sp2 sp1 sp2 sp3 sp4 baltica infrequens nevadensis sp1 margarita

Number of locations1 6 6 13 4 10 4 5 1 1 4 6 1 4 1 18 2 1 1 8 1 1 1 3 5 5 1 1

aureum badium caledonium caesaris claroideum

2 4 2 1 3

Ambispora

Diversispora

Entrophospora

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

Gigaspora Glomus

Genus

Species

Glomus (cont.)

claroideum group constrictum coronatum diaphanum etunicatum geosporum hoi hyderabadensis intraradices lamellosum luteum macrocarpum mosseae rubiformis versiforme viscusum sp. nov.1 sp1 sp2 sp3 sp4 sp5 sp6 sp7 sp8 sp9 dominikii franciscana scintillans

Number of locations1 3 18 4 2 18 5 1 2 15 5 3 17 7 11 5 1 8 2 1 1 1 1 1 1 1 1 7 1 2

Paraglomus

occultum

1

Scutellospora

calospora

11

Pacispora

1

Number of locations where the fungus was detected as spores in the soil surrounding the roots of the target plants from the total of 34 locations studied. Sp. nov.: morphotype of the indicated genus that do not correspond to any of the species described up to now. Sp.: non-identified species.

The other AMF genera found in Sierra Nevada were represented by a low number of species: four in the case of Ambispora, Diversispora and Entrophospora, three of Pacispora and only one species of each of the genus Gigaspora, Paraglomus and Scutellospora (Table 5). Some of the AMF isolated from the soils around the target plant species are recorded in Figure 3.

The Mycorrhizal Symbiosis in Mediterranean Environment: Importance in Ecosystem Stability and in Soil Rehabilitation Strategies : Importance in

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

62

Concepción Azcón-Aguilar, Javier Palenzuela, Nuria Ferrol et al.

Figure 3. Some of the arbuscular mycorrhizal fungi isolated from the soil around the target plant species.

The Mycorrhizal Symbiosis in Mediterranean Environment: Importance in Ecosystem Stability and in Soil Rehabilitation Strategies : Importance in

Mycorrhizal Status and Arbuscular Mycorrizal Fungal Diversity ...

63

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

Figure 4. Spores of the recently described AM fungus Entrophospora nevadensis isolated from Sierra Nevada National Park. a: Spore showing rests of the sporiferous saccule (sac) attached. b: Young spore showing the long spiny, thorn-like projections (orn) on the surface. Note also rest of the sporiferous saccule. c: Mature spore showing the spiny projections and organic debris on it surface. d: Spores exhibit a wide, circular cicatrix on the base proximal to the sporiferous saccule and a small cicatrix distal to the saccule. e: Planar view of a broken spore showing its characteristic ornamentation. f: Spores stain reddish-brown in Melzer‘s reagent. Scale bar = 25 mm.

One of the AMF new species isolated from the sampled soils around the target plants has already been described in the scientific literature (Palenzuela et al. 2010). It has been named Entrophospora nevadensis, referring to Sierra Nevada, where the new species has been found. Briefly, spores are yellow brown to brown, about 100 m diameter, and form singly in soil, in the neck of adherent sporiferous saccules. Spores have two three-layered walls and conspicuous, 6–12 mm long, spiny, thorn-like projections on the outer wall (Figure 4). The new fungus was found in soil near plants with different living strategies (P. nivalis, A. fontqueri, S. elodes and S. hybrida) but growing at high altitudes, in soils having acidic pH, high soil moisture and organic matter content, and close to streams. The different AMF found in Sierra Nevada show a different behaviour in relation to their distribution. Whereas some AMF species, mainly belonging to the Glomus genus, seem to be quite ubiquitous and are found indistinctly in acid, neutral or alkaline soils (as for instance G. etunicatum, G. constrictum, G. macrocarpum, G. intraradices and Am. gerdemannii), other species are only found in acid (as most of the Acaulospora species, E. nevadensis and E. baltica) or alkaline soils (as G. badium, G. coronatum and A. thomii). Most of the fungi were found in the soil near the roots of different plant species. Forty two AMF were successfully isolated and established in pure culture, constituting a first germplasm collection of Sierra Nevada native AMF. This collection includes 26 Glomus species, six of the Acaulospora genus, three of Diversispora, two from each of Entrophospora and Ambispora and one species of Pacispora, Scutellospora and Paraglomus. From all target plants at least one of the AMF present in the soil around their roots has been included in the germplasm collection. As it has been recommended for protected

The Mycorrhizal Symbiosis in Mediterranean Environment: Importance in Ecosystem Stability and in Soil Rehabilitation Strategies : Importance in

64

Concepción Azcón-Aguilar, Javier Palenzuela, Nuria Ferrol et al.

environments (Bothe et al. 2010), this collection will constitute the base for the formulation of tailored mycorrhizal inoculants using specific mixes of suitable, autochthonous mycorrhizal fungi for each one of the target plant species.

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

6. Effect of Mycorrhizal Inoculation on the Growth of Endangered Plants To check the effect of a tailored mycorrhizal inoculation with specific mixes of native mycorrhizal fungi on the growth of the endangered plant species several greenhouse experiments were carried out. Given that most of the target plant species are threatened with extinction, the main difficulty for these studies was to get enough number of quality plantlets to be used in the different experiments. Therefore, plants for these studies were selected according to the availability of either seeds or plant cuttings. As an example, the results obtained with Artemisia alba subsp. nevadensis are summarized below. Seeds of A. alba were surface-sterilized and germinated in a vermiculite:sand (1:1, v:v) mixture. Seedlings were grown in soil collected from around the plants growing in their natural habitat. Half of the soil was steam-sterilized to remove the indigenous AM fungi (sterile soil) and the other half was kept in natural conditions (Natural soil). In both cases (sterile and natural) half of the plants (a minimum of 10 per treatment) were inoculated with a mixture of AM fungi isolated from the soil around the plant growing in natural conditions and the other half was kept as uninoculated controls. The mycorrhizal inoculum consisted mainly of Glomus constrictum, Glomus etunicatum and Glomus sp. (claroideum group). All plants received an aliquot of a filtrate (