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6000 Broken Sound Parkway, NW
Suite 300, Boca Raton, FL 33487
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New York, NY 10017 9 781 466 59 468 5
an informa business 2 Park Square, Milton Park 9 781 466 59 468 5
Abingdon, Oxon OX14 4RN, UK 9 781 466 59 468 5 A SCIENCE PUBLISHERS BOOK
w w w. c rc p r e s s . c o m
Ectomycorrhizal Symbioses
in
Tropical and Neotropical Forests
Ectomycorrhizal Symbioses
in
Tropical and Neotropical Forests
Editors
Amadou M. Bâ
Université Antilles-Guyane
Guadeloupe (French West Indies)
France
Krista L. McGuire
Barnard College
Columbia University
New York
USA
Abdala G. Diédhiou
Université Cheikh Anta Diop
Dakar
Sénégal
p,
A SCIENCE PUBLISHERS BOOK
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Foreword
Ectomycorrhizal symbioses in tropical and neotropical forests: A major

step toward to a neglected research imperative.
In a 2009 paper in the New Phytologist, Ian Alexander and myself called
the mycorrhizal research in the tropics ‘a neglected research imperative’.
Although Janse’s ‘Les endophytes radicaux de quelques plantes javanaises’ in 1896
was published shortly after the description of ectomycorrhizas by Frank in
1885, the research on tropical mycorrhizas attracted too limited attention
in the next 100 years. Although papers dealing with mycorrhiza and tropic
continuously accumulated in the last decade (ca. +10% per year, according
to ISI Web of Knowledge), they represented only, each year between 2009
and 2012, a remarkably constant 0.6% fraction of the papers dealing with
mycorrhiza.
Yet, one should wish more than such a constant progression. Tropical
ecosystems represent more than 0.6% of land ecosystems and have pivotal
role in the Earth’s biogeochemical cycle and climate; moreover, threats on
biodiversity in tropical forests should encourage faster study in tropical
latitudes. In this framework, a book devoted to ‘Ectomycorrhizal symbioses
in tropical and neotropical forests’ is very timely.
Indeed, we are far from the simple view that the tropics are dominated
by arbuscular mycorrhizas, as popularized by Malloch et al. in PNAS in
1975. However, what determines the success of ectomycorrhizas in tropical
ecosystems remains unclear. One of the most striking features of tropical
ectomycorrhizal symbioses is their frequent occurrence in ‘monodominant’
forests, where a single species dominates 60 to 100% of the canopy.
The present book investigates these exciting monodominant forests by
comparing their mycorrhizal diversity with that of more host-diverse
ectomycorrhizal forests (Diédhiou et al., Chapter 1), and by questioning
the role of ectomycorrhizas among the positive feedbacks that maintain
the dominant species over generations (McGuire, Chapter 10).
Beyond such questions, basic descriptions of these insufficiently
studied ectomycorrhizal symbioses are still required. This book nicely
describes ectomycorrhizas of Nyctaginaceae (Haug et al., Chapter 2) and
vi Ectomycorrhizal Symbioses in Tropical and Neotropical Forests
Gnetum (a liana probably related to Pinaceae; Bechem, Chapter 8) on the

plant side, and of Sebacinales (Moyersoen, Chapter 5) on the fungal side,
in a timely revival of often overlooked morphological and microscopic
investigations. The Gnetum chapter illustrates a more functional approach
for studying ECM fungi based on physiology and ecophysiology. These
functional aspects are also central in the chapter evaluating the role of the
ectomycorrhizal symbiosis in protection of Coccoloba uvifera against sea salt
(Bâ et al., Chapter 9), and in plant adaptation to soils highly concentrated
in heavy metals (Jourand et al., Chapter 6).
More ecological questions, such as the response of the ectomycorrhizal
community to disturbance (Onguene et al., Chapter 3), open the door
to the use of ectomycorrhizal symbioses for reforestation, especially in
physiologically constraining environments. The possibility of facilitation
by ‘nurse plants’, which pre-cultivates a fungal community suitable
for installing a target tree species, receives supports in Madagascarian
sclerophyllous forests (Baohanta et al., Chapter 4) and the tree performances
after inoculation of Afzelia by Scleroderma and Thelephora raises good hopes
in Western Africa (Sanon et al., Chapter 7). In Asiatic dipterocarps forests,
inoculation, using mycoflora from soil sampled under conspecific parents
is promising for forest rehabilitation (Tata, Chapter 11). In an overview
of the inoculation methods and results, Duponnois et al. (Chapter 12)
rightly point out that ectomycorrhizal symbioses are very promising
tools—yet, more long-term assessments of inoculant survival and of host
growth promotion are required, especially after out planting to field sites.
Encouraging results obtained from some models should not dissimulate that
the method needs independent optimization for each plant-fungus model.
Anarchic use of any inoculant or any method could introduce undesirable
exotic fungi, and discredit ectomycorrhizal inoculations by yielding low
results. In this framework, the careful case studies reported in this book
are very relevant.
Finally, beyond trees and forests, edible fungal fruit bodies represent
often neglected and potentially threatened resources, with high cultural
and economical importance (Yorou et al., Chapter 13). ‘Ectomycorrhizal
symbioses in tropical and neotropical forests’ is a rich milestone in our
knowledge of fungal and ectomycorrhizal functioning diversity over all the
tropics, from Africa to America, including Pacific islands, with rich insights
into functional and applied questions. The reader clearly feels emergent
models for further studies and practices: Scleroderma and Pisolithus spp.
on the fungal side, Coccoloba, Afzelia, Gilbertiodendron, Uapaca, Dicymbe and
Shorea on the plant side. Researches must now go on, in order to develop
robust methods ensuring environmental protection and sustainability of
ecosystems services.
Foreword vii
As a ‘primarily temperate’ mycorrhizologist, I have always been

fascinated by the lessons and exquisite diversity offered by tropical
mycorrhizal symbioses, and I feel greatly honoured to write these
introductory lines. Closing the pages of ‘Ectomycorrhizal symbioses in
tropical and neotropical forests’, I feel more fascinated than ever, and hope
all readers will share this great excitement!
Marc-André SELOSSE
Professor at Muséum national d’Histoire naturelle and
President of the Société botanique de France
Observatoire de la Structure et de l’Evolution de la Biodiversité
(UMR7205 CNRS – Muséum), Département Systématique et Evolution,
45 rue Buffon, 75005 Paris, France
Preface
Ectomycorrhizal (EcM) symbioses are mutualistic plant-fungus associations

that play a major role in function, maintenance and evolution of biodiversity
and ecosystems stability and productivity. EcM associations are integral
to the biology and ecology of forest trees, affecting growth, water and
nutrient absorption and protection against pathogens. They are also a
research imperative in tropical and neotropical forest ecosystems because
they concern ecologically and economically important tree species (e.g.,
Ceasalpinioid subfamily in Africa, family of Dipterocarpaceae in Asia and
South America). The book is an overview of the knowledge of EcM symbioses
in tropical and neotropical ecosystem forests. The contents address diversity
and function of ectomycorrhiza (ECM) associated with forest plants, impacts
of ectomycorrhiza on plant diversity and composition, regeneration and
dynamics of ecosystems, and biomass production in forestry, adaptation of
EcM plants to nutrient deficient, salted and ultramafic soils.
Diédhiou et al. (Chapter 1) has provided valuable information on
the diversity and community structure of EcM fungi from mixed and
monodominant forests in tropical Africa. They found that monodominant
forest tends to harbor more EcM-forming lineages than the mixed forest,
while there was no significant difference in terms of richness of EcM
fungal species between these two forest types. In the monodominant
Dicymbe corymbosa forest in Guyana, it appears that positive plant-soil
feedbacks function to maintain EcM monodominance through seedling
facilitation and alterations of decomposer communities and nutrient
cycling pathways. It is also possible that the EcM monodominant trees
are able to support a greater diversity of EcM fungi compared to non-
dominant EcM trees, which may partially explain why these trees attain
such extreme dominance. With a greater diversity of symbionts, trees
have access to more diverse pools of soil resources (McGuire, Chapter 10).
EcM forest communities could contribute not only to the preservation of
monodominant clump refuges in South Cameroon with endemic species,
but also to the protection of the biodiversity hot spots of Africa’s rain
forests. Maintenance of such EcM forest communities with their associated
edible mushrooms also helps to preserve a valuable source of alternative
x Ectomycorrhizal Symbioses in Tropical and Neotropical Forests
protein-rich food for local communities who depend on non-timber forest

products (Onguene et al., Chapter 3).
Different approaches exist to characterize EcM. Morpho-anatomical
features are important information, together with ultrastructure and DNA
sequences of EcM are used to confirm the EcM status of Sebacinales species.
These results are not only important for the taxonomy of Sebacinales
EcM but also for the understanding of Sebacinales ecology (Moyersoen,
Chapter 5). Haug et al. (Chapter 2) showed that although the common
ancestor of Nyctaginaceae may have been involved with one thelephoroid
taxon, coevolution of Nyctaginaceae and ectomycorrhizal Thelephoraceae
in different habitats may have led to the evolution of several different
species.
On the other hand, EcM fungi are an integral part of plant physiology.
For example, there is a possibility that the fungus Scleroderma sinnamariense
can access some organic P sources in nature when inorganic P is
limiting, an ability which would be beneficial to the Gnetum africanum,
a notable liana for its edible leaves (Bechem, Chapter 8). Bâ et al.
(Chapter 9) that Scleroderma bermudense possess considerable resistance
to salinity. Tolerance to salt stress was considerably enhanced by
S. bermudense. Otherwise, the focus on EcM Pisolithus albus isolated from
soils in New Caledonia highlighted the identification of an ultramafic nickel-
tolerant ecotype, showing specific and adaptive molecular response to this
metal. In thus, this fungus plays a key role in plant host adaptation to toxic
nickel concentrations as found in these soils (Jourand et al., Chapter 6).
From ecological point of view, the EcM fungi could be from an adult tree
of Uapaca bojeri, an endemic tree of the Malagasy sclerophyllous forest, where
various putative EcM fungi were observed, or from endemic and pioneer
shrub species (Leptolenabojeriana and Sarcolaenaoblongifolia) that persist on
disturbed sites and facilitate the survival of EcM fungi propagules that
could potentially infect roots of U. bojeri seedlings (Baohanta et al., Chapter
4). Sanon et al. (Chapter 7) already propose to use two effective EcM fungi,
S. dictyosporum and Thelephora sp., to inoculate Afzelia africana, a timber tree
for the reforestation programme in West Africa. Similar results were obtained
in Asian forests, where diptercarp seedlings are inoculated by mycoflora
from soil sampled under conspecific parents in order to rehabilitate forests
(Tata, Chapter 11). Duponnois et al. (Chapter 12) present an overview
of the inoculation methods and results rightly point to ectomycorrhizal
symbioses as very promising tools, but more long-term assessments of
inoculant survival and of host growth promotion are required, especially
after outplanting to field sites. Finally, beyond trees and forests, edible fungal
fruitbodies represent often neglected and potentially threatened resources,
of high cultural and economic importance (Yorou et al., Chapter 13).
Preface xi
From this finding, it should be evident that further research is

necessary in tropical and neotropical forests to (i) assess and compare
the morphological and phylogenetic community composition between
monodominant and mixed forests, (ii) to determine the role of EcM fungi
in the recruitment and establishment of seedlings of EcM tree species, (iii)
to use EcM symbioses for reforestation programmes.
Amadou M. Bâ
Krista L. McGuire
Abdala G. Diédhiou
Contents
Foreword v
Preface ix
1. Diversity and Community Structure of Ectomycorrhizal 1
Fungi in Mixed and Monodominant African Tropical
Rainforests
Abdala Gamby Diedhiou, Helvyne Christelle Michaella Ebenye,
Marc-André Selosse, Nérée Onguene Awana and Amadou Mustapha Bâ
2. Ectomycorrhizas of Three Species of Nyctaginaceae in the 19
Tropical Mountain Rain Forest of South Ecuador
Ingeborg Haug, Ingrid Kottke and Juan Pablo Suárez
3. Diversity and Abundance of Ectomycorrhizal Associations 29
in Rain Forests of Cameroon under Different Disturbance
Regimes
Onguene Awana Nérée, Judith Marthiale Tsamo, Christelle Michaella
Ebenye, Amadou Mustapha Bâ and Thomas Kuyper
4. Mycorrhizal Fungi Diversity and their Importance on the 51
Establishment of Native Species Seedlings within
Madagascarian Degraded Sclerophyllous Forest
Rondro Harinisainana Baohanta, Herizo Andrianantoandro
Randriambanona, Marc Ducousso, Christophe Nirina Rakotoarimanga,
Yves Prin, Heriniaina Ramanankierana and Robin Duponnois
5. Morpho-anatomical Characterization of Three 79
Sebacinales Ectomycorrhizal Species from a Pakaraimaea
dipterocarpacea ssp. nitida (Dipterocarpaceae) Forest in
Southern Venezuela
Bernard Moyersoen
xiv Ectomycorrhizal Symbioses in Tropical and Neotropical Forests
6. Abundance, Distribution, and Function of Pisolithus albus 100

and other Ectomycorrhizal Fungi of Ultramafic Soils in New
Caledonia
Philippe Jourand, Fabian Carriconde, Marc Ducousso,
Clarisse Majorel, Laure Hannibal, Yves Prin and Michel Lebrun
7. Diversity and Function of Ectomycorrhiza between 126
Scleroderma and Afzelia Species in Burkina Faso
(West Africa)
Kadidia Bibata Sanon, Amadou Mustapha Bâ and Robin Duponnois
8. The Physiology of Scleroderma sinnamariense Mont. 147
(Sclerodermaceae), an Ectomycorrhizal Fungus Associated
with Gnetum spp. (Gnetaceae)
Eneke Esoeyang Tambe Bechem
9. Alleviation of Salt Stress by Scleroderma bermudense in 164
Coccoloba uvifera Seedlings in the French West Indies
Amadou Mustapha Bâ, Raymond Avril, Eric Bandou, Seynabou Sène,
Robin Duponnois, Régis Courtecuisse, Samba Sylla and
Abdala Diédhiou
10. The Contribution of Ectomycorrhizal Fungal Feedbacks to 185
the Maintenance of Tropical Monodominant Rain Forests
Krista L. McGuire
11. Ectomycorrhiza in Forest Rehabilitation in Indonesia 200
Hesti L. Tata
12. The Controlled Ectomycorrhization Practices in Tropical 215
Areas: Fungal Inoculum Biotechnology, Field Results and
Research Perspectives
Robin Duponnois, Hervé Sanguin, Amadou Mustapha Bâ,
Antoine Galiana, Marc Ducousso, Ezékiel Baudoin, Michel Lebrun
and Yves Prin
13. Biodiversity and Sustainable Use of Wild Edible Fungi in 241
the Sudanian Centre of Endemism: A Plea for Valorisation
Nourou Soulemane Yorou, N’Golo Abdoulaye Koné, Marie-Laure
Guissou, Atsu Kudzo Guelly, Dao Lamèga Maba, Marius R.M. Ekué
and André De Kesel
Index 271
Color Plate Section 275
CHAPTER
1
Diversity and Community
Structure of Ectomycorrhizal
Fungi in Mixed and
Monodominant African
Tropical Rainforests
Abdala Gamby Diedhiou,1,2,* Helvyne Christelle
Michaella Ebenye,2,3,4 Marc-André Selosse,3,4 Nérée
Onguene Awana5 and Amadou Mustapha Bâ2,6
1. Introduction
Mycorrhizal symbioses play a prominent role in the biology and ecology
of forest trees. They involve soil fungi and roots of trees, which together as
a symbiosis provide the fungi with carbohydrates and enhance the uptake
of water and nutrients for the trees, and also have a major protective role
for the roots (Smith and Read 2008). Forest trees are primarily associated
with two types of mycorrhizas: arbuscular mycorrhizas which include fungi
from the phylum of Glomeromycota and ectomycorrhizas (EcMs) mainly
formed by members of Ascomycota and Basidiomycota. It is currently
estimated that 6,000–10,000 plant species (Smith and Read 2008, Brundrett
2009) and 20,000–25,000 fungal species (Rinaldi et al. 2008) are involved in
Authors’ affiliations given at the end of the chapter.

2 Ectomycorrhizal Symbioses in Tropical and Neotropical Forests
ectomycorrhizal symbioses. EcMs are the most widespread mycorrhizal

type in the forests of cool-temperate and boreal latitudes (Molina et al.
1992). Conversely, tropical forests are dominated by arbuscular mycorrhizas
(Malloch et al. 1980), a situation that once constrained the introduction of
EcM Pinus species in the tropics (see Pringle et al. 2009 for review). EcMs
being found only in a minority of ecologically and economically important
tree species that can form stands where they dominate (Hart et al. 1989,
Alexander 1989, 2006, Alexander and Lee 2005, McGuire 2007). Moreover,
the species richness of EcM fungi appears to have a unimodal relationship
with latitudinal gradient (Tedersoo and Nara 2010, Tedersoo et al. 2012).
In tropical Africa, EcMs are mainly distributed in open forests, gallery
forests, and rainforests of the Guineo-Congolian basin, Zambezian Miombo
woodlands of East and South-Central Africa, and Sudanian savannah
woodlands of the sub-Sahara (Fig. 1, Bâ et al. 2012).
Fig. 1. Distribution of EcM trees in tropical Africa: (1) rainforests in the Guinea-Congo region; (2)
open forests in the Sudanian and Zambezian regions; (3) savanna woodlands in the Sudanian
and Zambezian regions (Bâ et al. 2012).
Color image of this figure appears in the color plate section at the end of the book.
Ectomycorrhizal Fungi in African Tropical Rainforests 3
EcM associations are found mainly on Caesalpinioideae, Dipterocarpaceae,

Phyllantaceae, Sarcolaenaceae, Asteropeiaceae, Sapotaceae, Papilionoideae,
Gnetaceae (now considered to be close to Pinaceae; Burleigh and Mathews
2004), and Proteaceae (Bâ et al. 2012). In their natural habitats, some African
EcM tree species tend to aggregate in patches forming monodominant or
mixed stands where they significantly contribute to the forest basal area
(Newbery et al. 2004). For instance, Microberlinia bisulcata, Tetraberlinia
bifoliolata, and T. moreliana form up to 70% of local patches in the Korup
National Park in Cameroon (Newbery et al. 1997). Similarly, in the Southern
Guinea rainforests (West Africa), Caesalpinioideae and Phyllanthaceae trees
are the most abundant native EcM species, growing in mixed patches with
high regeneration of seedlings (Diédhiou et al. 2010). In the Congo basin,
Gilbertiodendron dewevrei forms monodominant forests where it represents
more than 90% of trees in some stands, with abundant seedlings and
saplings (Hart et al. 1989, Hart 1995).
It has been documented that EcM fungi contribute to the establishment
and structure of these African EcM plant communities (Högberg and Piearce
1986, Newbery et al. 2000, Onguene and Kuyper 2001). However, the
diversity and specificity of African EcM fungi have remained inadequately
understood. Until recently, available African EcM fungi data was mainly
obtained from sporocarp collections, ex situ fungal baiting, inoculation of
seedlings, and pure culture synthesis trials (Thoen and Bâ 1989, Thoen
and Ducousso 1989, Bâ and Thoen 1990, Bâ et al. 1991, Buyck et al. 1996,
Sanon et al. 1997, Diédhiou et al. 2004, 2005). The use of molecular tools,
such as barcoding with ITS (Nilsson et al. 2008) for in situ identification
and phylogenetic analysis substantially improved our knowledge of the
diversity and specificity of African EcM fungi (Rivière et al. 2007, Tedersoo
et al. 2007, 2010a, 2011, Diédhiou et al. 2010, Jairus et al. 2011).
Indeed, a high diversity of EcM fungal species belonging to more
than 25 phylogenetic lineages has been revealed from Continental Africa,
Madagascar, and the Seychelles. As in temperate forests, the /russula–
lactarius and /tomentella–thelephora lineages dominated the EcM fungal
flora in these African forests. Furthermore, a low level of host preference
and dominance of multi-host fungal species have been revealed from mixed
EcM forests (Diédhiou et al. 2010, Tedersoo et al. 2011), suggesting that
some EcM tree species may facilitate the recruitment and establishment of
conspecific and non-conspecific seedlings. However, little is known about
EcM fungal diversity in African tropical monodominant forests. Based on
these different observations we addressed the following question: Do mixed
forests and monodominant forests in tropical Africa have similar diversity
and community structure patterns of EcM fungi? In a monodominant forest
of South-Eastern Cameroon we described the EcM and sporocarp diversity,

and compared these results to those found from a mixed forest of Southern
Guinea (Rivière et al. 2007, Diédhiou et al. 2010).
2. Site Description
The mixed forest was located in Southern Guinea, one of the last regions
of West Africa retaining a primary tropical rainforest. Above- and below-
ground EcM fungal diversity surveys were conducted in typical evergreen
rainforests covering hills and mountains ranging in altitude from 500
m in the Ziama forest (8°51′N, 9°31'W) to 1,752 m on the Mount Nimba
forest (7°60'N, 8°49'W). The evergreen rainforests are characterized by a
mean annual rainfall of 2,500–3,000 mm and a dry season (mean rainfall
<15 mm) from January to March. Temperatures are generally above 24°C
with relative humidity up to 80%. The soils are generally poor, lateritic
and prone to heavy leaching (McGinley 2008). Canopy trees are at least 30
m tall, with some emergent individuals reaching 50–60 m in height. The
canopy is dominated by Cryptosepalum tetraphyllum (EcM Caesalpinioideae,
Fabales), as well as the AM trees Erythrophleum ivorense (Fabaceae) and
Heritiera utilis (Malvaceae, Malvales). The other EcM trees are represented
by Caesalpinioideae (Afzelia bella, Anthonotha fragans, A. macrophylla,
Gilbertiodendron limba, Paramacrolobium coeruleum and Pelligriniodendron
diphyllum), and Phyllanthaceae (a family formerly classified within
Euphorbiaceae: Uapaca esculenta, U. chevalieri, U. guineensis and Uapaca
heudelotii). Some of these Caesalpinioideae and Phyllanthaceae tree species
grow in mixed patches where they dominate (Rivière et al. 2007, Diédhiou et
al. 2010). Sporocarps (fruit-bodies) and ECMs of mature trees and seedlings
were collected within 3 plots of 0.5–1 ha each in the mixed forest. For the
mature trees, root tips were sampled around trees by tracing roots from the
base of trunk whenever possible.
The monodominant forest of G. dewevrei is located in the Dja Faunal
Reserve (2°49’ to 3°23’N; 12°25’ to 13°35’E) in the Southeastern region of
Cameroon, which extends from Mouloundou to south of Bertoua. The
climate is characterized by two wet seasons from March to June and from
August to November with a mean annual rainfall varying from 1600 to 1700
mm. The two dry seasons are December–February (18–59 mm) and July (54
mm). Mean annual temperature varies from 23.7° to 24.3°C. Soils are clayey
ferralitic, acidic, and poor in nutrients (Peh et al. 2011). The vegetation has a
main canopy of 30–40 m with highest trees rising to 60 m (Letouzey 1985).
The monodominant forests are surrounded by mixed forests including
many tree and shrub species, which form mainly arbuscular mycorrhiza
(e.g., Afrostyrax lepidophyllus, Afzelia bipendensis, Anthonotha ferruginea,
Baphia pubescens, Beilschmiedia louisii, Cryptosepalum congolum, Drypetes paxii,
Entangrophragma congoensi, Erismadelphus exsul var. platyphyllus, Fernandoa

ferdinandii and Hesteria trillesiana). Sporocarps and EcMs of mature trees,
saplings and seedlings of G. dewevrei were collected within 3 plots of 0.5 ha
each in a monodominant stand. EcMs from mature trees and saplings were
sampled by extracting cylindrical soil cores from around trees.
3. General Traits of the EcM Fungal Communities from the

Two Forest Types
A total of 856 samples, 463 from the monodominant forest and 393 from the
mixed forest, were collected and analyzed by morphological and molecular
approaches. Of the 856 samples 343 (145 and 198 for the monodominant
forest and mixed forest, respectively) were represented by sporocarps and
513 (354 and 159 for the monodominant forest and mixed forest, respectively)
were represented by EcM tips. Most of the morphologically identified
sporocarps sampled from the mixed forest were vouchered and stored in
the Museum National d’Histoire Naturelle, France (Rivière et al. 2007). The
voucher specimens of sporocarps from the monodominant forest are kept
at the herbarium of the Institute of Agricultural Research for Development
(IRAD), Yaounde, Cameroon. EcMs were classified into morphotypes (MTs)
for molecular analyses based on distinctive macroscopic and microscopic
features: branching, color and texture of the mantle, presence or absence of
emanating hyphae, rhizomorphs, and sclerotia linked to EcMs (Diédhiou et
al. 2004, 2009). The molecular identification of EcM fungi from sporocarps
and MTs was conducted by DNA sequencing of the internal transcribed
spacer (ITS) region (Diédhiou et al. 2010, H.C.M. Michaella Ebenye
unpublished data) and a portion of mitochondrial rDNA large subunit
(mtLSU) (Rivière et al. 2007). Hence, from the 856 samples collected, 613 were
successfully sequenced and the sequences were aligned with the Clustal X
program (Thompson et al. 1997). Alignments were manually optimized with
the Genedoc program (Nicholas et al. 1997). BlastN searches of obtained
sequences against the INSD were conducted to determine their taxonomical
affiliation (Altschul et al. 1990). ITS sequences (OTUs, operational taxonomic
units) displaying >90% and >97% full-length similarity to identified fungal
taxa were considered congeneric and conspecific, respectively. These
sequence similarities are arbitrary, but were deemed as adequate screening
for DNA barcoding DNA barcoding thresholds for fungal taxonomy at
genus and species levels, respectively (Smith et al. 2007, Nilsson et al. 2008,
Hughes et al. 2009, Tedersoo et al. 2010b).
Hence, 625 samples were successfully identified at the family, genus
or species level by combining DNA barcoding and morphological analyses
of sporocarps and EcMs collected from the two forest types. The identified
fungi fell into three phyla: Ascomycota, Basidiomycota and Zygomycota

(Table 1). The Basidiomycota was the most abundant phylum and the
Zygomycota the least abundant. The identified fungi were then assigned
to the phylogenetic lineages of EcM fungi predefined by Tedersoo et al.
(2010b); however, we excluded from the analysis the genera Chalciporus,
Geastrum and Leptodontidium as their EcM status is still debatable (see Rinaldi
et al. 2008, Tedersoo et al. 2010b, Comandini et al. 2012). The names of the
identified EcM-forming lineages are written in lower case, non-italicized
font with a slash (e.g., /russula-lactarius; Moncalvo et al. 2002). Thus, 18
EcM-forming lineages (Table 1) accounting for 88% of the total abundance
of identified samples were recorded from both African tropical forests. This
result is consistent with those obtained from other tropical forests where a
considerable number of fungi of unknown trophic status have been revealed
from EcMs (Tedersoo et al. 2010a, Peay et al. 2010, Smith et al. 2011, 2013).
The fungi of unknown trophic status included members of Agaricomycetes,
Sordariomycetes, Zygomycetes, Leotiomycetes, Dothideomycetes and
Tremellomycetes (Table 1). Although these latter fungi were molecularly
identified from EcMs, they are traditionally considered to be parasitic,
saprophytic, endophytic, or of unknown trophic status. However, some
of these fungi, particularly Mortierellaceae (Zygomycetes), Hypocreaceae
(Sordariomycetes) and Polyporaceae (Agaricomycetes) found on many
healthy EcMs (data not shown) may represent new EcM-forming lineages
in tropical ecosystems (Peay et al. 2010, Smith et al. 2011, 2013) or even on
larger scales. Further work is thus necessary to clearly determine the EcM
lifestyle of not only these tropical fungal taxa but also of many unexplored
fungi which were often revealed in EcMs by DNA barcoding. Indeed, it
was only ten years ago that important EcM taxa such as Sebacinales were
confirmed to associate with tree roots (Selosse et al. 2002). The EcM-forming
lineage inventory is a continuous challenge and requires extensive careful
work from field to laboratory, particularly in the fungal taxa where EcM
associations are uncommon (de Roman et al. 2005, Rinaldi et al. 2008,
Tedersoo et al. 2010b, Comandini et al. 2012).
Among the 18 phylogenetic lineages of EcM fungi revealed from the
mixed and monodominant forests, only one (/tuber-helvella) has not yet
been reported from other African tropical forests (Tedersoo et al. 2007, 2010a,
2011, Jairus et al. 2011, Bâ et al. 2012). This corroborates the hypothesis of
a lower diversity of phylogenetic lineages of EcM fungi in tropical forests
relative to temperate forests where individual sites usually support more
than 20 lineages (Tedersoo and Nara 2010). Besides the genus Tuber, known
only from the Holarctic realm (Bonito et al. 2010), the /tuber-helvella
lineage includes many separate taxa which are widely distributed in the
Austral regions (Tedersoo et al. 2010b). However, the members of this EcM-
forming lineage would be poorly represented in the African tropical forests
Table 1. Fungal species identified from EcMs and sporocarps collected from the two forest types (EcM-forming lineages are written in lower case,
non-italicized font preceded with a slash (e.g.,/russula-lactarius)).
Type of forest: Mixed Monodominant

Fungi:
Phylum Class/EcM lineage Family/Genus sporocarps EcMs sporocarps EcMs
Basidiomycota /russula-lactarius Russula + + + +
Lactarius + + + +
/tomentella-thelephora Tomentella + + + +
Thelephora + +
/boletus Boletus + + + +
Leccinum + +
Strobilomyces + +
Xerocomus + +
Tubosaeta +
/sebacina Sebacina +
/pisolithus-scleroderma Scleroderma + + +
/amanita Amanita + + + +
/tricholoma Tricholoma + +
Pseudobaeospora +
/clavulina Clavulina + + +
/coltricia Coltricia +
/cantharellus Cantharellus + +
Sistotrema +
/cortinarius Cortinarius +
/inocybe Inocybe + +
Table 1. contd....
8
Table 1. contd.
Type of forest: Mixed Monodominant
Fungi:
Phylum Class/EcM lineage Family/Genus sporocarps EcMs sporocarps EcMs
/ramaria-gautieria Ramaria +
/suillus-rhizopogon Truncocolumella +
Agaricomycetes Chalciporus + +
Geastrum +
Polyporaceae +
Tremellomycetes Trichosporon +
Tremellaceae +
Ascomycota /marcelleina-peziza Marcelleina +
gerardii
/tuber-helvella Helvella +
/helotiales Helotiales +
/elaphomyces Elaphomyces +
Sordariomycetes Nectriaceae +
Chaetosphaeria +
Cercophora +
Sordariomycete +
Ectomycorrhizal Symbioses in Tropical and Neotropical Forests
Hypocreaceae +
Leotiomycetes Leptodontidium +
Leohumicola +
Hyaloscyphaceae +
Dothideomycetes Botryosphaericeae +
Leptosphaeria +
Zygomycota Zygomycetes Mortierellaceae +
as observed in the suillus-rhizopogon lineage assumed to be specific to

Pinaceae (Molina et al. 1992, Bruns et al. 2002). On the other hand, there is
a noticeable absence of some Holarctic and Austral EcM-forming lineages
such as /cenococcum and /laccaria, and some panglobal EcM-forming
lineages (e.g., /entoloma, /hebeloma-alnicola and /hysterangium) as
well.
The/russula-lactarius, and /tomentella-thelephora, were the most
abundant EcM-forming lineages, accounting for 43.17%, and 17.49% of
the total abundance of the identified EcM fungi, respectively (Fig. 2). The
relative abundances of the /boletus, /amanita, /sebacina and /pisolithus-
scleroderma lineages were 10.38%, 6.58%, 5.65% and 5.28% respectively.
The remaining EcM-forming lineages accounted for 11.47% of the total
abundance, each contributing less than 4% (Fig. 2).
In addition, the below-ground fungal diversity and abundance differed
from those observed above-ground. For instance, the /russula-lactarius,
/boletus and /amanita lineages dominated the sporocarps, while the
/russula-lactarius, /tomentella-thelephora, and /sebacina lineages
dominated the EcMs (data not shown). Other EcM-forming lineages such
as the /elaphomyces, /helotiales, and /marcelleina-peziza gerardii were
Fig. 2. Relative abundance of the EcM-forming lineages revealed from EcMs and sporocarps
collected from each forest type.
found only in EcMs, whereas the/cantharellus,/coltricia, and/inocybe were

identified only from sporocarps (Table 1). This result confirms the benefit
of combining sporocarp and EcM surveys to predict the EcM association
patterns at the root level (Richard et al. 2005, Bâ et al. 2012).
4. Comparisons of the EcM Community between the Mixed

and Monodominant Forests
Of the 549 samples representing the EcM-forming lineages, 332 were
collected from the monodominant forest of Cameroon and 217 from the
mixed forest of Guinea. The samples from the mixed forest were dominated
by sporocarps, which represented 63.30%, whereas the samples from the
monodominant forest were dominated by EcMs (74.69%). Of the 18 EcM-
forming lineages, nine were found in both forest types, eight were found
exclusively in the monodominant forest, and one in the mixed forest
(Table 1, Fig. 2). Rarefaction analysis performed using the software EstimateS
ver. 8.0.0 (Colwell 2006) suggested that, for the minimal samples size
n = 217, the richness of EcM-forming lineages was lower in the mixed forest
(Chao2, Jackknife 2 and ICE were 22.09, 24.57 and 26.27, respectively for the
monodominant forest vs 10, 11 and 10.49, respectively for the mixed forest).
Three non-exclusive reasons can account for the lower richness in terms of
EcM-forming lineages in the mixed forest. First, given the seasonal shifts and
spatial heterogeneity that exist in EcM fungal communities, the difference
in the sampling strategy (e.g., tracing roots vs. extracting soil cores) may
provide two different pictures of the EcM fungal communities. Second,
the age and history of hosts may influence the EcM communities through
competition and selection of more adapted EcM fungal taxa (Selosse et al.
2006). Third, the lower root density of EcM hosts in the mixed forest (our
personal observations) may reduce the population size of EcM fungi and
subsequently result in lower richness of EcM-forming lineages (Tedersoo
and Nara 2010).
Furthermore, with the exception of the /sebacina lineage which
accounted for 9.34% of the total abundance of EcM fungi in the
monodominant forest, the other EcM-forming lineages found exclusively
in a single forest type were represented by either one or two species and
displayed relative abundances ≤ 1% (Fig. 2). In this context, we cannot
exclude that the members of these latter EcM-forming lineages might have
been simply overlooked in one type of forest due to their apparent scarcity.
The members of the /sebacina lineage are among the most common EcM
fungal species in temperate and Mediterranean forests (Selosse et al. 2002,
2007, Avis et al. 2003, Weiß et al. 2004, Richard et al. 2005) and have been
reported from the Neotropics (Moyersoen 2006, Morris et al. 2009, Selosse
et al. 2009, Henkel et al. 2012) and the Paleotropics as well (Peay et al. 2010,
Tedersoo et al. 2010a, 2011, Jairus et al. 2011). In regard to this observation
and because the members of this lineage produce inconspicuous sporocarps,
one could argue that they might have been missed in the mixed forest where
relatively few samples were collected. However, some EcM-forming lineages
such as /pisolithus-scleroderma and /tricholoma were more abundant
in the mixed forest (accounting for 11.98% and 5.53%, respectively) than
in the monodominant forest where they accounted for < 1% each (Fig.
2). Thus, although possibly underestimating the number and abundance
of EcM-forming lineages in the mixed forest, our results show that the
monodominant forest tends to harbor more EcM-forming lineages.
In terms of species richness, the lineages of /russula-lactarius (86 spp.),
/tomentella-thelephora (30 spp.), /sebacina (19 spp.), and /boletus (13 spp.)
dominated in the monodominant forest, while the /russula-lactarius (42
spp.), /tomentella-thelephora (21 spp.), /boletus (20 spp.), and /amanita (15
spp.) dominated in the mixed forest. Other species-rich lineages included
the /pisolithus-scleroderma (10 spp.) and /tricholoma (10 spp.) in the
mixed forest and the /clavulina (11 spp.) and /amanita (10 spp.) in the
monodominant forest (Fig. 3).
The EcM-forming lineages included 189 and 126 putative EcM species
for the monodominant forest and mixed forest, respectively (Table 2).
The high EcM species richness observed from these African forests is a
Fig. 3. Number of putative EcM fungal species identified from EcMs and sporocarps collected
from each forest type.
Table 2. Number (No.) of EcM samples observed and estimated EcM species richness in each
forest type. *The numbers appearing in bold were obtained after rarefaction to 217.
Monodominant forest Mixed forest

No. of EcM samples 332 217
No. of putative EcM species 189 126
137.37* 126
Chao2 (95% CI) 575.42 (419.98–835.47) 249.08 (193.87–349.18)
478.06 (321.39–768.92) 249.08 (193.87–349.18)
Jackknife 2 440.97 259.25
329.82 259.25
ICE 675.07 282.2
523.9 282.2
Fisher’s alpha ± SD 182.1 ± 17.87 125.51 ± 15.38
161.82 ± 20.93 125.51 ± 15.38
Shannon ± SD 4.86 4.62
4.64 ± 0.05 4.62
common trend in Neotropical (Morris et al. 2009, Smith et al. 2011, 2013,
Henkel et al. 2012) and Paleotropical forests (Peay et al. 2010, Tedersoo et
al. 2010b, 2011). The calculation of sample-based rarefaction curves and
minimal species richness estimates showed that the sampling efforts were
insufficient in both types of forests. Indeed, the rarefied accumulation
curves of species and minimal species richness estimates (Chao2, Jack2 and
ICE) did not reach a clear asymptote with increasing sample size (Fig. 4).
The Chao2, Jack2 and ICE richness estimators predicted 249.08, 259.25 and
282.2 putative EcM species, respectively for the mixed forest and 575.42,
440.97 and 675.07 putative EcM species, respectively for the monodominant
forest (Table 2).
Surprisingly, when rarefying the samples from the monodominant
forest to n = 217 to match the sample size of the mix from the mixed forest,
we found no significant difference in terms of richness of putative EcM
fungal species between the two forests types (Table 2, Fig. 4). Indeed, the
95% CI (confidence intervals) for Chao2 are overlapping and the indices of
species diversity, Fisher’s alpha and Shannon are not significantly different
between the two types of forests (Table 2). This lack of difference in richness
of EcM fungal species between the mixed forest and monodominant forest
may be in part related to the prevalence of multi-host EcM fungi (Onguene
and Kuyper 2002, Diédhiou et al. 2010) which could mask the influence of
plant host species on EcM fungal community structure. Interestingly, this
rejects the generalization of the idea that host diversity is the sole driver
of diversity in EcM community (Dickie 2007, Ishida et al. 2007, Tedersoo
Fig. 4. Rarefied accumulation curves of (a) EcM fungal species and (b) minimal species richness
estimates for the monodominant forest (MoF, black) and mixed forest (MiF, grey). Dotted lines
in (a) indicate 95% CI.
et al. 2008). This idea may be supported by the finding that African EcM
fungal communities are not strongly structured by soil horizon and host
at the plant species and family levels (Tedersoo et al. 2011).
5. Conclusion
This study has provided valuable information on the diversity and
community structure of EcM fungi from mixed and monodominant forests
in tropical Africa. The monodominant forest tends to harbor more EcM-
forming lineages than the mixed forest, while there was no significant
difference in terms of richness of EcM fungal species between these two
forest types. The dominant EcM-forming lineages are similar in the two
forest types. On the other hand, a large number of fungi of unknown trophic
status were recovered from healthy EcMs; some of them may represent new,
overlooked tropical EcM-forming lineages. From this finding, it should be
evident that further research is necessary to (i) determine the EcM lifestyle
of the latter fungal taxa, (ii) rigorously assess and compare the phylogenetic
community composition between the two forest types, and (ii) highlight
the role of EcM fungi in the recruitment and establishment of seedlings of
EcM tree species in monodominant and mixed forests.
Acknowledegments
We thank the anonymous referees for their valuable comments on this study,
and Krista L. McGuire and Caitlyn Gillikin for improving the language.
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1
Département de Biologie Végétale, Université Cheikh Anta Diop (UCAD), BP 5005 Dakar,
Sénégal.
2
Laboratoire Commun de Microbiologie IRD/UCAD/ISRA, Centre de Recherche de Bel Air,
BP. 1386, Dakar, Sénégal.
3
Centre d’Ecologie Fonctionnelle et Evolutive (CNRS, UMR 5175), 1919 Route de Mende, 34
293 Montpellier cedex 5, France.
4
Muséum national d’Histoire naturelle, Département Systématique et Evolution (UMR 7205
OSEB), 45 rue Buffon, 75005 Paris, France.
5
Institut de Recherche Agronomique pour le Développement, BP. 2123, Yaoundé,
Cameroun.
6
Laboratoire des Symbioses Tropicales et Méditerranéennes (LSTM), UMR113, Campus de
Baillarguet, A10/J, 34398 Montpellier, Cedex 5, France.
*Corresponding author: fungalmolecol@gmail.com
CHAPTER
2
Ectomycorrhizas of Three
Species of Nyctaginaceae in
the Tropical Mountain Rain
Forest of South Ecuador
Ingeborg Haug,1,* Ingrid Kottke1 and Juan Pablo Suárez2
1. Introduction
Arbuscular mycorrhizas are the most ancient mycorrhizal association
(Taylor 1995) and are found in more than 80% of the plant species (Smith
and Read 2008). In a limited number of plant groups, the arbuscular
association was replaced by an ectomycorrhizal association (Smith and
Read 2008). Certain families such as Pinaceae and Fagaceae exclusively have
ectomycorrhizal associations, but there are also several isolated lineages
of ectomycorrhization in other families (Brundrett 2002). For instance, in
the Nyctaginaceae, only a few ectomycorrhiza forming species are known;
these are found in Neea Ruiz & Pav. (Harley and Smith 1983), Guapira Aubl.
(syn Torrubia, Harley and Smith 1983) and Pisonia L. (Ashford and Allaway
1982). All three genera belong to the tribe Pisonieae (Bittrich and Kühn
1
Institute of Evolution and Ecology, Plant Evolutionary Ecolology, Eberhard-Karls-University
Tübingen, Auf der Morgenstelle 1, D-72076 Tübingen, Germany.
2
Departamento de Ciencias Naturales, Universidad Técnica Particular de Loja, Loja,
Ecuador.
*Corresponding author: ingeborg.haug@uni-tuebingen.de
1993). Species of Neea were found to be ectomycorrhizal in the Amazonian

rainforest (Singer and Araujo 1979, Janos 1980a,b, Tedersoo et al. 2010) and
in Peru (Alexander and Högberg 1986) or ecto- and arbuscular mycorrhizal
in an Amazonian lowland rain forest (St. John 1980a,b), in the South of
Venezuela (Moyersoen 1993), and in French Guiana (Béreau et al. 1997).
Species of Guapira were found to be ecto- and arbuscular mycorrhizal in
the South of Venezuela (Moyersoen 1993) and ectomycorrhizal in Western
Amazonia (Tedersoo et al. 2010). Ectomycorrhizas of Pisonia grandis R.Br.
were described from coral cays in the Great Barrier Reef (Ashford and
Allaway 1982, Chambers et al. 2005) and from the Seychelles (Ashford and
Allaway 1985, Suvi et al. 2010). The mycorrhizas associated with Pisonia
grandis have a unique structure with a hyphal mantle, Hartig net, and transfer
cells (Ashford and Allaway 1982, 1985). Arbuscular mycorrhizal infection
was detected in other species of Nyctaginaceae, namely Boerhavia repens,
Bougainvillea spectabilis, Mirabilis jalapa, Pisonia umbellifera (Koske et al. 1992)
and Pisonia seychellarum (Suvi et al. 2010). A low degree of mycorrhization
with only vesicles and mycelium was reported for Boerhavia diffusa (Rachel
et al. 1989) and no mycorrhization was reported for Mirabilis jalapa, Boerhavia
diffusa (Muthukumar and Udaiyan 2000) and Boerhavia coccinea (Khan
1974). Based on these results, the Nyctaginaceae were considered to be a
predominantly non-mycorrhizal family with isolated ectomycorrhizal and
arbuscular mycorrhizal species (Tester et al. 1987, Brundrett 2002, Wang
and Qiu 2006). However, a very small percentage of the ca. 400 species
included in the family (Daly and Roberts 2004) have been investigated so
far (Wang and Qiu 2006). In summary, the available data of ectomycorrhizas
of Nyctaginaceae show typical and unique structures. Only a few fungal
partners are identified with molecular methods. In this study we aim to
enhance the knowledge of morphological features of ectomycorrhizas in
the neotropics and to identify their fungal partners.
2. Ectomycorrhizas of Nyctaginaceae in the Tropical Mountain

Rain Forest of South Ecuador
Two species of Neea and one species of Pisonia sampled in the species-rich
Neotropical mountain rain forest in South Ecuador (Haug et al. 2005) were
found to be ectomycorrhizal. While Neea sp. 1 has typical ectomycorrhizal
structures, Neea aff. floribunda Poepp. & Endl. and Pisonia sp. showed
distinctive and unique anatomical and morphological features.
Ectomycorrhizas of Nyctaginaceae 21
2.1 Ectomycorrhizas of Neea species 1
The root system consists of long and short roots. Ectomycorrhizas are
formed on short roots (diameter 0.2 to 0.6 mm). Five different mycorrhizal
morphotypes were distinguished (Fig. 1a–e). All morphotypes revealed the
typical features of ectomycorrhizas with a hyphal mantle and Hartig net
located between rhizodermal cells (Haug et al. 2005).
Fig. 1. Micrographs of mycorrhizas of Neea species 1 (a-e), Neea aff. floribunda, Pisonia sp. (g-h),
a. Russula puiggarii − Neea species 1, b. Russula-Lactarius − Neea species 1, c. Tomentella-Thelephora
species 1 − Neea species 1, d. Tomentella-Thelephora species 2 − Neea species 1, e. Ascomycete −
Neea species 1, f. Tomentella-Thelephora species 3 − Neea aff. floribunda, g.,h. Tomentella-Thelephora
species 3 − Pisonia sp., g. overview: long roots partially with hyphal mantle (arrows), partially
with root hairs and no hyphal mantle (*), h. one root shown at higher magnification: distal
portion with root hairs (*), proximal portion with hyphal mantle (arrows). Scale bars: a.,b.,f.,h.
1 mm; c.-e. 0,5 mm; g. 5 mm. Data from Haug et al. 2005.
Molecular analyses of the ectomycorrhizal partners of Neea species 1

revealed six different fungi: two Thelephoraceae belonging to the genera
Tomentella-Thelephora (AY667418, AY667419), two Russulaceae (Russula-
Lactarius, AY667426, AY667427), one species of Clavulina (Tedersoo et
al. 2010), and one ascomycete. Most frequent were the thelephoraceous
ectomycorrhizas.
2.2 Ectomycorrhizas of Neea aff. floribunda Poepp. & Endl.
The root system of Neea aff. floribunda consists of brown, straight long
roots which bear bright, unramified fine roots. The fine roots (diameter
0.3 to 0.6 mm) are partially covered with a hyphal mantle (Fig. 1f). Only
one morphotype with a brown and smooth hyphal mantle was found. The
outer mantle layers are loosely plectenchymatous (Fig. 2a) while the middle
Fig. 2. Longitudinal sections of mycorrhizas of Neea aff. floribunda. (a) Outer hyphal mantle:
loose plectenchyma, (b) middle and inner hyphal mantle: dense plectenchyma, (c) tangential
section of the epidermal layer: epidermal outgrowths (*) and Hartig net, d,e. median section
through mycorrhiza: hyphal mantle, epidermal cells with outgrowths (*) and Hartig net, f.
hyphal mantle, Hartig net and intracellular hyphae (with clamps, →) in epidermal and cortical
cells (scale bars: a. - f. 15 µm). Data from Haug et al. 2005.
and inner layers are compactly plectenchymatous (Fig. 2b). A prominent

Hartig net develops between root-hair-like outgrowths of the epidermal
cells (Figs. 2c–e). The root-hair-like outgrowths are attached to the root
surface (Figs. 2d,e) and are elongated cells with a rounded base in tangential
section (Fig. 2c). In median sections, connections of the outgrowths to
epidermal cells are evident (Fig. 2d,*), which occasionally join up and
look like a septum (Fig. 2d). Epidermal cells, cortical cells, and hyphae are
plasmatic. There are regions with intracellular hyphae in the epidermal and
cortical cells and these hyphae form clamps (Fig. 2f). Molecular analyses of
different mycorrhizal tips of Neea aff. floribunda yielded the same sequence
(AY667424). A BLAST search and a NJ-tree clearly showed a membership
within the genera Thelephora/Tomentella (Haug et al. 2005).
2.3 Ectomycorrhizas of Pisonia sp.
The fine root systems of Pisonia consist of only long roots. There are sections
of the long roots (diameter 0.3 to 0.6 mm) covered with a hyphal mantle
and sections without hyphal mantle but with root hairs (Figs. 1g–h). Root
tips covered with a hyphal mantle are rare (Fig. 1h) and many roots have
no hyphal mantle at all. Only one morphotype is distinguishable. The
hyphal mantle is dark brown with silvery patches on the surface (Figs. 1g,
h). Emanating hyphae are colourless and bear clamps. The hyphal mantle
is plectenchymatous throughout, where the outer layers are loosely (Fig.
3a) and the middle layers are densely plectenchymatous (Fig. 3b). The
epidermal layer develops a Hartig net (Figs. 3c–e). Root segments without
hyphal mantle have root hairs. Loose hyphae and intracellular colonization
in the root hairs and in the epidermal cells are visible (Figs. 3f–g). Since
ultrastructural details are the same as in the hyphae of the Hartig net and
hyphal mantle (Figs. 3h-i), we conclude that the intracellular infection is due
to the ectomycorrhizal fungus. All mycorrhizas from four individuals of the
species of Pisonia had identical ITS sequences (AY667420-423). Phylogenetic
analyses of the rDNA ITS region revealed a membership within the genera
Thelephora/Tomentella (Haug et al. 2005).
Morphological and molecular results from ectomycorrhizas of the three
Nyctaginacean species are summarized in Table 1.
2.4 Ectomycorrhizas of Nyctaginaceae
The morphology of ectomycorrhizas found on the short roots of Neea species

1 was also seen in Neea obovata (Moyersoen 1993). Fine root systems of only
long roots such as those of Neea aff. floribunda and the species of Pisonia
were reported from Neea robusta (Moyersoen 1993) and Pisonia grandis
Fig. 3. Longitudinal sections of mycorrhizas of Pisonia sp. (a) Outer hyphal mantle: loose
plectenchyma, (b) middle hyphal mantle: dense plectenchyma, (c,d). tangential sections of the
Hartig net layer adjacent to the outer walls of the epidermal cells, (e) median section through
mycorrhiza: hyphal mantle and prominent Hartig net, (f) tangential section of the epidermal
layer: intracellular hyphae, (g) median section: intracellular hyphae in epidermal cells and
root hairs; (h,i) transmission electron micrographs of hyphal mantle hyphae, intracellular
hyphae in cortical cells and root hairs (scale bars: a. - g. 15 µm, h. - i. 2,5 µm). Data from Haug
et al. 2005.
(Ashford and Allaway 1985). The differences in the root systems correspond
to differing degrees of mycorrhization. While mycorrhization of rootlets
was close to 100% in species forming short root systems (Neea species 1,
Neea obovata, Guapira sancarlosiana; Moyersoen 1993 and results of this
study), species with long root systems showed an incomplete development
of the hyphal mantle and no suppression of root hair formation (Pisonia
grandis, Neea robusta, Neea species 2, Pisonia sp., Ashford and Allaway 1985,
Moyersoen 1993, and results presented here). The combination of long root
systems that are only partly transformed into ectomycorrhizas, with root
Table 1. Ectomycorrhizal types of three Nyctaginacean species in South Ecuador.
Host plant Fungal partner Morphology Sequences

Neea species 1 Russula puiggarii typical ectomycorrhiza AY667426
Neea species 1 Russula-Lactarius typical ectomycorrhiza AY667427
Neea species 1 Tomentella-Thelephora 1 typical ectomycorrhiza AY667411 AY667418
Neea species 1 Tomentella-Thelephora 2 typical ectomycorrhiza AY667412 AY667419
Neea species 1 Ascomycete typical ectomycorrhiza no sequence
Neea species 1 Clavulina sp. typical ectomycorrhiza Tedersoo et al. 2010
Neea aff. floribunda Tomentella-Thelephora 3 long roots covered AY667424
partly with a hyphal
mantle,
root-hair-like epidermal
outgrowths integrated
in the hyphal mantle
Pisonia sp. Tomentella-Thelephora 4 long roots covered AY667420-423
partly with a hyphal
mantle
hair formation that is not suppressed, occasional intracellular penetration

of hyphae, and sporadic formation of transfer cell-like structures has not
been described from any other plant family so far. We hypothesize that this
set of characters represents an early evolutionary step in ectomycorrhiza
formation. Subsequently, the typical ectomycorrhizal state evolved as found
in the short root-forming Nyctaginacean species. Hence, the Nyctaginaceae
may be a model for the evolutionary change from arbuscular mycorrhization
to ectomycorrhization.
Our molecular analyses of the Nyctaginacean fungal partners revealed
two tree species (Neea aff. floribunda, Pisonia sp.) that were associated with
only one thelephoracean taxon. For Neea species 1, six different fungal
partners were found belonging mainly to Thelephoraceae and Russulaceae.
Further investigations of ectomycorrhizal fungi of Neea spp. and Guapira
spp. were done by Tedersoo et al. (2010) in Yasuni National Park in North
East Ecuador. Guapira harboured three to six fungal partners belonging
to five lineages (/cantharellus, /clavulina, /inocybe, /russula-lactarius,
/tomentella-thelephora). Neea had one to ten fungal partners belonging
to four lineages (/amanita, /clavulina, /russula-lactarius, /tomentella-
thelephora).
Pisonia grandis is probably only associated with species of Thelephoraceae
throughout the area and five species of Thelephora-Tomentella are known to
form ectomycorrhizas (Chambers et al. 1998, Chambers et al. 2005, Suvi et
al. 2010).
All investigations revealed relatively low richness of ectomycorrhizal
fungi when compared with the richness of temperate regions (Smith and
Read 2008, Bahram et al. 2012). This may be because of the low amount of
ectomycorrhizal trees in these tropical forests (3 out of 115 investigated tree
species; Kottke and Haug 2004) and because of the scattered distribution
of individual trees. The sampling effort is still low, but species of Neea,
Pisonia, and Guapira have fungal partners that mainly belong to three
lineages (Tomentella-Thelephora, Russula-Lactarius, Clavulina) and several
of the ectomycorrhizal fungi preferred one host genus (Haug et al. 2005,
Tedersoo et al. 2010), a feature uncommon in boreal forests.
The high frequency of Thelephoraceae as fungal partners is
conspicuous. Many species of Thelephoraceae are saprotrophic (Brundrett
et al. 1996) and it is possible that ectomycorrhizal species may also exhibit
saprotrophic activity in order to survive periods without a host. Some of
the thelephoraceous sequences of the different Nyctaginacean hosts were
scattered throughout the phylogram of Thelephoraceae but others were
clustered as sister species (Suvi et al. 2010).
3. Conclusion
Although the common ancestor of Nyctaginaceae may have been
involved with one thelephoroid taxon, coevolution of Nyctaginaceae and
ectomycorrhizal Thelephoraceae in different habitats may have led to the
evolution of several different species. It will be a future challenge to study
the mycorrhizal status of additional species of Nyctaginaceae in South
America and identify their mycobionts to clarify the question of potential
co-evolution.
Acknowledgements
T h i s re s e a rc h w a s g e n e ro u s l y s u p p o r t e d b y t h e D e u t s c h e
Forschungsgemeinschaft (DFG project FOR 402, 816). We thank the
Fundacíon Científica San Francisco, Ecuador, the NCI for providing
research facilities, Jürgen Homeier and Jeremy Hayward for identification
of Nyctaginaceae, and New Phytologist for the permission to reprint the
figures (copyright 2005). The skilful technical assistance of Jutta Bloschies
is much appreciated. We would like to thank Tanja Schuster for editing
the manuscript. We also thank Krista L. McGuire and Caitlyn Gillikin for
improving the English.
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New York, pp. 3–9.
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and high host preference of ectomycorrhizal fungi in Western Amazonia, a neotropical
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plants. Mycorrhiza 16: 299–363.
CHAPTER
3
Diversity and Abundance of
Ectomycorrhizal Associations
in Rain Forests of Cameroon
under Different Disturbance
Regimes
Onguene Awana Nérée,1,* Judith Marthiale Tsamo,2
Christelle Michaella Ebenye,3 Amadou Mustapha Bâ3
and Thomas Kuyper4
1. Introduction
In the rainforests of tropical Africa, two kinds of mycorrhizal associations
occur: arbuscular mycorrhiza (AM) and ectomycorrhiza (Alexander 1989,
Onguene and Kuyper 2001, Bâ et al. 2011). Whereas most timber trees form
AM, the ectomycorrhizal (ECM) habit occurs in a very limited number of
plant families, viz. Asterpeiaceae, Caesalpiniaceae (a family that is partly
arbuscular mycorrhizal), Dipterocarpaceae, Papilionoideae, Phyllanthaceae
1
Institute of Agricultural Research for Development, Soil, Water and Atmosphere department,
Yaoundé, Cameroun.
2
Department of Plant biology and physiology, University of Yaoundé I, Cameroon.
3
Laboratoire Commun de Microbiologie (LCM) IRD/UCAD/ISRA, Centre de Recherche de
Bel Air, BP. 1386, CP. 18524 Dakar, Senegal.
4
Department of soil quality, University of Wageningen, the Netherlands.
*Corresponding author: nereeoa678@yahoo.fr
(formerly Uapacaceae), Sarcolaenaceae, Sapotaceae, and Gnetaceae. This

may explain the “high latitudinal bias” of the “ecological insignificance”
of ECM in the tropics found in earlier literature (Alexander 1989, Tedersoo
et al. 2009, Bâ et al. 2011). ECM associations prevail in both the Paleo- and
Neotropics (Fassi and Moser 1991). In the Congo basin, they occur in the
forest continuum from the Cameroonian Atlantic Ocean coast to the open
forest in the DR Congo (Buyck 1994), through the Congo, parts of the
Central African Republic and most of the Gabon (De Saint Aubin 1963).
These forests could be the hotspots of soil fungal biodiversity in Central
Africa. Yet, little information and limited data exists on their occurrence,
regeneration requirements, and present functioning under such threats as
mining, climate change, agriculture, and logging.
Recent inventories of ectomycorrhizal fungi based on molecular
characterization had revealed that native ECM fungal genera in tropical
Africa belong, to a large extent, to the same Basidiomycete taxa as those
that provide the fungal partners of temperate ECM plants (Boa 2006, Rivière
et al. 2007). Common ECM taxa in the tropics belong to the Amanitaceae,
Boletaceae, Cantharellaceae, Russulaceae and Sclerodermataceae (De
Kessel et al. 2002, Smits 1994). However, on species level, hardly any ECM
fungal species (except when introduced) are shared between both regions
(Boa 2006, Rivière et al. 2007). Furthermore, many ECM fungal species are
poorly documented or have their microscopic details poorly illustrated:
a reliable key to all African ECM fungal species is lacking and many
species remained unnamed. Various species of Amanitaceae, Boletaceae,
Cantharellaceae and Russulaceae have been described from West African
rainforests, with virtually no members of the Cortinariaceae (Buyck et al.
1996). Mycoinventories in forests of Korup National Park in SW Cameroon
yielded close to 40 species of suspected mycorrhizal fungi. In forests of SW
Burkina Faso, 27 ECM fungal species were described beneath indigenous
Afzelia and Uapaca trees (Sanon et al. 1997). Using a fragment of the
mitochondrial large subunit rRNA gene, more than 100 ECM sporocarps
were typed from Ceasalpiniaceae and Phyllantaceae in a Guinean tropical
rain forest (Rivière et al. 2007). It was also concluded that from the same
Guinean forest, six taxonomic groups such as boletaceae, sclerodermataceae,
russulaceae, telephoraceae, amanitaceae and trichomolotaceae predominate
and are shared between canopy trees and seedlings (Diédhiou et al. 2010).
The presence of these multi-host ECM fungi in a Guinean rainforest suggest
the formation of common ectomycorrhizal networks between differently
aged trees just as observed in the rainforest of south Cameroon (Onguene
and Kuyper 2002).
Our primary goals were to characterize the below-ground ectomycorrhizal
diversity and taxonomic structure of ECM forests in Cameroon, and to
test whether ectomycorrhizal fungal community composition changed
Ectomycorrhizal Associations in Rain Forests of Cameroon 31
following land use changes. The objectives of this study were to report on
the inventory of ectomycorrhizal associations in humid forests of Cameroon
including their habitats (soil and litter characteristics), host tree species,
ectomycorrhizas and sporocarps, and assessing the effect of logging and
agriculture practices on ECM inoculum potential.
2. Study Sites and Selection of Vegetation Types

The study was carried out in western portions of the Atlantic Biafrean forest
of south Cameroon, a humid, tropical climate region with two distinct wet
seasons (March-June and August–November) and two dry seasons. Rainfall
decreases in an easterly direction from Ebimimbang (1500 mm rainfall)
to Nyangong and Bityili (1900 mm rainfall). Along the same gradient,
pH and phosphorus availability decrease, as well as land use intensity
and consequently forest vegetation (Table 1). In the lowlands, only a few
undisturbed, near-primary forest fragments remain while a large part of the
area is deforested; in the hilly areas, undisturbed late-secondary forests are
more common. Four experimental sites were selected in Ebimimbang (low
elevation), Ebom (mid elevation), Nyangong, and Bityili (high elevation).
In each site, field plots were selected in seven vegetation types, viz.
ectomycorrhizal forest clumps (EF), late-successional forest stands outside
the crown projection of ectomycorrhizal clumps (LS), early-successional
forest stands (ES), agricultural fields of food crops with plantain (Musa spp.),
cocoyam (Xanthosomas esculenta), groundnut (Peanut hypogea), and cassava
(Manihot esculenta) as the major crops (FI), Chromolaena odorata fallows
(FA), C. odorata fallows with the liana Gnetum (GN), and sites of forestry
practices such as skid trails and bare landings. The presence of Gnetum spp.,
Table 1. Localization, elevation, rainfall and soil physicochemical characteristics of forest

clumps at the four research sites.
Research sites Ebimimbang Ebom Nyangong Bityili

Localization 3°02.67’N; 3°04.73’N; 2°58.11’N; 2°56.07’N;
10°28.25’E 10°41.24’E 10°45.18’E 10°49.55’E
Altitude (m.a.s.l) 0–350 350–500 500–800 >800
Rainfall (mm) 1556 1987 1677 1800

Soil texture Sandy Highly clayey Highly clayey Very highly
clayey
pH 5–6 4–5 3–4 3–4
Carbon (%) 1.70 2.30 3.28 5.70
Nitrogen (%) 0.11 0.14 0.20 0.36
C/N ratio 15,5 16,4 16,4 15,9
Phosphorus (µm/ml soil) 0.01 0.005 0.002 0.000
an ectomycorrhizal plant, was considered important as this liana might

provide ECM inoculum that facilitates establishment of ectomycorrhizal
seedlings in former agricultural lands.
3. Sampling and Identification of Sporocarps

Along forest trails leading to permanent sampling plots, sporocarps
collections were carried out during both wet seasons with the help of local
assistants, one week per month, for three years. Collected specimens were
macroscopically described in fresh state before twilight. After description,
mushrooms were dried for 2–3 days at about 40°C. Dried mushrooms were
temporarily preserved in a local herbarium in Kribi, Cameroon (2°57’N;
9°55’E) before being sent to Wageningen, the Netherlands, for microscopic
examination and identification.
Distinction between Amanita was based on margin striation, presence
of a saccate volva, cap color, hygrophany, and spore amyloidy; Distinction
between Cortinarius species was based on cap hygrophany, translucence,
color and texture of hymenium margins, and presence of a cortina or
fibrillose dry ring; Species of Inocybe were separated on the basis of pileus
texture (radially fibrillose cap), and shape and texture of stipe; Genera of
Boletales varied in pileus color and shape, and were distinguished on the
basis of poroid hymenium, presence of large cystidia with or without clamp
connections and spore ornamentation. Russula species varied in pileus size,
color and shape, brittleness of gills, presence or absence of lamelullae and
an evanescent ring on stipe; they were differentiated from Lactarius on the
basis of exudation upon flesh injuries and the extension of the lactiferous
system into the hymenium. Species of Cantharellus differed in pileus texture,
hymenium color, and spore ornamentation. Several provisional keys were
elaborated based on morphological and anatomical characters of sporocarps
(Onguene 2000). Voucher specimens are kept at the National Herbarium
of the Netherlands (Leiden Branch) and some duplicates are preserved at
the agricultural research station in Kribi, Cameroon. Several manuals were
used for species identification (Onguene and Kuyper 2012).
4. Morphological and Anatomical Description of Root Tips

and Ectomycorrhizae
After identification and selection of ectomycorrhizal host trees, four root
samples were collected around the base of each host tree, washed gently
in water, and preserved in 50% alcohol or wrapped in aluminum foil with
litter and soil (when alcohol was exhausted in the field). In the laboratory,
root systems were cleared of soil debris by careful washing under a water
flow while immersed in tap water. Seven to ten selected root tips for each
morphotype representative were removed from the root sample and
morphologically and anatomically observed. The key morphological and
anatomical features examined under a dissecting microscope at 40x included
root tip branching, shape of branches, mantle colour and surface texture,
presence of rhizomorphs, emanating hyphae. Tips were also observed
under a photonic microscope between 25 and 40x to confirm the presence
of a mantle and a Hartig net, various layers of hyphal arrangement, and
to determine the presence of specialized cells, rhizomorphs and cystidia,
using both cross—and longitudinal sections (Agerer 1995).
5. Effects of Agriculture and Logging Practices on ECM

Inoculum Potential
To assess whether agriculture or logging practices impact ECM inoculum
potential and whether arbuscular mycorrhizal (AM) and ectomycorrhizal
fungi share the same niche, three bioassays were carried out using intact
soil cores collected from the seven disturbance stages.
In 1-ha forest plots, 100 m² quadrats were laid down. In each quadrat,
soil cores were dug out of three spots, each 50 m apart. Relatively
undisturbed, intact, cylindrical soil monoliths were collected (about 4.2–4.5
kg; wet weight basis) by driving a 15 cm diameter x 45 cm long PVC tube
into the ground with a hammer dropped from a constant height (10–20
cm) onto a flat steel plate, placed on top of the PVC tubes. In agricultural
food crops, fallow, and forestry plots, plot sizes were 100 m² and 100 m
long, respectively, and intact cores were removed as previously described.
Triplicate core samples were removed intact from each land-use per site.
However, due to different fruiting patterns of both trees, the bioassays were
carried out at different periods of the year: March to June for Tetraberlinia
and July to October for Afzelia.
Two native timber species, both belonging to the Ceasalpiniaceae, were
used for the bio-assay. Tetraberlinia bifoliolata is an ectomycorrhizal tree
that is valuable as a potential novel timber tree. Afzelia bipindensis is a dual
mycorrhizal tree that provides a highly priced redwood timber. Hereafter,
trees will be designated by their generic names only. Tetraberlinia typically
occurs in clumps together with other caesalp species, while Afzelia usually
occurs isolated in matrices of AM trees and has not been observed in
clumps with other ECM trees. Both tree species have large pods (10–20 x
5–8 cm) with a small number of large and heavy seeds; average seed size of
Tetraberlinia was 20–30 x 15–25 x 5–7 mm and that of Afzelia 30–40 x 20–30
x 10–20 mm; average seed mass of Tetraberlinia was 1.5 g (0.8–2.7 g) and
that of Afzelia 11.5 g (6.2–17.4 g). Seedlings of both species possess coarsely
branched roots with few root hairs (Onguene and Kuyper 2001).
Seeds were germinated for a week in steam-sterilized sand without
pregermination treatment. One 1-week old seedling of each tree was
placed in a small hole at the center of the soil core. Cores were placed on
benches and plants grown for four months under natural light conditions
in a greenhouse in Kribi in a randomized complete block design, watered
every three days to maintain soils at field capacity, without nutrient
amendments.
To determine whether ECM and EM fungal propagules share the same
niche, a local variety of cowpea, Vigna unguiculata (Fabaceae), was grown in
soil cores from ECM clumps. In addition, four intact soil cores were collected
around the stem base of ECM tree species, at 5 m and 10 m distance away
from the stem base of A. bipindensis and Brachystegia cynometroides at the
Ebom site, T. bifoliolata and Paraberlinia bifoliolata at the Bityili site. Cowpea
plants were raised for a month in the greenhouse under the same conditions
as previously described. At harvest, ECM fractional colonization was
assessed in water without staining while portions of Afzelia root samples
stained with acid fuchsine were assessed by the gridline intersect method
(Onguene and Kuyper 2001).
6. Experimental Design and Statistical Analysis

For Tetraberlinia, the experiment was a full factorial with two factors, site
(3 levels) and disturbance stage (7 levels). For Afzelia, a partial factorial
experiment was executed with soils from three sites and three forest types
(ECM clumps, late-successional stands, early-successional stands) owing
to seed shortage. The full set of disturbance stages was only investigated
for the Ebom soil, where the tree was fairly common and widespread.
The SAS package (SAS Inc. 2004) was used for statistical analysis. Data
were first tested for normality and homogeneity of variances using the
Levene test in the one-way analysis of variance (ANOVA). Data on ECM
fractional root colonization by Tetraberlinia contained many zeroes and
did not meet the requirements of normal distribution and homogeneous
variances. Therefore, the non-parametric Kruskall-Wallis test was applied.
When data analysis was restricted to the three forest stands (ECM clumps,
late- and early-successional stands), data of fractional ECM root colonization,
after arcsin square root transformation did meet the requirements for
ANOVA. Data on ECM and AM root colonization by Afzelia were also arcsin
square-root transformed.
7. Habitat Types of Ectomycorrhizal Associations in Cameroon

Ectomycorrhizal forest clumps occurred on sandy, highly clayey and very
highly clayey soils, throughout the entire landscape, even on swamps
like Gilbertiodendron clumps. They were acidic to very strongly acidic. The
mineral contents of N and available P in ECM forest clumps were very
low, sometimes undetectable such as in the Bityli soils, irrespective of sites
and soil texture. C/N ratio was always larger than 10 (Table 1). The layers
of litter were thin (one to two), tough, and varied from one forest clump
type to the other.
In the humid forests of Cameroon, ectomycorrhizal associations
occurred as small to large forest clumps of five types: Gilbertiodendron
monodominant clumps, Uapaca monodominant clumps, monodominant
Microberlinia clumps in the Korup National Park (Newbery et al. 1988),
oligo-dominant ceasalp clumps locally called “Ekop,” and mixed clumps
of “Ekop” with Uapaca spp.
Twenty-four tree species were found to be symbiotically associated with
ectomycorrhizal fungal species on the basis of root tip colonization. They
belonged to two botanical families: Caesalpiniaceae and Phyllantaceae.
Whereas most ECM host tree species occurred in one of the five ECM
clumps, six host tree species including Afzelia, Anthonotha and Berlinia
were always isolated in matrices of arbuscular mycorrhizal host plants
(Table 2).
The contribution of ECM host tree species to basal area of forest clumps
varied from 20 to 80%, and 100% in Gilbertiodendron clumps. The basal area
diameter of ECM trees was low in young and high in adult ECM trees,
respectively (Table 3).
8. Cameroonian Ectomycorrhizae and Sporocarp Diversity

Nineteen ectomycorrhizal morphotypes were described and found differing
by mantle color (Black, reddish, whitish, brown black, chestnut), external
textures (smooth, strongly rhizomorphic), thickness, and presence or
absence of cystidia (Table 4).
Found throughout all land types, including swampland, ECM forest
clumps were the only habitats for most Cameroonian ECM fungal diversity:
more than 1000 sporocarps were collected over three years and nearly 200
ECM fungal species were identified, belonging to eight families and 27
genera, mostly new to science. Only Lactarius gymnocarpus and all Cantharella
species were locally edible (Table 5).
Table 2. List of ectomycorrhizal tree species by types of ectomycorrhizal forest habitats in

Cameroon.
Habitat types Family Species Pilot name

Not observed in Caesalpiniaceae Afzelia bipindensis Doussie rouge
clumps (Tribe Detareae) A. pachyloba Doussie blanc
Caesalpiniaceae Anthonotha fragans Enak
(Tribe Amherstieae) A.macrophylla Enak
Berlinia bracteosa Ebiara
B. confusa Ebiara
Gilbertiodendron Caesalpiniaceae Gilbertiodendron dewevrei Abem
monodominant G. brachystegioides Abem
clumps
Uapaca Phyllantaceae Uapaca acuminata Rikio
monodominant or U. buchholizianum Rikio
mixed clumps U. guineensis Rikio
U. vanhoutei Rikio
Microberlinia Caesalpiniaceae Microberlinia bisulcuta* Zingana
monodominant
clumps
Mixed Caesalpiniaceae (Tribe Brachystegia cynometrioides Ekop naga
oligodominant Amherstieae) B. euricoma Ekop evene
ceasalp clumps B. zenkeri Ekop gombe
Didelotia africana Ekop rouge
Didelotia letouzeyi Ekop
Julbernadia seretii Ekop
Monopethalanthus letestui Ekop blanc
M. microphyllus Ekop mayo
Paraberlinia bifoliolata Ekop mayo ngang
Tetraberlinia bifoliolata Ekop beli
Touabouate brevipaniculata Ekop ribi
Ekop zing
Mixed Uapaca and Phyllantaceae and Various Uapaca and Ekop Rikio and Ekop
ceasalp clumps Caesalpiniaceae (Tribe species
Amherstieae)
Notes: Pilot names of “Ekop” were taken in Letouzey and Mouranche (1952). *Data from
Newbery et al. (1988).
Table 3. Basal stem diameter in different forest clump types in 1000 m² area (Average
observations of three years during the dry season).
Basal stem Mixed Gilbertiodendron Uapaca Microberlinia

diameter size oligodominant monodominant monodominant monodominant
(cm) ceasalp clumps clumps
<10 7 0 10 0
10–49 15 0 57 0
50–99 45 50 157 176
>99 164 250 98 >350
Table 4. Morphological and anatomical characteristics of some Cameroonian ectomycorrhizae.
Ectomycorrhizal Color Shape Texture Texture of mantle Thickness Presence of Presence of

morphotype External Internal (µm) rhizomorphs cystidia
collection number mantle mantle
CM 400 Yellow gold monopennate Thread like Felty Synenchyma 100.8–201.5 Present Absent
Gnetum spp. prosenchyma
CM 401 Milky white monopennate Smooth Felty Synenchyma 255–408 Absent Absent
Paraberlinia prosenchyma
bifoliolata
CM 402 Orange brown monopyramidal Smooth Felty Synenchyma 100.5 Absent Present
Uapaca guineensis prosenchyma
CM 403 Brown monopyramidal Smooth Regular Net 61.2 Absent Absent
Gilbertiodendron Synenchyma prosenchyma
dewevrei Irregular
Synenchyma
CM 404 Brown monopyramidal Spiny Felty Synenchyma 100.2 Absent Present
Uapaca guineensis prosenchyma
CM 405 White monopyramidal Felty or velvety Felty Synenchyma >533 Absent Absent
Didelotia letouzeyi prosenchyma
CM 406 Brown monopyramidal Cotton like Felty Synenchyma 408–510 Absent Absent
Julbernadia seretii prosenchyma
CM 407 Brown monopyramidal Smooth Felty Synenchyma 25–30 Absent Present
Uapaca acuminata prosenchyma
CM 408 White monopyramidal Felty Felty Synenchyma 100.8–183.9 Absent Present
Monopethalanthus prosenchyma
letestui
CM 409 White monopyramidal Felty Felty Net 162–200.4 Present Absent
prosenchyma prosenchyma
Table 4. contd....
Table 4. contd.
38
Ectomycorrhizal Color Shape Texture Texture of mantle Thickness Presence of Presence of

morphotype External Internal (µm) rhizomorphs cystidia
collection number mantle mantle
CM 410 White monopyramidal Cotton like Felty Synenchyma 255 Present Absent
Touabouate prosenchyma
brevipaniculata
CM 411 White monopennate Cotton like Felty Synenchyma 390–650 Present Absent
Gilbertiodendron prosenchyma
dewevrei
CM 412 Brown monopennate woolly Felty Synenchyma 110–119.2 Present Absent
Paraberlinia prosenchyma
bifoliolata
CM 413 Brown monopyramidal woolly Felty Synenchyma 306–561 Present Absent
Monopethalanthus prosenchyma
letestui
CM 414 Brown monopyramidal woolly Felty Synenchyma 540–615 Present Absent
Didelotia letouzeyi prosenchyma
CM 415 Brown or monopennate woolly Felty Irregular 510–765 Present Absent
Tetraberlinia maroon or prosenchyma Synenchyma
bifoliolata brown maroon
CM 416 Brown monopyramidal woolly Felty Net 306–459 Present Absent
Uapaca guineensis prosenchyma prosenchyma
CM 417 Brown maroon monopennate Cotton like to Felty Net 104 Absent Absent
Uapaca acuminata à maroon woolly prosenchyma prosenchyma
CM 418 Brown monopyramidal Cotton like Felty Synenchyma 403–502.3 Absent Present
Touabouate prosenchyma
brevipaniculata
Table 5. List of putative ectomycorrhizal mushroom species by family, genus and species in
humid forests of South Cameroon.
Amanitaceae Boletaceae Russulaceae

Amanita afzeliae sp. Afroboletus luteolus (Heinem) Russula afronigricans Buyck
A. albidodisca sp. P. & Y. R. albospissa Buyck
A. albopulverulenta sp. Boletus boletiformis sp. R. annulata Heim
A. afrobispora sp. B. macrocystis sp. R. annulatobadia Beeli
A. afroflavescens sp. B. nyangongensis sp. R. apsila Buyck
A. afrorubescens sp. B. pustulatus Beeli R. intrica Lizoii
A. afrovaginata sp. B. suspinulosus sp. R. areolata Buyck
A. annulatovaginata Beeli Boletellus sulcatipes Hein. & Goo. R. aurantiofloccosa Buyck
A. aureofloccosa Bas Chalciporus clypeatus sp. R. camerunensis sp.
A. bingensis Beeli Gyrodon aberrans sp. R. cellulata Buyck
A. brachystegiae sp. Gyroporus microsporus (Sing. & R. chrysotricha sp.
A. calopus Beeli Grinl.) Heinem & Rammeloo R. declinata Buyck
A. crassiconus Bas Leccinum excedens sp. R. diffusa var. fissurans
A. elegans Beeli Paxillus brunneotomentosus sp. R. discopus Heim
A. flavovirens sp. Paxillus camerunensis sp. R. echnosperma R. Heim &
A. fulvosquamulosa Beeli Phlebopus braunii (Bres.) Heinem Gilles
A. gigavolvata sp. P. silvaticus Heinem R. fulvoochrascens Buyck
A. griseofarinosa Hongo Phlebopus sp. R. heliochroma Heim
A. lanosa Beeli Phylloporus depressus Heinem R. intricate Buyck
A. leucoagaricioides sp. Pulveroboletus aberrans Heinem R. kivuensis Buyck
A. luteoflava Beeli P. viridis Heinem R. lamprocystidia (Nakasone)
A. monopetalanthi sp. Rubinoboletus luteopurpureus R. liberiensis Buyck
A. nigropyramis sp. (Beeli) R. macrocystis sp.
A. pseudoafroalba sp. Strobilomyces echinatus Beeli R. mimetic sp.
A. pseudolanosa sp. S. luteolus Heinem R. pausiaca Buyck
A. pustulata sp. S. strobilaceus (Berk.) R. pseudocarmesina Buyck
A. roseocinnamomea nov. S. velutipes Corner R. pseudopurpurea Buyck
A. rubescens (Pers.:Fr) G Tubosaeta alveolata Heinem R. speudostriatoviridis Buyck
A. strobilaceovolvata Beeli T. brunneosetosa (Singer) E. Horak R. striatoviridis Buyck
A. subviscosa Beeli T. goosseniae sp. R. testaceoaurantiaca Beeli
A. sulphurea sp. Tylopus violaceus sp. R. velutina (DC per Pers.:Fr)P
A. virella Beeli R. yaeneroensis Buyck
Clavulinaceae
Clavulina vanderstii sp.
Sclerodermataceae
Scleroderma sinnamariense Mont
S. roseacarneum sp.
Coltriciaceae
Coltricia spathulata (Hooker)
Murill
C. pyrophila (Wakef) Ryvarden
Table 5. contd....
Table 5. contd.
Inocybe Cantharellaceae Lactarius

Inocybe afronodulosa sp. Gomphus brunneus* (Heinem) Lactarius acutus Heim
I. afrostellata sp. G. clavatus (Pers.) Gray L. annalatoangustifolius sp.
I. korupensis sp. Craterullus crispus Fr L. claricolor sp.
I. bipindensis sp. C. cornucopioides Persoon L. denigricans Ver. & Kar.
I. perpusilla Velen Cantharellus camerunensis sp. L. densifolius Ver. & Kar.
I. zingii sp. C. cibarius var.roseocanus Arora & L. gymnocarpus Heim
Dunham L. kivuensis deWitte
Cortinarius C. congolensis Beeli L. medusa Verbeken
Cortinarius afroconicus C. dichrous (Fr.) Bres L. pumilus Verbeken
sp. C. densifolius Heinem L. pulcrispermus sp.
C. diobensis sp. C. floridulus Heinem L. sesemotani Beeli
C. ionopygmaeus sp. C. isabellinus Heinem L. undulates Verbeken
C. luteopunctatus (Beeli) Heinem
C. miniatescens Heinem
C. microcibarius Heinem
C. pseudocibarius Hennings
C. rufopunctatus (Beeli) Heinem
Note: Gomphus brunneus is a cantharella species restricted to Cameroon, RD Congo and
Uganda.
Ten provisionary keys to Cameroonian species were developed for

Amanitaceae, Boletaceae, Cantharellaceae, Clavulinaceae, Coltriciaceae,
Cortinariaceae, Inocybaceae, Russulaceae (Russula and Lactarius) and
Sclerodermaceae (Onguene 2000).
9. Effect of Disturbance Regimes on Ectomycorrhizal

Inoculum Potential
Non-parametric analysis of variance indicated that fractional ectomycorrhizal
colonization of Tetraberlinia was significantly affected by disturbance stage (p
< 0.001), but not by site (p = 0.125). Four-month seedlings grown in soils from
forestry practices, agricultural fields and fallow without Gnetum remained
free of ECM colonization. Seedlings in soils from fallow with Gnetum
from all three sites were colonized to some extent. A two-way analysis of
variance for ECM colonization of the three forest stands (EC, LS, and ES)
indicated that both site and disturbance stages were statistically significant,
whereas their interaction was not (Table 6). A rank by Mann-Whitney
U-test indicated that colonization was highest in soils from ECM clumps
and late-successional forests (Fig. 1A). ECM inoculum increased during
succession, with forest clumps showing a significantly higher colonization
than late-successional stands, but being low in early-successional stands.
ECM colonization was the highest in soils from Nyangong and lowest in
soils from Ebom (Table 7).
Table 6. Two-way analysis of variance of site and disturbance stages on ectomycorrhizal

colonization of four-month old seedlings of Tetraberlinia bifoliolata.
Sources of variation Df P F
Site 2 52.7 0.000
Disturbance stage 2 88.9 0.000
Site x disturbance stage 4 0.4 0.836
Table 7. Ectomycorrhizal colonization (percent root length colonized) of seedlings of

Tetraberlinia bifoliolata (T) and Afzelia bipindensis (A) in various forest stands. Letters indicate
significant differences according to Duncan’s Multiple Range Test at p < 0.05. Late-SF: late
successional forest; Early-SF: Early successional forest.
Forest types Ebimimbang Ebom Nyangong Bityili

T A T A T A T A
Forest clump 48bc 22a 32cd 5c 81a 34a 89a 53a
Late-SF 28d 26a 12e 0c 52b 13b 47b 28b
Early-SF 4f 1c 0f 11b 20dc 0c 15c 31c
A non-parametric analysis of variance indicated that ectomycorrhizal

colonization of Afzelia in soils from the three forest stands (EC, LS, and
ES) was neither significantly influenced by site nor by disturbance stage
(p > 0.1). No or very little colonization by ectomycorrhizal fungi was
observed in soils from a Gilbertiodendron clump in Ebom and in soils from
early-successional forest from Ebimimbang and Nyangong. For the Ebom
soils only, cores taken under a mature Afzelia (CO) resulted in the highest
fractional ectomycorrhizal root colonization. Colonization was high in sites
of agricultural practices (fields, fallow) and declined in soils from later-
successional stages. In soils from forestry practices and ectomycorrhizal
clumps, no ectomycorrhizal colonization was observed (Fig. 1B). In
the three forested disturbance stages, there was no correlation between
ectomycorrhizal inoculum potential as assessed by Tetraberlinia and Afzelia
(r = 0.50, n = 9; p > 0.1).
Analysis of variance for ectomycorrhizal colonization of Afzelia in
soils of forest stands (EC, LS, and ES) indicated only the interaction of
disturbance stage and site was statistically significant (Table 6). No or
very little colonization by native ECM fungi was observed in soils from
a Gilbertiodendron monodominant clump in Ebom and in soils from early-
successional forest from Ebimimbang and Nyangong. For the Ebom soils
only, ECM colonization was highest in sites of agricultural practices (fields,
100
A
)
80
Percent ECM root tips (%)
%
(
sp
it
to
or 60 a
M
C
E
tn 40
ec a
re
P
20
b
b
0 b b b
FP FI FA FG ES LS EF
Disturbance stages
100
B
80
Percent ECM root tips (%)
a
60
ab
40
ab
20
bc
c
d d
0
FP CO FI FA ES LS EF
Disturbance stages
Fig. 1. Ectomycorrhizal fractional colonization of seedlings of Tetraberlinia (A) and Afzelia

(B) grown in soils from different disturbance stages (average from three sites). Significant
differences between disturbance stages (Mann-Whitney U-test; p < 0.05) are indicated by
different letters. Abbreviations are as follows: EF = ectomycorrhizal forest clumps; LS =
late-successional forest stands outside the crown projection of ectomycorrhizal clumps; ES =
early-successional forest stands; FI = agricultural fields of food crops with plantain (Musa spp.),
cocoyam (Xanthosomas esculenta), groundnut (Peanut hypogea), and cassava (Manihot esculenta)
as the major crops; FA = Chromolaena odorata fallows; FG = C. odorata fallows with the liana
Gnetum; FP = sites of forestry practices such as skid trails and bare landings.
fallow) and decreased in soils from late-successional stages. Soil cores taken
under mature Afzelia trees resulted in even higher fractional ECM root
colonization. In soils from forestry practices and ECM clumps, no ECM
colonization was observed (Fig. 1). Fractional AM colonization was always
lower than 5%; no colonization by indigenous AM fungi was observed in
soil cores from forestry practices and from ECM clumps.
Most soil cores did not produce abundant arbuscular mycorrhizal
fungal colonization on roots of Vigna unguiculata. AM colonization was
detected in 56% (54 out of 96) soil cores from forest clumps. The sparse AM
colonization varied with sites: very low to low in clumps in Ebimimbang
and Ebom, and completely absent in Nyangong. No AM colonization was
observed in soil cores around the stem base of Afzelia, Brachystegia, and
Paraberlinia, but AM colonization varied from 2 to 22% in the vicinity of
Tetraberlinia trees.
In humid forests of south Cameroon, five types of ectomycorrhizal
forest clumps exist but no longer regenerate, as observed by the dominance
of only the adult size class of individuals: Gilbertiodendron monodominant,
Microberlinia monodominant in the Korup National Park (Newbery et
al. 1988), Uapaca monodominant, Uapaca oligo-dominant, mixed oligo-
dominant ceasalps, mixed ceasalp and Uapaca trees. If six ECM timber
species out of 24 occur isolated in the midst of arbuscular mycorrhizal plant
species, most ECM host species formed clumps, either canopy dominant
or oligo-dominant. They varied in size from small to medium in the
Bipindi-Lolodorf-AkomII zone, large in the Korup National Park and very
large in the forests of south-east Cameroon, like the 10 km long stretch of
monodominant Gilbertiondendron clumps along the road from Dja River to
Ngoïla (Onguene, Amadou and Ebenye, pers.obs.). Botanical inventories
in Gabon, Congo, Central African Republic, and the Democratic Republic
of Congo also depicted such clumps of ceasalp and Uapaca tree species (De
Saint Aubin 1963, Ndong et al. 2011). On the other hand, ECM associations
forming clumps are limited elsewhere in the Neotropics. Only two genera,
Dycimbe and Aldinia have consistently been shown to form such associations
(Singer et al. 1983, Smith et al. 2011). ECM fungi have also been described
in humid forest relics of West Africa (Diédhiou et al. 2010) and in the open
forest of the Zambesian region (Buyck 1994). Such a variety of habitat
conditions cannot be explained by environmental context and floristics
alone (Smith et al. 2011).
9.1 Ectomycorrhizal forest clumps grow only in specific

regeneration niches
In tropical Africa, most timber trees form arbuscular mycorrhiza (AM) but
the ectomycorrhizal habit occurs in a very limited number of plant families.
Co-occurrence of both mycorrhizal types has raised the question of whether

trees with different kinds of mycorrhiza show niche partitioning. Niche
partitioning could occur along various axes. ECM and AM trees could show
edaphic specialization. This niche differentiation has been suggested for
forests in the Amazonian region, with forests on podzolic or white sands
being dominated by ECM trees and forests on brown sand being dominated
by AM trees (Singer and Araujo 1986). Some supporting evidence for this
kind of niche differentiation has also been put forward for the rainforest of
Korup National Park, in south-west Cameroon (Newbery et al. 1988).
However, spatial separation of ECM and AM trees does not necessarily
provide support for a hypothesis on edaphic niche differentiation. Tropical
ECM trees often show conspicuous gregarious behavior in monospecific
or at least plant species-poor stands (Connell and Lowman 1989) and this
habit is well-known from ECM trees in the African rainforest (Letouzey 1968,
Newbery et al. 1997, Onguene and Kuyper 2001). In such clumps, taxa from
the Caesalpiniaceae, tribe Amherstieae, constitute the largest contribution
to basal area. The occurrence of related tree taxa in these clumps has been
previously noted by tree prospectors in Cameroon and Gabon, where these
unnamed caesalps were collectively known as “ekop” and “andoung”,
respectively (Letouzey and Mouranche 1952, De Saint Aubin 1963).
Richards (1996) stated that the gregarious behaviour might be a
consequence of the limited dispersal ability of either partner forming the
ECM symbiosis. As both ECM tree and ECM fungus do not possess the
capacity to grow and reproduce independently of the other symbiotic
partner, mycorrhizal establishment on new sites might be a rare chance
event (Janos 1996). After the ECM symbiosis is initiated, such trees could
serve as focal points for the establishment of other trees that are compatible
with the ECM fungus, thereby setting up the regeneration niche of ECM
trees. Our data provide better support to this hypothesis. All over the area,
ECM clumps did not show strong regeneration of intermediate plant size
(Table 3).
Our data provide support for the hypothesis that ectomycorrhizal
and arbuscular mycorrhizal trees differ in their regeneration niche (Grubb
1977). In a separate study, it was found that inoculum potential of AM fungi
was about twice as high in early-successional as in late-successional forest
(Onguene 2000). In that study, the effects of ECM clumps on inoculum
potential of native AM fungi had not been addressed. Mycorrhizal
colonization of Vigna unguiculata indicated that in ECM clumps the
inoculum potential of AM fungi is very low. Apparently, the buildup of
ECM inoculum together with AM inoculum decline. Direct competitive
interactions between ECM and AM fungi have also been postulated by
Moyersoen et al. (1998). Inoculum of ECM fungi in forested sites was highest
in Bityili and lowest in Ebom. These data are consistent with the relatively
large contribution of ECM trees to basal area (Onguene and Kuyper 2001)
and to inventories of ECM fungi (Onguene and Kuyper 2012), where Bityili
was highest and Ebom lowest.
9.2 Ectomycorrhizal regeneration niches are fungal biodiversity

hotspots
Nineteen ECM anatomo-morphotypes were described for the first time,

though not from Microberlinia forests. They differed in external texture,
mantle thickness, and presence or absence of rhizomorphs and/or cystidia.
They also differed markedly from the conspicuous yellow gold ECM of
Gnetum species (Eneke and Alexander 2012). Therefore, it appears that
Gnetum plants found in fallows of Chromolaena odorata could not serve as
focal points for the regeneration of ECM trees owing to differences of ECM
specificity. Though most ECM from different host tree species could not be
specific, thereby confirming the multi-host fungi observed in the Guinean
forest (Diédhiou et al. 2010), the only ECM observed in Gilbertiodendron
clumps were whitish and large rhizomorphs. Apparently, rhizomorphs
could play a role in water relations of host trees (Agerer 1995). Hence, ECM
clumps and isolated ECM tree species depict a particular biodiversity that
needs to be preserved.
Ectomycorrhizal forest habitats were the only sites where more than
200 ECM sporocarp species in 27 genera and eight families were collected
and identified. It is the first time in Africa that such a highly diverse and
abundant putative ECM sporocarp collection has been reported from the
same area. In the Pakaraima Mountains of Guyana, 75 morphotypes of
putatively ECM fungi were identified from discrete groves formed by
Dycimbe and Aldina species (Henkel et al. 2002). In the Miombo savanna
and humid forest of West Africa, only five families of ECM sporocarps were
recorded (Rivière et al. 2007, Sanon et al. 1997). The same high number of
members of the Russulaceae (44 species) from this study supports existing
data (Buyck et al. 1996). Species-rich genera included Amanita, Russula, and
members of Boletales and Cantheralles. Less frequent ECM fungal species,
Clavulina, Coltricia and Scleroderma were also collected for the first time
as well as rare ECM sporocarp species, viz. two, three and six species of
Paxillus, Cortinarius and Inocybe, respectively (Table 3), confirming earlier
observations made elsewhere in West Africa, the Neotropics and the Laojun
Mountain region in southwestern China (Rivière et al. 2007, Henkel et al.
2002). Lack of frequent inventories and experienced mycologists in Africa
could explain the poor accounts for tropical ECM sporocarp diversity.
Only 546 records of wild macrofungi have been made from South Saharan
countries (Boa 2006). In Benin, one species of Craterellus, Hebeloma, Inocybe,
Russula and eight Lactarius species were recorded (De Kessel et al. 2002). Five
out of six macrofungi in Burkina Faso were identified as Amanita hemibapha,

Cantharellus pseudofriessi, Lactarius gymnocarpus, Phaeogyroporous sudanicus
and Tubosaeta brunneosetosa (Sanon et al. 1997, Rammeloo and Walleyn 1993)
as well as an unidentified Calvatia (Walleyn and Rammeloo 1994). Recently,
more than two dozen ECM fungi have been recorded (Ndong et al. 2011),
with most of them also found in the rain forest of Cameroon (Onguene and
Kuyper 2012). No less than 40 ECM fungi have been recorded from the
Republic of Congo including 15 Cantharellus species and dozens of Russula
and Lactarius. The highest number of Lactarius species (19 species) has been
thus recorded in Tanzania. Other African countries are less prolific in ECM
fungi (Boa 2006).
Ectomycorrhizal forest communities could contribute not only to the
preservation of forest refuges in South Cameroon with endemic species, but
also to the protection of the biodiversity hot spots of Africa’s rainforests.
Maintenance of such ECM forest communities with their associated edible
mushrooms such as chanterelles also helps to preserve a valuable source
of alternative protein-rich food for local communities who depend on non-
timber forest products (Malaisse 1967, Van Dijk et al. 2003), in addition to
creating new ecological jobs for mushroom collectors. Thus, ECM forest
clumps should be protected as biodiversity sanctuaries, owing to the present
lack of knowledge on their regeneration requirements.
Fractional ectomycorrhizal colonization of Tetraberlinia was significantly
reduced by both shifting agriculture and selective logging. ECM colonization
increased during succession, with forest clumps showing a significantly
higher colonization than late-successional stands, but being low in early-
successional stands. ECM colonization was the highest in soils from Bityili
and lowest in soils from Ebom (Table 7). No or very little colonization
by native ECM fungi was observed in soils from a Gilbertiodendron
monodominant clump in Ebom and in soils from early-successional forest
from Ebimimbang and Nyangong. Apparently, each ECM clump may have
different ECM fungal consortia, confirming the observation of multi-species
fungal forests (Diédhiou et al. 2010). The question remains whether the
initial soil and climatic conditions regulate ECM symbiosis.
Various types of propagules, such as basidiospores, hyphal fragments,
or rhizomorphs and dying roots, can contribute to the mycorrhizal inoculum
potential. We were not able to evaluate the relative importance of various
propagule sources in our soil cores. Baiting techniques to assess ECM
inoculum might yield different results if plants are baited in the field within
reach of live mature ECM plants or are baited in the greenhouse (Diédhiou
et al. 2010, Onguene and Kuyper 2002, 2005). Studies where seedlings
were baited in intact vegetation in the field supported the view that the
species colonizing naturally regenerating seedlings in natural vegetation
were similar to that of the ectomycorrhizas of that surrounding vegetation
(Jonsson et al. 1999), whereas in the absence of surrounding vegetation of

ECM plants, a different suite of ECM fungi will be encountered (Taylor
and Bruns 1999).
In soils from forestry practices, both seedlings of Tetraberlinia and
Afzelia remained devoid of ECM. Although selective logging at present
concentrates on AM trees (most ECM trees are considered potential timber
species), lack of ECM inoculum on skid trails and landings suggests that
dispersal and survival of ECM propagules is limited. Lack of ECM inoculum
after severe disturbances is consistent with the results of earlier studies
(Boerner et al. 1996, Janos 1996).
Agricultural practices also affected ectomycorrhizal inoculum
potential of Tetraberlinia but not of Afzelia. The most likely explanation of
the differential behavior of the two ceasalps is that both taxa differ in their
specificity towards ECM fungi, thus confirming the above remark. The
issue of host plant specificity of ECM fungi has been repeatedly discussed.
Smits (1994) emphasized specificity of ECM fungi in dipterocarp forests in
Kalimantan (Indonesia) and implied that tropical ECM fungi were different
in that respect from ECM fungi in temperate areas. Kuyper (unpublished
observations) revised the taxonomy of ECM fungi from these forests and
concluded that ECM specificity was not different between tropical and
temperate forests.
Our results demonstrate that if most ectomycorrhizal tree species
behave similarly to Tetraberlinia, conservation of forest patches and clumps
where these trees occur is urgently needed. In the framework of sustainable
management of tropical rainforests, it would be important to preserve seed-
bearing trees and to assess whether addition of soil with ECM inoculum
to sites where selective logging has occurred would increase chances for
seedlings of ECM ceasalps to become ectomycorrhizal and hence contribute
to the maintenance of the diversity of ECM trees and fungi.
10. Conclusion
Cameroonian ectomycorrhizal fungi abundantly fruited mostly in five
types of clumps including Gilbertiodendron monodominant, Uapaca
monodominant, monodominant Microbelinia in Korup National Park,
oligo-dominant ceasalp clumps locally called “Ekop” and mixed clumps
of “Ekop” with Uapaca spp., independent of elevation, rainfall, topography
and soil texture. Though poor in plant diversity and no longer or barely
regenerating, these habitats recruited abundant and various ECM fungal
species on which native ECM tree species depend for survival, and some
others such as chanterelles which serve as a ready source of protein for local
people during harsh periods and could be a cash flow. Therefore, forest
clumps should be conserved as biodiversity sanctuaries. The presence
of ECM fungi could serve as indicators for sustainable management of

African humid forests and climate change. Hence, the presence of such
tree species and stands should be made known to all forest stakeholders
for their ecological specificity and relevance in the management of humid
forests of the Congo basin.
Acknowledgements
We thank the anonymous referees for their valuable comments on this
study, and Krista L. McGuire and Caitlyn Gillikin for improving the
English. Financial support from NWO (Priority programme—Biodiversity
in disturbed ecosystems) and logistic support by the Tropenbos Cameroon
Programme and the Government of Cameroon through financial support
to the Institute of Agricultural Research for Development (IRAD) are
gratefully acknowledged. Field assistance was provided by Jean-Baptiste
Mva, Edouard Nsomoto, Serge Aba’a Aba’s, Roger Eyene, and Thomas
Mba. The technical assistance by Veronique O. Anaba during the project is
very much appreciated. Thanks to the reviewer.
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CHAPTER
4
Mycorrhizal Fungi Diversity
and their Importance on
the Establishment of Native
Species Seedlings within
Madagascarian Degraded
Sclerophyllous Forest
Rondro Harinisainana Baohanta,1,* Herizo
Andrianantoandro Randriambanona,1 Marc Ducousso,2
Christophe Nirina Rakotoarimanga,1 Yves Prin,2
Heriniaina Ramanankierana1 and Robin Duponnois1
1. Introduction
The impact of human activities on tropical ecosystems has increased
dramatically in recent decades leading to a global reduction of primary
forests (Laurance 1999, Morris 2010). For tree species, fragmentation
of forests into patches has led to degradation of both their habitat and
1
Laboratoire de Microbiologie de l’Environnement, Centre National de Recherches sur
l’Environnement BP 1739 Fiadanana Antananarivo 101, Madagascar.
2
Laboratoire des Symbioses Tropicales et Méditerranéennes (LSTM), UMR 113 CIRAD/INRA/
IRD/SupAgro/UM2, Campus International de Baillarguet, TA A-82/J, Montpellier, France.
*Corresponding author: ninish.rondro@yahoo.fr
ecological processes such as biogeochemical cycling and community

dynamics (Weaver and Kellman 1981). The forest ecosystems of Madagascar
are recognized as being among the most species-rich around the world
(Myers et al. 2000). Approximately 80% of the plant species of Madagascar
are endemic and the island contains a wealth of fauna and flora (Mittermeier
et al. 2004), but they have been seriously impacted by a combination of
agricultural practices such as slash and burn culture, livestock production
and logging, and ancient climate changes. More recently, during the last
two centuries, the extent of the Malagasy forest has decreased dramatically.
Today, primary vegetation probably covers only about 10% of the original
area and dense forest has been reduced to a fragmented landscape (Myers et
al. 2000). Similarly, in the highland of Madagascar, sclerophylous woodland
dominated by Uapaca bojeri (Tapia forest) is the last remnant of primary
forest. The main components of this formation are small endemic trees
of Uapaca bojeri (family Euphorbiaceae), in some patches endemic shrubs
such as Sarcolaena sp. (family Sarcolaenaceae), or Asteropeia sp. (family
Asteropeiaceae) which also grow as subdominants. The Tapia forest plays
an important role in local community livelihood as it provides fuel, wood
and timber, medicinal plants, non-timber forest products, and many other
sources of food and income (Kull et al. 2002). Additionally, Tapia forest
provides important environmental services: e.g., protection against erosion,
protection of water sources, and carbon sequestration. However, Tapia
trees are threatened by human destruction through bush fires, firewood
collection, and charcoal production, and restoration efforts using native
trees of this forest formation should be undertaken to preserve the entire
ecosystem.
For several years, the trend for ecological restoration has been the use of
mycorrhizal fungi, considered an important component that stabilizes the
soil and enhances plant growth by alleviating nutrient and drought stress
(Mikola 1980, Högberg 1982, Williams et al. 2012). Mycorrhizal inoculation
has been used to promote native plant growth and reestablishment in
various ecosystems (Smith et al. 1998). Likewise, in restoration programs
of vulnerable or endangered native species, it is important to know the
mycorrhizal status of the plants. Cooke and Lefor (1998) reported that the
presence or absence of mycorrhizae in individuals of plant species used in
wetland restorations might be an important factor in the reestablishment
of wetland plant associations. Classified as biological tools in ecological
restoration (Ruiz-Jaen and Aide 2005), mycorrhizae colonization can
significantly affect plant growth and patterns of succession after a
disturbance (Haselwandter 1997).
During the last decade, an increasing number of studies have also
emphasized the importance of mycorrhizal facilitation in driving plant
community succession and vegetation restoration (Callaway 1995, 1997,
Mycorrhizal Fungi Diversity and Native Plant Regeneration in Madagascar 53
Brooker et al. 2008). In Madagascar, the description of mycorrhizal status of

native species and the role of facilitative interactions in plant communities
have received attention over the last few years, enhancing the knowledge
of the ecology of those threatened ecosystems (Ramanankierana et al. 2007,
Baohanta et al. 2012). For example, in the Tapia forest, it has been reported
that Leptolaena bojeriana (Bail.) Cavaco. (family Sarcolaenaceae), an endemic
shrub, can facilitate the ectomycorrhizal (ECM) colonization of Uapaca. bojeri
and mitigates the negative effects of the introduction of exotic tree species
(Pinus patula, Eucalyptus camaldulensis) on the early growth of the native tree
(Baohanta et al. 2012). The current tendency is to utilize the native plants
to restore degraded ecosystems (Castro et al. 2006). According to Ren et al.
(2008), the best nurse plants are the native species that offer microhabitats for
target plant establishment or recruitment on degraded environment. In this
chapter, we aim to review some of the recent advances in the understanding
of the implication of ECM symbioses in the development and regeneration
of native shrub and tree species in Madagascarian highland sclerophyllous
forest. We will focus on (i) the sporocarp survey within Madagascarian
sclerophyllous forest, (ii) the importance of mycorrhizal inoculation on early
development of U. bojeri, an endemic tree species, and (iii) the nurse plant
phenomenon between endemic shrub and tree species within sclerophyllous
forest in the Malagasy high plateau.
2. Sporocarp Survey within Sclerophyllous Forests in Central

Highland of Madagascar
The sclerophyllous forest dominated by U. bojeri constitutes one of the
few remaining patches of forest and woodland on the central highland
of Madagascar more than 1000 m above sea level. The sclerophyllous
woodlands are valuable examples of a very restricted vegetation type
that has mostly been replaced by artificial, fire-maintained grassland
(Dupuy and Moat 1996). It is estimated that U. bojeri forest size has been
reduced by approximately 43% since the 1970s (Moat and Smith 2007).
This forest formation is characterized by a low specific richness of plants,
but dominated by a few ECM endemic trees of the Euphorbiaceae family
and shrub species of Sarcolaenaceae and Asteropeiaceae. Three sites of this
sclerophyllous forest were visited by Ramanankierana et al. (2007) at 2 to 3
week intervals during the sampling seasons, mid-November 1992 to early
February 1993, July–August 1994, and July to mid-September 1995 in order
to collect ECM fungi fruiting under ectomycorrhizal tree and shrub species.
The forests were located at 50 km to the west of Antananarivo (site A), 20 km
to the south of Antsirabe (Ambositra site, site B), and 100 km to the east of
Toliara (Isalo site, site C). The vegetation sampled included savannas (sites
A and B) and deciduous forest (site C). The main chemical characteristics
of the upper soil layer (2–20 cm) of these sites are shown in Table 1.
These investigations showed that sclerophyllous woodlands dominated
by U. bojeri contain a wide range of sporocarps belonging to at least four
different fungal families: Russulaceae, Cantharellaceae, Boletaceae, and
Amanitaceae. Overall, 94 sporocarps of putative epigeous ECM fungi
were collected in three survey sites. They were identified as belonging
to the ECM genera Afroboletus, Amanita, Boletus, Cantharellus, Leccinum,
Gyroporus, Rubinoboletus, Russula, Scleroderma, Suillus, Tricholoma, and
Xerocomus (Table 2). Russula was the most frequent ECM genus recorded
(32.9% of the above-ground sporocarp diversity) followed by the genera
Amanita (17.1%) and Cantharellus (Fig. 1a). At species level, 21 species were
recorded for Russula followed by 14 Amanita species,10 Cantharellus species,
and 10 Boletus species (Fig. 1a, b). The fungal richness of the above-ground
sporocarps decreased from site A (40 species) to site C and B with 29 and
27 species, respectively.
The results of Ramanankierana et al. (2007) showed a large diversity of
sporophores recorded under ectomycorrhizal tree and shrub species in the
sclerophyllous forests of Madagascarian highlands. Different fungal families
(Russulaceae, Cantharellaceae, Boletaceae, and Amanitaceae) observed at
this forest have been recorded in other tropical forests under Afzelia africana,
Monotes kerstingii, Uapaca guineensis, and U. somon in Africa (Thoen and Bâ
1989, Sanon et al. 1997) and under dipterocarps in Asia (Lee 1998). It has
been already demonstrated that Russulaceae are often dominant in the
tropical rainforests of Africa, Asia, and Madagascar (Buyck et al. 1996, Lee
et al. 1997, Walting and Lee 1998, Rivière et al. 2006).
In the northern part of this sclerophyllous forest (Site A), three
ectotrophic plant species (U. bojeri, Leptolaena pauciflora Baker and Leptolaena
bojeriana (Baill.) Cavaco. were identified by Baohanta et al. (2012). The
highest fungal diversity of the above-ground sporocarpswas recorded at
Table 1. Main chemical characteristics of the upper soil layer (2–20 cm). Data from
Ramanankierana et al. (2007).
Site Site A Site B Site C

pH (H2O) 4.96 5.37 4.54
pH (KCl) 4.75 5.23 4.45
Total C (%) 1.12 3.09 1.33
Total N (%) 0.07 0.15 0.91
Total organic matter (%) 1.92 5.31 2.28
C/N 16.0 21.0 14.6
Total P (mg g–1) 15.2 15.2 17.3
Available P (mg g–1) 3.42 7.01 5.24
Table 2. Description of putative ectomycorrhizal fungi collected from the three studied sites beneath ectomycorrhizal tree and shrub species within
sclerophyllous forest in Madagascarian highlands. Data from Ramanankierana et al. (2007).
Species Prominent features Habitat Sites

Amanitaceae Site A Site B Site C
Amanita rubescens Gray White pinkish cap (8 cm diameter) covered with white powdered Solitary, scarce x
and flat scales, remnant veil visible at the margin, white stem
reddening by wound, often eaten by insect larvae
Amanita virosa (Fr.) White yellowish fruiting body (7 to 12 cm diameter), white and Patch of 5 to 6 x x x
Bertillon chinated stem (1.2 cm diameter) with ring and cup at the base individuals
Amanita phalloides var. verna White fruiting body (5.5 to 11 cm diameter), stem (0.6 diameter by Patch of 5 to 7 x x x
Bull 9.5 cm high) with a large pendant ring and a bulbous cup at the individuals
base
Amanita strobiliformis White and big fruiting body (10 to 12 cm diameter), fleecy remnant Solitary, scarce x
Bertillon veil on the cap, club-shaped stem (2.2 cm diameter) with a ring
Amanita cf. Baccata (Fr.) Big white fruiting body similar features than previous species but Solitary, scarce x
Gillet with no ring, stem (2 cm diameter by 7 cm high)
Amanita sp1 White finely scaled fruiting body (4 to 6 cm diameter) turning Solitary, scarce x
yellowish when aging or by wound, concolored gills and flesh
Amanita cf. strobiloceovolvata White fruiting body (8.5 to 11cm diameter), stem (1.2 cm diameter Patch of 3 to 4 x x x
Beeli by 10.5 cm high) without ring, well-developed bulbous cup at the individuals
base
Amanita sp2 White and big species with a convex scaly cap (10 to 13 cm Solitary, scarce x
diameter by 9 to 10 cm high), strong bulbous stem (3 to 4cm
diameter) with a pendant ring
Amanita sp3 Pale grey cap (4.5 cm diameter) with few veil remanences on Solitary, scarce x
surface, bulbous stem (0.7 to 6cm) with grey chinates
Amanita sp4 Yellow conical and mucronated cap (2.5 to 3 cm diameter), paler to Solitary, scarce x
whitish gills and stem (0.5 cm diameter by 12 cm high), white scaly
basal cup
Table 2. contd....
Table 2 .contd.
56

Amanita cf. cecilia (Berk. et Yellow grey cap (4 to 5 cm diameter) with rised scales, white gills Solitary, scarce x
Broome) Bas and concoloured stem (0.7 cm diameter to 6 cm high), bulbous base
covered by grey chinates and veil remanences
Amanita sp5 Convex and grey purplish-blue cap (4 to 4.5 cm diameter) with Solitary, scarce x
grey flat scales at the centre and hairy ones at the margin, white
flesh and gills, white bulbous stem (0.9 cm diameter by 6 cm high)
turning to grey by touch with a pendant ring
Amanita sp6 Small white species (2 to 3 cm diameter) with yellowish scales, Solitary, scarce x
bulbous based stem with pendant ring
Amanita sp7 Big white flat cap species (9 to 13 cm diameter) with veil Patch of 2 to 3 x x x
remanences at the margin, strong bulbous stem (3 to 4 cm diameter) individuals
with a ring
Boletaceae
Rubinoboletus griseus Big red-pink and grey-brownish dry and smooth cap (10 to 12 cm Patch of 5 to 6 x x x
diameter by 8 to 9 cm high), white flesh (1.8 cm thick) partially individuals
burnishing after sectioning, pale reticulated hairy scaled stem,
burnishing like pores by touch
Gyroporus cf. cyanescens Big white yellowish smooth cap (10 to 12 diameter by 8-9 cm high), Patch of 3 to 4 x x x
(Bulliard Fr.) Quélet concolored tubes and stem turning to blue by wound individuals
Boletus sp1 Brownish to brown cap, with large darker flat scales, cylindrical Patch of 3 to 4 x
and dark stem, red-reticulated becoming yellow at the base like individuals
rhizomorph, flesh and pores turning blue by wound
Leccinum sp1 Small grey boletus (1.8 to 3 cm diameter by 3 to 4cm high), yellow Patch of 3 to 4 x x x
pores, red hairy scales on the stem, base of the stem yellow like the individuals
rhizomophs
Boletus sp2 Big brownish-brown wet cap (7 to 8 cm diameter to 12 to 15 cm Patch of 5 to 6 x
high), white and smooth flesh individuals
Xerocomus sp1 Brown scaly cap (8.5 cm diameter) showing white flesh between Solitary, scarce x
scales, white stem (1.4 cm diameter by 5 to 6 cm high) with some
red zone
Leccinum sp2 Yellow grey scaled boletus (4.5 to 6 cm diameter by 6 to 7 cm high), Solitary, scarce x
stem yellow at the base and red in its upper part, yellow blueishing
pores
Boletus sp3 Brown cap (7.5 cm diameter) with red-pink pigments, yellow and Solitary, scarce x
red pores, greenishing and blueishing tubes, yellowish stem with
some red pigments
Leccinum sp3 Red purplish-blue wet cap (7 cm diameter), yellow burnishing stem Solitary, scarce x
(0.8 cm diameter by 6 cm high), concolored yellow flesh and pores,
blueishing after air exposure
Boletus sp4 Big smooth and shiny red boletus (8 to 12 cm diameter by 7 to 8 cm Patch of 2 to 3 x x x
high), yellowish stem with some pink pigments, concolored flesh individuals
(1.6 cm thick)
Xerocomus sp2 Pale to dark brown scaly dry cap (5 cm diameter), white dirty stem Solitary, scarce x
(0.8 cm diameter by 4 cm high) with a white-yellowish flesh, yellow
greenish and pink pores
Boletus sp5 Yellowish brown cap (8 cm diameter) with flat partially pink scales, Solitary, scarce x
yellow pores and stem (1.2 cm diameter by 6 cm high), white flesh
(1.6 cm thick)
Boletus sp6 Dark brown scaly cap showing yellow flesh, pale concolored pores Solitary, scarce x
and stem
Boletus sp7 Brown boletus with dry and silky cap (4.5 cm diameter), concolored Solitary, scarce x
dark stem (2.2 cm diameter by 5.2 cm high), white flesh (1.6 cm
thick) rapidly turning to red, then black after air exposure
Boletus sp8 Pale brown boletus with silky cap (5 cm diameter), white stem Solitary, scarce x
(1.5 cm diameter by 5.2 cm high) and flesh (1.3 cm thick) turning
purplish-blue after air exposure
Table 2 .contd....
Table 2 .contd.
58

Leccinum sp4 Yellow and wet cap (3.5 cm) with hairy grey scales, yellow pores, Solitary, scarce x
yellow and red stem (0.5 cm diameter) with dark scales and a
narrow base
Leccinum sp5 Yellowish-brown dry cap (3.5 cm diameter), red pores and lighter Solitary, scarce x
stem (0.6 cm diameter by 4 cm high) turning to dark-brownish in
section, white flesh turning burnish after air exposure
Suillus sp2 Yellow and grey scaly cap (5 cm diameter), yellow pores covered by Patch of 2 to 3 x
a yellow partial veil when young, yellow stem (1.4 cm diameter by individuals
4.5 cm high) with greenish grey scales, becoming very slimy
Boletus sp9 Yellow brownish boletus (7 to 8 cm diameter) with a sticky surface, Solitary, scarce x
yellow pores and stem, yellowish flesh (1.7 cm thick)
Leccinum sp6 Pale brown cap (5 to 4 cm diameter) with red brownish scales at Solitary, scarce x
the centre, white pores and white flesh turning rapidly to red, then
black by wound
Boletus sp10 Yellow brown boletus (4.5 to 5.7 cm diameter) with wet and smooth Solitary, scarce x
surface, yellow pores, yellow stem (1.2 diameter by 3 cm high),
white flesh (1 cm thick)
Cantharellaceae
Cantharellus sp1 Tall thick and lobed fasciculate bright yellow caps (4 to 6 cm Patch of 8 to 10 x x x
diameter) forming patches of 4 to 5 individuals (12 cm), grained individuals
gills, pale yellow stem (1.8 cm), white flesh

Cantharellus sp2 Small orange-brownish cap (2 to 2.2 cm diameter), white pinkish Solitary, scarce x
gills, pink stem and white flesh
Cantharellus sp3 Yellowish to pale brown cap (3.5 to 3.2 cm diameter), yellow Solitary, scarce x x x
grained gills, pale yellow stem (0.6 to 2.5 cm)
Cantharellus sp4 Red orange cap (3.2 to 3.5 cm diameter), largely spaced yellowish Patch of 8 to 10 x x x
grained gills, pale yellow to reddish stem (0.9 cm) individuals
Cantharellus sp5 Pale brown cap (3.2 to 3.5 cm diameter), pale pink grained gills, Solitary, scarce x x x
white stem and flesh, turning to yellow by touch or sectioning
Cantharellus sp6 Red pinkish fasciculate caps (2.5 cm diameter) forming small patch Patchy x
(3.5 to 4 cm), yellowish grained gills, pink orange stem and white
fibrous flesh
Cantharellus sp7 Small and fragile bright yellow cap (2 to 3.2 cm diameter), pale Solitary, scarce x
yellow gills, concolored short stem (0.3 cm)
Cantharellus cf decolorans Small pink orange cap (0.7 to 1.5 cm diameter, 2.5 to 3.5 cm high), Patch of 5 to 6 x
Eyss. et Buyck concolored gills and short stem (0.2 cm) individuals
Cantharellus cf. Yellow and purple cap (4 cm diameter), pale pink grained gills, pale Patch of 2 to 3 x
cyanoxanthus R. Heim yellow stem (1.8 cm), fibrous flesh individuals
Cantharellus rubber R. Heim Pale pink cap (3.5 to 4 cm diameter), concoloured stem and gills Patch of 2 to 3 x
individuals
Russulaceae
Russula subfistulosa Buyck White-greyish (darker at the centre) umbilicated cap (3 to 12 cm Solitary to patch of x x x
diameter). 3 individuals
Russula ochraceorivulosa Greyish to purplish-blue grey cap (7 to 8 cm diameter). Convex cap Solitary x x x
with an undulating margin
Russula patouiillardi Pale yellow and purple (darker at the centre) dry scaly cap, white Solitary to patch of x x x
and purple stem 5 individuals
Russula liberiensis Buyck White-greyish fibrillose cap (3 to 12 cm diameter) turning brown Solitary to patch of x x x
when ageing, closely spaced decurrent gills 3 individuals
Russula cf. cyanoxantha Pink to purple-red cap (5 to 15 cm diameter), white stem Patch of 2 to 3 x
individuals
Russula cellulata Buyck Brown scaly cap (3 to 9 cm diameter), closely spaced decurrent gills Patch of 2 to 3 x
individuals
Table 2 .contd....
Table 2 .contd.
60

Russula cf. archae R. Heim White smooth and flat cap (4.5 to 6 cm diameter) Solitary x
Russula cf. nigricans White-greyish cap turning to brown when ageing, white flesh turn Solitary x
rapidly pink to red by air exposure
Russula cf. subfistulosa White-greyish convex cap (3 to 8 cm diameter) Solitary to patch of x
4 individuals
Russula sp3 White to pale yellow gluey and convex cap (3 to 13 cm diameter) Solitary to patch of x
3 individuals
Russula sp5 Yellow smooth umbilicated cap (6 to 12 cm diameter), with a very Patch of 2 to 3 x x x
regular margin individuals
Russula sp6 White-yellowish flat or slightly umbilicated cap (4 to 10 cm Patch of 3 to 5 x
diameter), white flesh turning reddish after air exposure individuals
Russula sp7 Dark grey to brown convex cap (3.5 to 8 cm diameter), involucrated Solitary, rarely x
margin, wet surface covered by orange to yellow layers, white- patchy
yellowish flesh
Russula sp8 White convex to slightly umbilicate cap (4 to 13 cm diameter) Solitary, scarce x
turning brown when ageing, smooth surface with involucrated
margin, white flesh turning reddish after air exposure
Russula sp10 Dark grey to brown when fully mature convex to flat cap (4 to 9 cm Patch of 2 to 4 x
diameter), white flesh individuals
Russula sp11 Small purple to purple-reddish umbilicate when young to flat when Solitary to patch of x
ageing cap (2 to 7 cm diameter), sticky surface, regular margin, 3 individuals
adnate white to yellowish closely spaced gills, white flesh
Russula sp13 Brown-reddish convex and smooth glutinous cap (6 to 15 cm Solitary x
diameter), decurrent gills, white flesh turning greyish by air
exposure
Russula sp14 Dark yellow to brown convex to flat sticky cap (4 to 10 cm Patch of 3 to 5 x
diameter), adnate closely spaced gills individuals
Russula sp15 Yellow to orange-yellow flat slightly umbilicated with an Patch of 2 to 5 x
involucrated yellow margin cap (2 to 8 cm diameter) with a smooth individuals
surface with small strias
Russula sp16 Pink to reddish (darker at the centre) fragile convex glutinous cap, Patch of 2 to 4 x
(2 to 6 cm diameter) with a smooth or dusty surface, white flesh individuals
Russula sp17 Slightly umbilicated convex and glutinous cap (4 to 10 cm Solitary to patch of x
diameter), dark yellow tending to brown, yellow to pale orange 4 individuals
closely spaced gills
Strobilomycetacea
Afroboletus sp1 Brown-purple scaly cap (3 to12 cm diameter), fibrous stem, pale Patch of 3 to 5 x
yellow flesh turning purplish by air exposure individuals
Afroboletus sp2 Flat-convex dusty cap (3 to 10 cm diameter) with dark-brown to Patch of 2 to 3 x
black scales, fibrous stem inflated at the base, greyish-yellow flesh individuals
Sclerodermataceae
Scleroderma sp1 Whitish to yellowish small pyriformic fruit bodies, size below 3 cm Solitary to patch of x
in diameter, dark grey gleba 5 individuals
Scleroderma sp2 Whitish to yellowish 3 to 7cm diameter fruit bodies with grey spots Solitary, rarely x
at the top, dark grey gleba patchy
Tricholoma sp2 Yellow cap (3 to 9 cm diameter), dry surface, involucrated margin, Solitary to patch of x
thick widely spaced gills, yellow flesh keeping yellow even after 4 individuals
exposure to air
Tricholoma sp3 Yellow-greyish cap (3 to 12 cm diameter), dry surface, white Solitary to patch of x
yellowish stalk, white flesh 4 individuals
Tricholoma sp4 Dark-grey cap (3 to 15 cm diameter), smooth dry surface, thick gills, Solitary x
white flesh
Relative frequency per genus (%)
us
let
us
o
or
ob
op
n
bi
yr
Ru
G
b
25
20
Species per genus
15
10
5
0 us
us
ph
et
ol
om
ob
G
in
ub
R
Genus
Fig. 1. Structure of the ectomycorrhizal community (above-ground species richness) expressed
as genus relative frequency of the most abundant genera (a) and total species per genus (b).
this site (40 species). With other tropical ectomycorrhizal tree species, Lee
et al. (1997) recorded only 28 fungal species under Shorea leprosula, while
Sanon et al. (1997) have identified 14 fungal species under U. guineensis
and 11 species under U. somon in Burkina Faso. However, numerous
studies in temperate areas indicate little correlation between above-ground
(sporocarps) and below-ground (morphotypes of ectomycorrhizas) fungal
diversity (Buscot et al. 2000, Horton and Burns 2001). Further investigations
using molecular approaches are needed to entirely describe the fungal
community associated with plants.
3. Time Sequence of Mycorrhizal Colonization on

Uapaca bojeri
The arbuscular mycorrhizal (AM)-ectomycorrhizal succession may be linked
to spatial competition for colonization sites and differential colonization
rates by the two types of fungi (Last et al. 1983, Chilvers et al. 1987, Last et
al. 1987, Duchesne et al. 1988).
Uapaca bojeri constitutes a fundamental species in the sclerophyllous
forests of the Madagascarian highlands. It has been illustrated that this
endemic Euphorbiaceae of Madagascar is associated both with arbuscular
mycorrhizas and ectomycorrhizas (Ramanankierana et al. 2007). Using soils
collected beneath an adult tree of U. bojeri, Ramanankierana et al. (2007)
demonstrated that ECM and AM structures were recorded on root systems
of this plant after two months growing time (Fig. 2). Three native ECM
fungi colonized approximately 50% of the lateral roots sampled from this
tree species after 5 months growing time in pot culture.
In Madagascar, the result of Ramanankierana et al. (2007) illustrated,
for the first time, the occurrence of two types of mycorrhizal fungi and their
importance on seedling development of U. bojeri, an endemic tree species.
These investigations highlighted the succession of three ECM morphotypes
on root system during the early development of this tree (Fig. 2). However,
the dominance of mycorrhizal structures on Malagasy tree and shrub
species has been already shown within five coastal humid forests located
along the eastern coast of Madagascar (Ducousso et al. 2008). These authors
demonstrated that all of tree and shrub species belonging to Asteropeiacea
100
Mycorrhizal colonization (%)
90
80
70
60
50
40
30
20
10
0
0 1 2 3 4 5 6
Time (months)
Fig. 2. Mycorrhizal colonization and sequence of ectomycorrhizal morphotypes on U. bojeri
seedlings during 5 months growing time on soil collected from the native stand of this tree,
: AM colonization, : Total ectomycorrhizal colonization, ▲: morphotype 1,•: morphotype
2, x : morphotype 3. Data from Ramanankierana et al. (2007).
and Sarcolaenaceae, two botanical families entirely endemic of Madagascar,

were found to have both ectomycorrhizal and arbuscular mycorrhizal
structures. However, the succession of each mycorrhizal structure on the
root system of these plants was not described. In other tropical ecosystems,
more attention has been focused on mycorrhizal colonization patterns of
commercial forest tree species (Oliveira et al. 1997, Dos Santos et al. 2001,
Founoune et al. 2002) and of forest-dominating tropical trees (McGuire et
al. 2008). Because of the many desirable characteristics of these trees, such
as rapid growth, high cellulose production, and resistance to disease and
adverse environmental conditions, their use in reforestation programs in
the tropics has been increasing steadily by using advanced mycorrhizal
inoculation technologies such as controlled mycorrhization in nursery. In
Madagascar, ecological restoration and reforestation programs using native
plants have been undertaken in recent years in order to preserve, valorize,
and sustainably manage the high biodiversity of this island. In this way,
U. bojeri constitutes a potential candidate for reforestation programs in the
highland region of Madagascar by managing the associated mycorrhizal
fungi of this tree. Furthermore, research on mycorrhizal inoculation
technology has been expanded to other valuable endemic trees in recent
years, such as Dalbergia trichocarpa Jum. Adansonia za Jum & H. Perrier.
4. Mycorrhizal Dependency of U. bojeri

New opportunities to stimulate the growth of these plants or to reduce their
mortality rate after transplantation to natural sites have arisen by using
seedlings inoculated with both arbuscular mycorrhizas and ectomycorrhizas.
This dual symbiotic association is well documented for Populus (Lodge
and Wentworth 1990), Salix (Dhillion 1994), Eucalyptus (Lapeyrie and
Chilvers 1985), Alnus (Molina et al. 1994), Pinaceae (Cazares and Trappe
1993), Quercus (Egerton-Warburton and Allen 2001), and Casuarinaceae
(Duponnois et al. 2003). Using two types of mycorhizas (arbuscular
endomycorrhizas and ectomycorrhizas) to inoculate Acacia holosericea (A.
Cunn. Ex G. Don), Founoune et al. (2002) showed that inoculating with
both forms of mycorrhiza resulted in greater plant development than if only
one inoculant was used. However, the possibility of negative interactions
among mycorrhizal fungi has been suggested (Lodge and Wentworth 1990).
In Madagascar, single or dual mycorrhization of U. bojeri by Scleroderma
sp. SC1 and/or Glomus intraradices (Schenk and Smith DAOM 181602,
Ottawa Agricultural Herbarium) was conducted on sterilized sandy soil
in greenhouse conditions (daylight of approximately 12 h, average daily
temperature of 25°C) (Ramanankierana et al. 2007). Scleroderma sp. SC1
was a native ectomycorrhizal strain isolated from sporocarps collected
under an adult tree of U. bojeri. The shoot dry weight of plants inoculated
with G. intraradices or Scleroderma sp. SC1 was significantly higher than in

the control treatment (non-inoculated plants) (Table 3). The shoot growth
of inoculated plants was stimulated 1.9 times with Scleroderma sp. SC1,
whereas it was 1.7 times higher for plants inoculated with G. intraradices
compared to the control treatment. Inoculation with both fungal symbionts
significantly increased the shoot dry weight of plants over the single
inoculation treatments.
Table 3. Shoot growth, mycorrhizal development, and relative mycorrhizal dependency of

U. bojeri seedlings 5 months after G. intraradices and/or Scleroderma sp. SC1 inoculation in pot
culture. Data from Ramanankierana et al. (2007).
Treatments Shoot biomass Ectomycorrhizal AM colonization RMD (%)a

(mg per plant) colonization (%) (%)
Control 91.1ab 0a 0a -
Scleroderma sp. SC1 180.2b 8.7b 0a 47.6a
G. intraradices 160.0b 0a 77.5b 42.7a
Scleroderma sp. SC1 + 360.3c 11.5b 85.5b 70.7b
G. intraradices
5. Dominance of Ectotrophic Endemic Shrub Species in

Degraded Area of Sclerophyllous Forest in Malagasy
Highland
Human activities such as land use change, slash and burn agriculture,
or overexploitation of forest trees lead to soil degradation and loss of
biodiversity in tropical areas. These anthropogenic impacts not only degrade
natural plant communities (vegetation structure and species diversity)
but also physicochemical and soil biological properties such as nutrient
availability, microbial activity, and soil structure (Agarwal et al. 2005). It is
well known that changing the vegetation structure can modify the dynamics
of mycorrhizal fungi communities in soil (Dickie et al. 2002, Dickie and
Reich 2005). Studies were carried out within two disturbed sclerophyllous
forest ecosystems of Uapaca bojeri, located at Arivonimamo (S 19° 00’ 15’’;
E 47° 07’ 00’’) and Ambatofinandrahana (S 20° 39’ 41.5’’; E 047° 06’ 53.1’’)
in the Central part of Madagascar. The relative importance of mycorrhizal
associations on early established shrub species in the degraded areas of
these forests ecosystem was studied by using the transect method at three
sampling points which were situated at the forest edge (0 m), passed
through the area colonized mainly by shrubs species (25 m), and ended at
the most degraded area without vegetation influences (50 m). It has been
observed that a large part of these two respective natural forest ecosystems
was replaced by exotic tree plantations, shrub formations, and grassland,
and/or by degraded surface areas where the regeneration of native species

is quite limited or non-existent. Within the two study sites, the results
showed that most of the shrub species identified are mycotrophic. Indeed,
among all species inventoried, eight shrub species had AM infections and
three, represented by Leptolaena bojeriana, Leptolaena pauciflora, Sarcolaena
oblongifolia, were found with both AM and ECM colonization (Table 4).
In the Arivonimamo forest ecosystem, ten shrub species distributed
among three sampling points were recorded. They were identified as
belonging to the botanical families of Sarcolaenaceae, Ulmaceae, Ericaceae,
Flacourtiaceae, Anacardiaceae, Asteraceae, and Rosaceae. The shrub species
Leptolaena bojeriana was the most dominant at 25m from the U. bojeri adult
tree, followed by L. pauciflora (Figs. 3A, C) (Baohanta 2011). Most of those
shrub species were also found in the Ambatofinandrahana forest, which was
characterized by the dominance of the shrub species Sarcolaena oblongifolia.
These results corroborate those found by Pidwirny (2006) illustrating that
the degradation of natural forests often leads to the formation of bare areas
devoid of vegetation or of zones colonized by early colonizers such as
grasses or shrubs. Particularly, ectomycorrhizal shrub species (L. bojeriana
and/or S. oblongifoila) were widespread throughout degraded areas of the
Madagascarian highland sclerophyllous forest. Some authors such as Schatz
et al. (2001) and Ducousso et al. (2004, 2008) have already reported that most
of the genera belonging to the Sarcolaenaceae family are ectotrophic. These
properties could be the basis of their great capacity to tolerate environmental
stresses found at degraded areas.
Table 4. Mycorrhizal status of early established shrub species in the degraded areas of the
Arivonimamo and Ambatofinandrahana forests. Data from Baohanta (2011).
Shrub and tree species Family Mycorrhizal Study site

status
Leptolaena pauciflora Baker. Sarcolaenaceae ECM & AM Arv & Amb
Leptolaena bojeriana (Baill.) Cavaco. Sarcolaenaceae ECM & AM Arv & Amb
Trema sp. Cannabaceae AM Arv & Amb
Vaccinium emirnense Hook. Ericaceae AM Arv & Amb
Aphloia theaeformis (Vahl.) Benn. Aphloiaceae AM Arv & Amb
Rhus taratana (Baker.) H. Perrier Anacardiaceae AM Arv
Helychrysum russillonii Hochr. Asteraceae AM Arv & Amb
Psiadia altissima (D.C.) Drake Asteraceae AM Arv
Rubus apetalus Poir. Rosaceae AM Arv & Amb
Erica sp. Ericaceae AM Arv & Amb
Sarcolaena oblongifolia (Baill.) Cavaco. Sarcolaenaceae ECM & AM Amb
Fig. 3. Vegetation structure (A and C), global microbial activity and phosphatase activities of soil
(B and D) at degraded forest edge (from an individual adult tree of U. bojeri) of sclerophyllous
forest in high plateau of Madagascar.
6. Early Established Ectotrophic Shrub Species Affect Soil

Chemical and Microbial Properties
In some cases, the propagation of pioneer species in degraded areas
could affect soil restoration processes such as chemical and/or microbial
properties (Holl 2002, Gómez-Aparicio et al. 2005). Within the two study
sites in the high plateau of Madagascar, Baohanta (2011) observed the same
results by analyzing total microbial activities [by fluorescein diacetate
(FDA) hydrolysis assay] and soil phosphatase activities. Values of these
two parameters were significantly high on soil samples mainly colonized
by shrub species (25 m) compared to those recorded on bare soil (50 m)
and on U. bojeri soil (0 m) (Fig. 3B, D). Correlation between these two soil
parameters and the distribution of shrub species was observed in the
Arivonimamo forest. It has been shown that the abundance of L. pauciflora
and L. bojeriana was associated with a high value of global microbial and
phosphatase activities of soil (Fig. 3).
Some authors (Gómez-Aparicio et al. 2005, Montané et al. 2010) have
already reported that the propagation of some shrub species enhance litter
quality and the amount of organic matter in soil. Such situations could lead
to a definite increase in soil fertility. However, more experiments must be
undertaken in order to confirm that modified soil conditions by pioneer
shrub species facilitate both the establishment of tree species as well as
the development of late successional species in Madagascarian highland
sclerophyllous forest. On degraded soil, mycorrhizal fungi constitute one of
the well-known biological components that can improve host performance
by enhancing nutrient and water uptake from the soil and protecting host
roots from pathogens and toxic compound (Smith and Read 2008).
7. Importance of Ectotrophic Shrub Species on the

Establishment of Native Tree Species: The Case of
S. oblongifolia-L. bojeriana / U. bojeri
Seedling establishment may be both inhibited and facilitated by pre-
established plants. In this part of the chapter, we aim to review the recent
advances in the capacity of two pioneer shrub species (S. oblongifolia and
L. bojeriana) to facilitate seedling development of U. bojeri by sharing ECM
symbionts in Ambatofinandrahana and Arivonimamo sclerophyllous forests
respectively. By providing compatible fungal symbionts or by sharing
interspecific hyphal links, it has been demonstrated that mycorrhizal fungi
can mediate plant coexistence and succession (Dickie et al. 2004, Simard
and Durall 2004, Baohanta et al. 2012).
Numerous studies have reported that the introduction of exotic tree
species has an environmental impact on soil characteristics (i.e., soil
nutrient contents, water dynamics) (Smith et al. 2000, Sicardi et al. 2004).
By monitoring the development of U. bojeri seedlings in culture or not with
L. bojeriana under glasshouse bioassays during five months growing time,
Baohanta et al. (2012) illustrated that the occurrence of the shrub species
significantly enhances the development of tree seedlings in both bare and
U. bojeri origin soils or soil invaded by exotic plant species (Eucalyptus
camaldulensis or Pinus patula) from Arivonimamo forests (Table 5). This
form of facilitation was reported by several authors as the ability of a plant
species to promote the development of other plant species by enhancing soil
fertility or by stimulating soil beneficial microorganisms (Gómez-Aparicio
et al. 2005).
Table 5. Development and ectomycorrhizal colonization of U. bojeri seedlings in culture or not with L. bojeriana on degraded and bare soils or in soil
invaded by exotic plant species (Eucalyptus camaldulensis or Pinus patula).
Treatments SB4 RB5 RB/SB6 N7 P8 ECM9

Bulk soil
Control1 131 (11)10 a11 113 (12)a 0.88 ‘0.13)b 0.89 (0.06)a 71.1 (7.3)a 36 (2.1)a
2
L. bojeriana 277 (11)b 140 (10)ab 0.51 (0.04)a 3.02 (0.12)b 253.4 (10.9)b 42 (6)a
L. bojeriana WA3 309 (26)b 166 (3)b 0.55 (0.04)ab 3.08 (0.27)b 332.1 (29.1)b 90. 3 (3.2)b
U. bojeri soil
Control 125 (15)a 295 (35)a 2.37 (0.16)b 0.85 (0.1)a 94.1 (9.9)a 73.7 (3.2)a
L. bojeriana 222 (38)ab 242 (28)a 1.21 (0.33)a 2.14 (0.32)b 197.7 (34.1)b 78 (2.1)a
L. bojeriana WA 332 (19)b 219(39)a 0.67 (0.14)a 3.58 (0.19)c 303.9 (14.1)c 90.7 (2.4)b
E. camaldulensis soil
Control1 83 (0.9)a 27 (4)a 0.34 (0.08)a 0.65 (0.07)a 62.3 (7.3)a 16.3 (2.4)a
L. bojeriana2 233 (41)b 99 (6)b 0.45 (0.09)a 2.30 (0.41)b 194 (35.5)b 65.3 (3.3)b
L. bojerianaWA3 250 (42)b 129 (12)b 0.57 (0.17)a 3.17 (0.57)b 268 (44.9)b 79.3 (4.1)b
P. patula soil
Control 85 (12)a 119 (10)a 1.42 (0.12)b 0.65 (0.09)a 58.9 (8.7)a 29.3 (5.5)a
L. bojeriana 233 (9)b 146 (27)a 0.62 (0.11)a 2.28 (0.10)b 181.3 (5.7) b 30.3 (2.4)a
L. bojeriana WA 333 (66)b 127 (7)a 0.41 (0.08)a 3.90 (0.78)b 278.1 (53.9)b 65.3 (1.5)b
1
U. bojeri without pre- and dual cultivation with L. bojeriana. 2Pre-cultvation with L. bojeriana and dual cultivation with L. bojeriana seedlings with aerial
parts.3 Pre-cultivation with L. bojeriana and dual cultivation with L. bojeriana seedlings without aerial parts.4Shoot biomass (mgdry weight).5Root biomass
(mg dry weight).6Root:shoot ratio. 7N leaf mineral content (mg per plant). 8P leaf mineral content (mg per plant). 9Ectomycorrhizal colonization (%).
10
Standard error of the mean. 11Data in the same column and for each soil origin followed by the same letter are not significantly different according
to the Newman-Keuls test (p<0.05).
In another experiment without the shrub species, these authors showed

that seedling development of U. bojeri was less stimulated on soil invaded
by exotic plant species and bare soil than on U. bojeri origin soil of this
tree. The development of U. bojeri seedlings was significantly stimulated
on pre-colonized soil by L. bojeriana and dual cultivation with this shrub
species without aerial parts. Seeing that experiments were conducted in
pot culture, the nutrient resources of soil may constitute a limiting factor
for the development of the two plant species after 5 months growing
time. However, it has been demonstrated by the results of Baohanta et
al. (2012) that for each soil origin, the occurrence of L. bojeriana affected
soil characteristics, U. bojeri growth, and ECM communities. Co-cultured
with the shrub species (with or without aerial parts), the development of
U. bojeri seedlings was accompanied by higher ECM colonization rate and
high activity levels of total soil microbial [by fluorescein diacetate (FDA)
hydrolysis assay] and soil phosphatase enzymes (Fig. 4A, B). These results
suggest that ectotrophic early-successional shrub species such as L. bojeriana
could lower the negative effects provided by E. camaldulensis and P. patula
by facilitating ECM establishment and consequently improving U. bojeri
early growth.
As mentioned above, S. oblongifolia constitutes the most dominant shrub
species in degraded areas of sclerophyllous forest in the Ambatofinandrahana
region(20-39’-S, 47-06’-E). The morphological and molecular structure
of ECM communities associated with this shrub species and U. bojeri
was described in this part of the Malagasy highland in December 2006
(Ramanankierana H., unpublished data). Study sites were selected among
the surface areas where little to no anthropogenic disturbances had occurred
during at least the last five years. Ectomycorrhizas were collected with soil
cores from three microhabitats on the basis of ECM host plant composition:
(i) a homogenous population of U. bojeri trees devoid of S. oblongifolia or
other ECM host plants, (ii) a clumped population of S. oblongifolia saplings
devoid of other ECM host plants and, (iii) a mixed formation of U. bojeri
and S. oblongifolia. The main objective was to collect soil cores likely to
contain ectomycorrhizas from exclusively U. bojeri or S. oblongifolia and
ectomycorrhizas from both S. oblongifolia and U. bojeri saplings. To this
goal, a sampling scheme was developed in which the nearest undesirable
ECM host plant was located at least 20–25 m from the sampling point.
Ectomycorrhizas from each core were treated separately until RFLP type
identification and three ectomycorrhizas randomly selected from each
morphotype group were screened by RFLP analysis using a part of fungal
rDNA ITS region with 2 units of endonuclease, HaeIII and HinfI. Three
groups of RFLP types were then created following the host plants species:
U. bojeri, S. oblongifolia and mixture host plants. All RFLP types were
sequenced to specify their taxonomic position by using the same primers as
Fig. 4. Results of the PCA on the data table of soil, plant, and microbial activity parameters.
(A) Correlation circle of all the parameters. The 12 variables are: pH=pH, P=total phosphorus
(mg Kg–1), N=total nitrogen (%), OM=total organic matter (%), FDA= global microbial activity;
AcP=acid phosphatase, AlkP=alkaline phosphatase, SB=shoot biomass (g), RB= root biomass
(g), ER=ectomycorrhizal rate (%), PN=leaf nitrogen (%), PP=leaf phosphorus (mg Kg–1), H=
Shannon diversity index of ectomycorrhizal fungi. (B) Map of sample scores on the first two
principal components. Samples are coded as follows. The first three characters correspond to
the soil origin; Eca=soil collected under E. camaldulensis, Ppa= soil collected under P. patula,
Ubo=soil collected under U. bojeri, BaS=bare soil. The treatment applied to the U. bojeri seedlings
is coded as follows. U=Uapaca plant alone, UL=Uapaca plant + L. bojeriana, Ulc=Uapaca plant
+ L. bojeriana cut after 4 months cultivation. For example, a sample coded “EcaULc” is a U.
bojeri seedling grown in soil collected under E. camaldulensis in which a plant of L. bojeriana
was grown and cut after 4 months of cultivation.
in fungal rDNA amplification. A total of 2025 ECM root tips were collected
from 15 soil cores and classified into 165 ECM morphotypes without
mixing root samples from different soil cores. DNA extraction and RFLP
analysis were carried out on 495 ectomycorrhizas and revealed 137 RFLP
types (Table 6). Considering that each RFLP type corresponds to a single
ECM fungal species, the highest diversity of ectomycorrhizas expressed by
Simpson’s diversity index and by Shannon-Wiener information index was
found on mixed vegetation. These diversity indexes were similar between
S. oblongifolia and mixed host plant vegetation. However, diversity indexes
were lower for U. bojeri, suggesting that in homogeneous vegetation this tree
species harbors more rare species than it does with the multiple assemblage
community.
Overall, 52.3% of identified RFLP types were found in both U. bojeri
and S. oblongifolia root systems. The remaining RFLP types were found
exclusively on either U. bojeri (19%) or S. oblongifolia (28.5%) (Fig. 5). Previous
studies have reported that ECM fungi are not uniformly distributed in forest
ecosystems but rather with variability in terms of presence, abundance,
and community composition (Baylis 1980, Perry et al. 1989, Dickie et al.
2004, Dickie and Reich 2005). Environmental factors can influence patterns
of fungal distribution such as soil characteristics and soil microbiota
(Smith and Read 2008), but it is largely admitted that mycorrhized plant
communities are the main factor influencing the availability of ECM fungal
propagules in soil (Dickie and Reich 2005). These biological processes
provide large implications for the establishment of ECM plant cover and
for fungal community ecology.
Table 6. Sampling design, host plant colonization by ectomycorrhizal fungal and fungal
diversity.
Origins
U. bojeri S. oblongifolia Mixture
vegetation
Number of soil cores 5 5 5
Number of ectomycorrhizal root tips 665 580 780
Number of RFLP types (without soil core 38 46 53
comparison)
Percentage of shared identified taxa* 60 58,33 76,92
between the two host plant species (%)
Simpson’s diversity index 6.75 10.58 11.22
Shannon-Wiener information index 0.87 1.05 1.07
*Taxa are identified as RFLP types based on similarity of fragmented size in GenBank.
Fig. 5. Distribution of ectomycorrhizal fungi between Uapaca bojeri and Sarcolaenaoblongifolia

expressed by percentage of RFLP types and percentage of total number of sampled
ectomycorrhizal root tips.
In the present study, two ECM plant species (U. bojeri and S. oblongifolia)
can support patches of ECM fungi. The same study also illustrated that
the growth of U. bojeri seedlings was significantly higher in soils sampled
from areas with established adult trees of U. bojeri than in other soils
(Table 7). Well-developed seedlings on these soils were accompanied by
higher ECM colonization rate and by significant taxa richness of ECM
communities. The stimulation effect of external fungal mycelium radiating
from mature trees on young seedling ECM formation was evidenced in
a Guinean tropical forest (Diédhiou et al. 2010), in Mediterranean holm
oak (Richard et al. 2005), and in temperate coniferous forests (Jonsson
et al. 1999, Kranabetter and Friesen 2002). However, in the experimental
site, the ECM shrub S. oblongifolia displayed an important effect on ECM
fungal infection of young seedlings. In this part of the Madagascarian
sclerophyllous forest, S. oblongifolia was naturally established especially
at the forest edge or in degraded surface areas free of U. bojeri adult trees.
This investigation demonstrates that more than half of collected ECM taxa
were shared between S. oblongifolia and U. bojeri grown separately or in
mixed formation. It suggests that S. oblongifolia, as a pioneering species,
may persist on disturbed sites and facilitate the survival of ECM fungi
that could potentially infect U. bojeri seedling roots. This nurse effect was
particularly efficient by enhancing the ECM fungal diversity associated with
young U. bojeri seedlings and the rate of ECM infection. These results are
similar to reports on facilitation of ectomycorrhizal infection of Pseudotsuga
Table 7. Growth and ectomycorrhizal development of U. bojeri seedlings after 5 months of

culture in soil samples collected at different distances from established U. bojeri adult tree.
Distances from U. bojeri adult tree

Control 20 m 10 m 5m 1m 0m
Seedling development
Shoot biomass (mg per plant) 92 a (1) 120 a 313 c 335 c 199 b 183 b
Root biomass (mg per plant) 61 a 75 a 168 bc 211 c 128 b 137 b
Total biomass (mg per plant) 154 a 195 a 481 c 546 c 328 b 320 b
Shoot / Root mass ratio 1.53 ab 1.65 ab 1.86 b 1.59 ab 1.64 ab 1.42 a
Number of root tips per mg of root 0.83 b 0.73 b 0.51 ab 0.39 a 0.63 ab 0.49 a
biomass
Ectomycorrhizal colonization (%) 3.19 a 5.88 ab 21.06 c 22.05 c 11.70 bc 10.49
bc
Ectomycorrhizal diversity
Taxa richness (expressed as RFLP 0.5 a 0.8 a 3c 3.5 c 1.6 ab 2b
type)
(1)
Data in the same line followed by the same letter are not significantly different according
to the Newman-Keul test (p< 0.05).
menziesii seedlings by pioneering hardwoods (Borchers and Perry 1987) or

the positive effect of Helianthemum bicknellii on ECM infection of Quercus
spp. (Dickie et al. 2004).
8. Conclusion
This chapter shows that the development of U. bojeri, an endemic tree of
the Malagasysclerophyllous forest, strongly requires the occurrence of
compatible ECM fungi. These ECM symbionts could be from an adult
tree of this plant where various putative ECM fungi were observed, or
from pioneer shrub species that persist on disturbed sites and facilitate
the survival of ECM fungi propagules that could potentially infect roots
of U. bojeri seedling. In this way, two species of endemic shrub species
(L. bojeriana and S. oblongifolia) could be of great interest. These data
suggest that these nurse plant species should be considered to enhance
the performance of reforestation efforts in Madagascar by providing ECM
inoculum potential.
Acknowledgments
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CHAPTER
5
Morpho-anatomical
Characterization of Three
Sebacinales Ectomycorrhizal
Species from a Pakaraimaea
dipterocarpacea ssp. nitida
(Dipterocarpaceae) Forest in
Southern Venezuela
Bernard Moyersoen
1. Introduction
Sebacinales are a genetically diverse group of basidiomycetes with a broad
range of lifestyles including a diversity of mycorrhizal and endophytic
associations (Weiß et al. 2011). These fungi are widespread and the same
species can colonize a wide range of host plants and be involved in different
symbiotic associations (Selosse et al. 2002a, Weiß et al. 2011). Anatomically,
Sebacinales are characterized by longitudinally septate basidia, imperforate
parenthesomes, and a lack of both clamp connections and hymenial cystidia
(Weiß et al. 2004). A very high diversity within the Sebacinales has been
School of Biological Sciences, University of Aberdeen, AB243UU, United Kingdom.

Email: bmoyersoen@hotmail.com
described from host plant and soil DNA samples and the observed global
diversity was estimated to be 365 ITS taxonomic units (Setaro et al. 2012).
Molecular-phylogenetically, Sebacinales are divided into two groups
called A and B (Weiß et al. 2004). Up to date, most ectomycorrhizal (EcM)
associations have been documented from group A (Weiß et al. 2004, 2011)
and only one clade in group B included EcM strains (Hynson et al. 2013).
Only a few EcM fruitbody species belonging to Sebacina, Tremellodendron
and Tremelloscypha have been described to date (Weiß et al. 2004). These
morphospecies include the cosmopolitan cryptic species S. incrustans
(Pers.) Tul. & C. Tul. and S. epigea (Berk. & Broome) Bourdot & Gazin and
the polymorphic Tremelloscypha gelatinosa (Murrill) Oberw. & K. Wells (Weiß
et al. 2004, Wells and Oberwinkler 1982). In addition to the fruitbodies,
EcM anatomotypes can be used to hypothesize phylogenetic relationships
(Agerer 2006). Amongst Sebacinales, only seven EcM anatomotypes
have been described to date and all of these EcMs were from temperate
areas (Selosse et al. 2002b, Urban et al. 2003, Azul et al. 2006, Twieg and
Durall 2009, Wei and Agerer 2011). A precise taxonomy should rely on a
combination of characters (Dayrat 2005). Morphological and anatomical
descriptions, in combination with other data, are a prerequisite for accurate
EcM biodiversity surveys. A greater knowledge of Sebacinales EcM morpho-
anatomy could be particularly useful for the taxonomy and biodiversity
surveys of this fungal group with cryptic or unknown fruitbody species.
This study is part of a phylogeographic and taxonomic analysis
of Pakaraimaea dipterocarpacea Maguire & Ashton ssp. nitida Maguire &
Steyerm. EcM fungi in Southern Venezuela (Moyersoen 2006, 2012a,b,c,d). P.
dipterocarpacea, an endemic tree species from Guayana region (Maguire et al.
1977, Maguire and Ashton 1980, Moyersoen 2006), is one of the few known
locally dominant EcM tree species in the Neotropical lowland forests. This tree
is phylogenetically related to the most important EcM tropical tree family in
SE Asia: the Dipterocarpaceae. The phylogeny of P. dipterocarpacea EcM fungi
might reflect the disjunct distribution of this endemic tree species. A diverse
EcM fungal community was observed in a 400 m2 P. dipterocarpacea forest plot
(Moyersoen 2012a). In total, 40 EcM fungal species were found and up to 12
different EcM morphotypes could colonize a single 125cm3 soil core. Floristic
comparisons and a molecular-phylogenetic analysis of two newly reported
Inocybe species (Moyersoen 2012a,d) indicated that host jump and fungal
dispersion (Pirozynski 1983) were probably important radiation mechanisms
for P. dipterocarpacea fungi (Moyersoen 2012a). Possible EcM endemic fungi
in the forest indicated that P. dipterocarpacea fungal communities might also
include old relictual species (Moyersoen 2012a). Similar trends regarding the
great EcM fungal diversity, the dominance of host-generalist fungi and the
presence of possible unique Agaricales were observed in a P. dipterocarpacea
ssp. dipterocarpacea forest in Guyana (Smith et al. 2013).
Tropical Sebacinales Ectomycorrhizas 81
Sebacinales were selected to extend the P. dipterocarpacea EcM fungi

phylogeographic study. Tremellodendron ocreatum (Berk.) P. Roberts and
S. incrustans fruitbodies were first described in a Dicymbe rain forest in
Guayana region (Henkel et al. 2004). The first report of Sebacinales EcM in
the tropics was made on P. dipterocarpacea (Moyersoen 2006). In total, three
Sebacinales species (specimens BM03M3, PD10 and 6MM2) were found in
a P. dipterocarpacea forest plot (Moyersoen 2012a). Of the 97 sequences from
a total of 113 EcM DNA samples, 10 percent (11 sequences) belonged to the
three Sebacinales species. This fungal group contributed to 11% of total
below ground fungal richness; it was frequent and was collected in five out
of nine soil cores scattered in the plot (B.M., unpublished data).
Tropical Sebacinales biodiversity was recorded mostly from DNA
analysis in a wide range of forests across the tropics (Moyersoen 2006,
2012a, Rivière et al. 2007, Morris et al. 2008, Morris et al. 2009, Peay et al.
2009, Roy et al. 2009, Tedersoo et al. 2010a,b, Jairus et al. 2011, Kennedy et
al. 2011, Smith et al. 2011, Tedersoo et al. 2011) and only one Sebacinales
EcM has been briefly described to date (Moyersoen 2006). The aims of
this study were to confirm that the three Sebacinales identified by DNA
analysis are EcM, to describe in detail the morpho-anatomy of these three
EcM anatomotypes and to compare them with published descriptions. The
molecular-phylogeny of the three Sebacinales species will be published
separately.
2. Fine Root Sampling, Morpho-anatomical and Ultrastructural

Descriptions
The specimens were sampled at 4º20’N, 61º48’W, altitude 500 m, near
Icabarú Village, in Gran Sabana, Estado Bolivar, Venezuela. The precise
location of the 400 m2 plot was selected based on a previous report of a
stand of Pakaraimaea dipterocarpacea Maguire & Ashton ssp. nitida Maguire
& Steyerm described by Maguire and Steyermark (1981) in the same area.
Four separate sampling expeditions were conducted, in November 2003
(one day), March 2006 (one day), July 2007 (five days) and July 2008 (nine
days).
Fine roots from the top organic soil layer were either traced from P.
dipterocarpacea trees (collections BM03M3, PD10) or collected from soil cores
(sample 6MM2). The sampling procedures are described in Moyersoen
(2006, 2012a). EcM were washed from the soil and clusters were assigned
to morphotypes using a field dissecting microscope (Novex AP-7) or a 10X
magnifying glass. EcM tips were stored in 2% cetyltrimethylammonium
bromide (CTAB). BM03M3 subsamples from the same cluster were stored in
water at 5ºC (subsample 1) or fixed in glycerol: 100% ethanol: water (1:1:1)
(subsample 2). Within three days of collection, for BM03M3 subsample 1,

the habit was recorded with a dissecting microscope (Leica MZ6) fitted with
a camera (Leica MPS60). The anatomical features of BM03M3 subsample
2, PD10 and 6MM2 were described from mantle peelings using Agerer’s
(1991) method of description. Drawings were performed using a Normarski
interference contrast microscope (Leica DM2500) equipped with a drawing
tube. Reference specimens of BM03M3 and PD10, and DNA extract of 6MM2
are stored in the University of Liège Herbarium (LG).
To examine specimen BM03M3 at the ultrastructural level, EcM tips
fixed in glycerol:ethanol:water were transferred in 2% gluteraldehyde in 0.1
M sodium cacodylate buffer (pH 7.2) at room temperature for 1 h. These tip
samples were then submitted to six transfers in 0.1 M sodium cacodylate
buffer and post fixed in 1% osmium tetroxide in the same buffer for 1 h in the
dark. They were then washed in distilled water and stained in 1% aqueous
uranyl acetate for 1h in the dark. After five washes in distilled water, samples
were dehydrated in acetone, using 10 min changes at 10, 25, 50, 70, 95%
and three times in 100% acetone. Samples were imbedded in Spurr’s plastic
(Spurr 1969) and sectioned with a glass knife for semi-thin sections and a
diamond knife for ultra-thin sections. Semi-thin sections were stained with
new fuchsin and crystal violet, mounted in Entellan and used for light
microscopy. Ultra-thin serial sections were mounted on Formvar coated,
single-slot copper grids, stained with lead citrate at room temperature for
5 min, and washed with distilled water. They were examined using a Zeiss
transmission electron microscope operating at 80 kV.
Sebacinales BM03M3 + Pakaraimaea dipterocarpacea
Short description: The mycorrhizae are characterized by a simple or irregularly
monopodially-pinnate system with a yellowish colour and a woolly surface.
Emanating hyphae and rhizomorphs are frequent and occur throughout.
The mantle outer layer is a transition between a dense plectenchyma
with ramified hyphae and a pseudoparenchyma with a matrix, covered
by an hyphal net; it is a plectenchyma in deeper layers. Septa are simple.
Septal pores are dolipore parenthesome type and the parenthesomes are
imperforate with electron-dense middle lamella. Walls are smooth and light
orange; they are thick in outer mantle layer, rhizomorphs and emanating
hyphae. Triangular inflations and short protrusions are present in the outer
mantle layer and emanating hyphae. Polytomies with three branches are
present in the net. Rhizomorphs are not differentiated and are uniformly
compact. Emanating hyphae are smooth, and some are similar to awl-shaped
cystidia. The mantle preparation is slightly dextrinoid. The mycorrhizae are
hydrophilic and belong to a transition between short distance and smooth
subtype of medium distance exploration type.
Morphological characters (Fig. 1a): Mycorrhizal systems simple or irregularly

monopodially-pinnate. Main axes 0.2–0.3 mm thick. Unramified ends straight,
0.5–1 mm long and 0.2–0.3 mm diam., yellowish (G in Colour Identification
Chart, Royal Botanic Garden Edinburgh, Flora of British Fungi, 1969) in
older parts, white at apex (Fig. 1B in Moyersoen 2006). Surface of unramified
ends woolly; emanating hyphae frequent, occurring throughout, colourless.
Rhizomorphs colourless to yellowish, approximately 10 µm wide, frequent,
circular in cross-section, occurring throughout, unramified. Soil particles
often sticking to the mantle surface and rhizomorphs. Hydrophilic.
Exploration type, a transition between the short distance exploration type
and the smooth subtype of the medium distance exploration type.
Fig. 1. Sebacinales BM03M3 + Pakaraimaea dipterocarpacea ssp. nitida. a. Habit of ectomycorrhiza,

woolly surface, emanating hyphae and rhizomorphs occurring throughout, soil particles
sticking to the mantle and rhizomorphs surface (Bar = 1 mm). b–c. Plan view of hyphal network
in outer mantle layer; the drawn features include: ramified, irregularly thick-walled hyphae,
ramifications Y-shaped or at 90°, polytomies with three branches (c), triangular inflations
at ramifications, short protrusions, distal hyphal ends sometimes aculeate, septa simple,
frequent, of the same thickness or thinner than the walls, sometimes incomplete. d–e. Plan
views of outer layer below the net, a transition between a dense plectenchyma of thick-walled
ramified hyphae and a pseudoparenchyma, with a matrix. f–g. Plan view of middle mantle
layer, dense plectenchyma of ramified hyphae, anastomoses short, open (g). h. Plan view of
inner mantle layer, dense plectenchyma of short, ramified hyphae, anastomosis short, open,
some incomplete septa. (Bars = 10 µm).
Anatomical characters of mantle in plan views (Figs. 1b–h): Outer mantle layers
(Figs. 1b–e), a transition between a dense plectenchyma of thick-walled
ramified hyphae and a pseudoparenchyma (type E/C/H, Agerer 2006),
with a matrix, covered by a loose network of ramified, thick-walled hyphae
from which emanating hyphae and rhizomorphs originate; hyphae from
the network mostly cylindrical, with frequent short protrusions and
triangular inflation at ramifications, sometimes aculeate; ramifications
Y-shaped or at 90°, sometimes polytomies with three branches; cells 2.2–3.5
µm diam.; cell walls light orange, smooth, irregularly thick (0.5–1.7 µm);
septa simple, frequent, of the same thickness or thinner than the walls,
sometimes incomplete; anastomoses lacking; hyphae deeper in the surface
layer variably shaped; walls difficult to distinguish from the matrix at 1000X
with transmission light microscope; walls and matrix yellowish. Middle
mantle layers (Figs. 1f–g) a dense plectenchyma of ramified, cylindrical
hyphae; cells 1.7–2.7 µm diam.; ramifications Y-shaped; cell walls smooth,
yellowish, thin; septa simple, frequent, of the same thickness or thicker
than the hyphal walls, sometimes incomplete; anastomoses scarce, short,
open. Inner mantle layers (Fig. 1h) a dense plectenchyma of short, ramified
hyphae, in a matrix; cells mostly cylindrical or of variable shape; walls thin,
yellowish; septa simple, frequent, of the same thickness or thicker than the
hyphal walls, sometimes incomplete; anastomoses scarce, short, open. Very
tip with the same arrangement as in other parts of mantle.
Anatomical characters of emanating elements (Figs. 2a–d): Rhizomorphs (Figs.
2a–b) abundant, 5–25 µm diam., uniform-compact (uniform hyphae, densely
packed and glued together, type B in Agerer 2006), often with ramified
hyphae at distal end; hyphae 2.6–3.6 µm diam.; hyphal ramifications at
rhizomorph distal end, Y-shaped, at 90°, or oriented backwards; walls
yellowish, smooth, 0.7–1.5 µm thick, thin at hyphal distal end; septa simple,
frequent, of the same thickness or thinner than the hyphal walls, sometimes
incomplete; rhizomorph apex simple or with ramified, tortuous hyphae
with protrusions, and with a protuberance at the tip; anastomoses open,
short or with a bridge, only observed at rhizomorph distal end. Emanating
hyphae (Figs. 2c–d) abundant, with or without short protrusions at proximal
end, 2.8–3.2 µm thick; septa simple, frequent, evenly distributed, generally
thinner than the hyphal walls, sometimes incomplete; hyphal ends simple;
walls yellowish, 1–1.5 µm thick, sometimes thinner at distal end, surface
smooth; ramifications lacking; anastomoses lacking; shorter emanating
hyphae similar to bent awl-shaped cystidia (type A, Agerer 2006), 52–135
µm long, diam. between 1.7–2.5 and 2.6–3.7 µm, and wall thickness between
1–1.5 and 0.5–1 µm at proximal and distal end, respectively.
Anatomical characters, longitudinal section (Fig. 3): Mantle plectenchymatous,
15–25 µm wide, with two layers; outer layer 7–9 µm thick, with thick-walled
Fig. 2. Sebacinales BM03M3 + Pakaraimaea dipterocarpacea ssp. nitida. a–b. Rhizomorphs, thick-
walled hyphae of uniform diameter running in parallel (a), apex simple (a) or with ramified,
tortuous hyphae with protrusions and protuberance at the tip (b), septa frequent, of the same
thickness or thinner than the walls (a, b), anastomosis open, short or with a hyphal bridge (a).
c–d. Emanating hyphae, thick-walled, with or without short protrusion at proximal end (c),
frequent simple septa, generally thinner than hyphal walls, shorter emanating hyphae similar
to bent awl-shaped cystidia (d). (Bars = 10 µm).
Fig. 3. Transmission light micrograph of Sebacinales BM03M3 + Pakaraimaea dipterocarpacea

ssp. nitida in longitudinal section. OL: outer mantle layer, thick-walled cells imbedded in a
matrix; IL: inner mantle layer, cells mostly cylindrical, matrix visible in innermost layer; HN:
Hartig net paraepidermal, hyphal cells in one row; E: epidermal cell. (Bar = 10µm).
cells of different shape, imbedded in a matrix; inner layer 10–15 µm thick,

with cells mostly cylindrical, tangentially 4–15 µm, radially 1.5–2 µm,
matrix visible in innermost layer; remnants of calyptra observed. Tannin
cells lacking. Epidermal cells rectangular, tangentially 7–20 µm, radially
24–33 µm, ECt (average tangential measure of epidermis cells) 12 µm, ECq
(average ratios between tangential and radial measure of epidermis cells)
0.5. Hartig net paraepidermal, hyphal cells in 1 row; in plan view of palmetti
type. Haustoria lacking.
Colour reactions with different reagents: Preparations of mantle: Melzer’s
reagent: cell walls orange brown.
Ultrastructure of ectomycorrhizae, longitudinal section (Figs. 4–8): Outer layer
with thick-walled hyphae imbedded in an electron-dense matrix; inner
layers with thinner-walled hyphae, matrix not very distinct; septal pores
Figs. 4–8. Tansmission electron micrographs of Sebacinales BM03M3 + Pakaraimaea

dipterocarpacea ssp. nitida. Figure 4: Section through the mantle. Thick-walled hyphae imbedded
in a matrix in outer layer and thinner-walled hyphae in inner layer. E: portion of epidermal cell;
M: mantle. Figure 5: Section through the Hartig net (HN), Palmetti type lobes. E: epidermal
cells. Figure 6: Section through the inner mantle layers, complete (S) and incomplete septa
(IS). Figure 7: detail of a dolipore. Figure 8: detail of imperforate parenthesome. Figures 4–5:
Bar = 2 µm., Figure 6: Bar = 1 µm. Figures 7–8: Bar = 0.2 µm.
in the mantle and Hartig net, dolipore-parenthesome type, parenthesome

imperforate with an electron-opaque middle lamella; Hartig net with thin-
walled hyphae, in one row, palmetti type in plan-view.
Reference specimen: Venezuela, 4º20’N, 61º48’W, altitude 500 m, near
Icabarú Village, in Gran Sabana, Estado Bolivar, tropical forest dominated
by Pakaraimaea dipterocarpacea Maguire & Ashton ssp. nitida Maguire &
Steyerm.; ectomycorrhizae collected by root tracing from a P. dipterocarpacea
ssp. nitida adult tree (Moyersoen 2006), in top of mineral soil, below the
litter, 25.11.2003, myc. coll. and isol. B. Moyersoen, reference specimen in
glycerol/ethanol/water in Herb. B. Moyersoen BM03M3 (in LG).
Sebacinales PD10 + Pakaraimaea dipterocarpacea
Short description: The mycorrhizae are characterized by an irregularly
monopodially-pinnate system with a smooth or cottony surface. Emanating
hyphae are frequent and rhizomorphs are lacking. The mantle outer layer is
plectenchymatous with squarrose branched hyphae; it is plectenchymatous
with a matrix in middle layers. Septa are simple. Walls are thick or thin in
outer mantle layer and they are colourless. Inflations are frequent in the
outer mantle layer and they are triangular at ramifications. Thick-walled
staghorn-like hyphae with polytomies with three to many finger-like
outgrowths are frequent in the outermost layer. Emanating hyphae are
thick-walled and smooth, with short protrusions. The mycorrhizae belong
to the short distance exploration type.
Morphological characters (Fig. 9a): Mycorrhizal systems monopodial, irregularly
pinnate. Main axes 0.2–0.3 mm thick. Unramified ends straight or bent, 0.5–2
mm long and 0.2–0.3 mm diam., colour not recorded. Surface of unramified
ends smooth or cottony, with some woolly patches; emanating hyphae
frequent, occurring throughout. Soil particles often sticking to the mantle
surface and the emanating hyphae. Exploration type, short distance.
Anatomical characters of mantle in plan views (Figs. 9b–d, 10a–b): Outer mantle
layers (Figs. 9b-d), a loose plectenchymatous network of squarrose branched
hyphae (hyphae multiply ramified with short, frequently Y-shaped or
almost rectangular branches, type E (Agerer 2006)) becoming more densely
arranged in deeper layers; frequent staghorn-like hyphae, with many
finger-like outgrowths and inflations in the outermost layer, hyphae mostly
cylindrical in deeper layers; ramifications Y-shaped or at 90°, often with
triangular inflation, polytomies with three to many finger-like outgrowths;
cylindrical cells 2.2–6.4 µm diam.; cell walls colourless, smooth, either
irregularly thick (0.5–2.5 µm), or thin; septa simple, not frequent, straight
or curved, sometimes incomplete, thinner or of the same thickness as the
hyphal walls; anastomoses not frequent, short, open; matrix lacking. Middle
Fig. 9. Sebacinales PD10 + Pakaraimaea dipterocarpacea ssp. nitida. a. Habit of ectomycorrhiza,

smooth or cottony surface with some woolly patches, emanating hyphae occurring throughout,
soil particles sticking to the mantle surface. b–d. Plan views of outer layer; ramified, staghorn-
like hyphae, with inflations either intercalary (b, c) or at ramifications (b), wall thickness
irregular and variable (c, d), ramifications Y-shaped or at 90° (d), polytomies with three to
many finger-like outgrowths (b, c), septa simple, not frequent, sometimes incomplete (c),
thinner or of the same thickness as the hyphal walls (c). (Bar = 10 µm).
mantle layers (Fig. 10a), a dense plectenchymatous network of ramified,

cylindrical hyphae, sometimes with constriction at septa; cells 1.3–2.6 µm
diam.; ramifications Y-shaped or at 90°, often with triangular inflation; cell
walls smooth, colourless, generally thin, some hyphae with thicker walls;
septa simple; anastomoses short, open; matrix present. Inner mantle layers
(Fig. 10b) a dense plectenchymatous network of ramified hyphae; hyphal
shape cylindrical, sometimes with constriction at septa, hyphae sometimes
with palmetti type lobes; cells 2.1–3.2 µm diam.; ramifications Y-shaped
or at 90°, often with triangular inflation; walls smooth, thin, colourless;
septa simple, frequent, sometimes incomplete; anastomoses not observed;
matrix not observed. Very tip with the same arrangement as in other parts
of the mantle.
Fig. 10. Sebacinales PD10 + Pakaraimaea dipterocarpacea ssp. nitida. a. Plan view of middle
mantle layer, dense plectenchyma of thinner-walled, ramified hyphae, with a matrix. b. Plan
view of inner mantle layer, dense plectenchyma of ramified hyphae, septa frequent, sometimes
incomplete. c–d. Emanating hyphae, thick-walled, distal end simple, sometimes thinner-
walled and with a protuberance at the tip (c), septa infrequent, thinner than the hyphal walls,
short protrusions occurring throughout (d), ramifications Y-shaped or at 90° with triangular
inflation (d). (Bar = 10 µm).
Anatomical characters of emanating elements (Figs. 10c–d): Rhizomorphs lacking.

Emanating hyphae abundant, 2.1–2.5 µm thick; septa simple, infrequent,
thinner than the hyphal walls; hyphal ends simple or with a protuberance;
walls 0.5–1 µm thick, even in thickness or thinner at distal end, surface
smooth; ramifications Y-shaped or at 90°, often with triangular inflation;
short protrusions occurring throughout; anastomoses lacking. Cystidia
(i.e., hyphae with constant length) lacking, but staghorn-like hyphae in the
outermost layer could be interpreted as cystidia.
Reference specimen for Pakaraimaea ectomycorrhiza: Venezuela, 4º20’ N, 61º48’
W, altitude 500 m, near Icabarú Village, in Gran Sabana, Estado Bolivar,
tropical forest dominated by Pakaraimaea dipterocarpacea Maguire & Ashton
ssp. nitida Maguire & Steyerm.; ectomycorrhizae collected by root tracing
from a P. dipterocarpacea ssp. nitida adult tree (Moyersoen 2006), in top of
mineral soil, below the litter, 20.07.2007, myc. coll. and isol. B. Moyersoen,
reference specimen in cetyltrimethylammonium bromide (CTAB) in Herb.
B. Moyersoen PD10 (in LG).
Sebacinales 6MM2 + Pakaraimaea dipterocarpacea

Short description: The mantle outer layer is a loose network of squarrose,
branched, mostly cylindrical hyphae, becoming densely arranged in deeper
layers and from which abundant emanating hyphae originate. A matrix is
lacking both in outer and deeper layers. Septa are simple and they are often
curved. Walls are thick in outer mantle layer, rhizomorphs and emanating
hyphae and they are colourless. Inflations including triangular ones at
ramifications and short protrusions are present both in outer mantle layer
and emanating hyphae. Polytomies with three branches are present in the
net. Rhizomorphs are frequent, not differentiated, and uniform compact.
Emanating hyphae are smooth and some are similar to awl-shaped cystidia;
clusters of hyphae are often observed emanating from the outer mantle
layer. The mycorrhizae belong to a transition between short distance and
smooth subtype of medium distance.
Morphological characters: not recorded. Exploration type, a transition between
the short distance exploration type and the smooth subtype of medium
distance exploration type (on basis of anatomy of emanating elements).
Anatomical characters of mantle in plan views (Figs. 11a–d): The mantle
anatomy is difficult to observe for the great amount of thick-walled
emanating hyphae covering the outer layer. Outer mantle layers (Figs. 11a–b),
a loose plectenchymatous network of squarrose branched hyphae (mantle
type E; Agerer 2006) becoming densely arranged in deeper layers, from
which abundant emanating hyphae originate; hyphae mostly cylindrical,
with frequent inflations and short protrusions; ramifications Y-shaped or
at 90°, often with triangular inflation, polytomies with three branches,
frequent clusters of hyphae emanating from the outermost layer; cells
2–3.6 µm diam.; cell walls colourless, smooth, irregularly thick (0.5–1.5
µm); septa simple, often thinner than the hyphal walls, often curved;
anastomoses lacking; matrix not observed. Middle mantle layers (Fig. 11c) a
dense plectenchyma of ramified, cylindrical hyphae; cells 2.2–3.4 µm diam.;
ramifications Y-shaped; cell walls smooth, colourless, frequently thin, some
hyphae with thicker walls (0.5–1 µm); septa simple, not frequent, of the same
thickness as the hyphal walls; anastomoses not observed. Inner mantle layers
(Fig. 11d) a transition between a plectenchyma and a pseudoparenchyma;
cells cylindrical or of variable shape, sometimes with palmetti type lobes;
between 21–24 cells in a square of 20 X 20 µm; walls thin, colourless; septa
simple, frequent, sometimes incomplete; anastomoses open. Very tip with
the same arrangement as in other parts of mantle.
Anatomical characters of emanating elements (Figs. 11e–i): Rhizomorphs
(Figs. 11e–g) 10–50 µm diam., uniform-compact (type B, Agerer 2006);
hyphae 2.4–3.2 µm diam.; hyphal ramifications infrequent, Y-shaped or
Fig. 11. Sebacinales 6MM2 + Pakaraimaea dipterocarpacea. a–b. Plan views of outer layer,
ramified, thick-walled hyphae (a) from which many emanating hyphae originate (b), inflations
intercalary or triangular at ramifications (a), short protrusions (b), ramifications Y-shaped or
at 90° (a), polytomies with three branches (b), septa simple, frequent, of the same thickness or
thinner than the hyphal walls (a–b). c. Plan view of middle mantle layer; dense plectenchyma,
walls generally thinner than in outer layer. d. Plan view of inner mantle layer, a transition
between a plectenchyma and a pseudoparenchyma; thin walls, palmetti type lobes, anastomosis
short, open. e–g. Rhizomorph, thick-walled hyphae of uniform diameter running in parallel,
apex simple, cell walls thinner at distal end, septa straight or curved, clustered at distal end,
ramification Y-shaped (f), anastomosis open, short (g). h–i. Emanating hyphae, thick-walled,
thinner-walled at distal end, with or without short protrusion at proximal end (i), simple septa
frequent, of the same thickness or thinner than the hyphal walls, straight or curved, ramification
Y-shaped, shorter emanating hyphae similar to awl-shaped cystidia (i). (Bar = 10 µm).
at 90°; walls colourless, smooth, 0.5–1 µm thick, thinner at hyphal distal

end; septa simple, infrequent, of the same thickness as the hyphal walls,
straight or curved, often one to three septa at hyphal distal end; hyphal
end at rhizomorph apex simple; anastomosis short, open. Emanating hyphae
(Figs. 11h–i) abundant, 2.6–3.1 µm thick; septa simple, frequent, evenly
distributed, of the same thickness or thinner than the hyphal walls; hyphal
ends simple; walls colourless, 0.7–1 µm thick, thinner at distal end, surface
smooth; ramifications Y-shaped or at 90°, often with triangular inflation;
anastomoses lacking; intrahyphal hyphae present. Cystidia lacking, but

emanating hyphae originating from the superficial net similar to awl-
shaped cystidia with basal dichotomous or trichotomous ramifications
(Agerer 2006).
Reference specimen for Pakaraimaea ectomycorrhiza: Venezuela, 4º20’ N, 61º48’
W, altitude 500 m, near Icabarú Village, in Gran Sabana, Estado Bolivar,
tropical forest dominated by Pakaraimaea dipterocarpacea Maguire & Ashton
ssp. nitida Maguire & Steyerm. ectomycorrhizae collected from a soil core
collected near a P. dipterocarpacea ssp. nitida adult tree (Moyersoen 2012a),
in first 5 cm organic soil, 27.07.2008, myc. coll. and isol. B. Moyersoen, DNA
extract in Herb. B. Moyersoen 6MM2 (in LG).
3. Molecular Identification with Sequences from GenBank and

UNITE
For the molecular identification of BM03M3, PD10 and 6MM2, DNA
protocols used have been described by Moyersoen (2006, 2012a). Fungal
sequences from both ITS and LSU were obtained using the primers
ITS1f, ITS4b, LROR, LR21 and LR6, depending upon the success of PCR
amplifications. GenBank accession number for ITS are JQ063056, JQ063057,
and JQ063058 for specimens BM03M3, PD10, and 6MM2, respectively.
The host of specimen 6MM2 was identified by DNA sequencing using the
primers rbcLN and rbcLR as described by Moyersoen (2006, 2012a) and
host identity of specimen BM03M3 was confirmed by rbcL DNA RFLP
using Hinf1 as restriction enzyme (Moyersoen 2006). ITS sequences were
assigned to molecular species on the basis of arbitrary 3% similarity cut-off
value (Nilsson et al. 2008). Sequence comparisons were performed using
the BlastN (Altschul et al. 1997) in GenBank and UNITE (Kõljalg et al. 2005,
http://unite.ut.ee).
None of the Pakaraimaea Sebacinales ITS and LSU sequences matched
identified Sebacinales species (data not shown, analysis 22 January 2013).
A significant match with non-described species was found between
specimen BM03M3 ITS sequence and uncultured Sebacinales voucher
EcM17 (JN168754) (497/514, 97%) and specimen 6MM2 ITS with uncultured
Sebacinales voucher EcM84 (JN168756) (251/258, 97%). Both matching
sequences were from a Dicymbe dominated forest in Guyana (Moyersoen
2012a). ITS best match for specimen PD10 was with Sebacinales isolate
L7604_Seb MAD02 (FR731328) (518/568, 91%).
4. Analysis of Specimens based on Morpho-anatomical and

Ultrastrutural Features Together with DNA Sequences
Ultrastructural characteristics (investigated in one anatomotype) and
morpho-anatomical features, together with DNA sequences indicated
that BM03M3, PD10 and 6MM2 Sebacinales species were involved in
P. dipterocarpacea EcM.
Ultrastructurally, the dolipore parenthesome architecture investigated
in specimen BM03M3 was consistent with that of Sebacinales (Urban et al.
2003). The ultrastructure of septa could not be studied in specimens PD10
and 6MM2, but the presence of only simple septa was in agreement with
the sequenced Sebacinales EcM.
Morphological and anatomical features observed on temperate
Sebacinales EcM are the following: a plectenchymatous mantle with
squarrose branched hyphae (type E/C, Agerer 2006) or a transition between
a plectenchyma of ramified hyphae and a pseudoparenchyma, a matrix in
the outer mantle layer, thick-walled hyphae (at least in the outer mantle
layer and/or the emanating elements), ramifications with more than
two branches (referred to as polytomies), inflations including triangular
inflations at ramifications, the absence of hydrophobicity, cystidia (when
present) awl-shaped, smooth and colourless cell walls. Rhizomorphs were
lacking in most temperate Sebacinales EcM anatomotypes, and the EcM
were classified as short distance exploration type by Agerer (2006). Most
recently, infrequent, undifferentiated rhizomorphs with loosely arranged
hyphae in a matrix have been reported in Pinirhiza nondextrinoidea (Wei
and Agerer 2011). A dextrinoid colour reaction was observed in Pinirhiza
multifurcata (Wei and Agerer 2011) and Quercirhiza dendrohyphidiomorpha +
Quercus suber L. (Azul et al. 2006).
Specimens BM03M3, PD10 and 6MM2 shared morphological and
anatomical features with temperate Sebacinales. The mantle was a
plectenchyma of squarrose branched hyphae in PD10 and 6MM2 or a
transitional type to a pseudoparenchyma in BM03M3. Hyphae in the outer
mantle layers and emanating elements were thick-walled. Cell walls were
smooth and hyphal inflations were conspicuous in the outer mantle layer and
emanating elements. A matrix was present in BM03M3 and PD10. Polytomies
were frequent either in the superficial hyphal net (BM03M3 and PD10) or in
the emanating hyphae (6MM2). Both BM03M3 and PD10 were hydrophilic
(this feature was not recorded in 6MM2). Colour reaction to Meltzer was only
recorded in BM03M3 and a slight dextrinoid reaction was observed.
Specimens BM03M3, PD10 and 6MM2 could be distinguished by
several morphological and anatomical features. BM03M3 was woolly and
it was cottony in PD10. Cell walls were yellowish in BM03M3 whereas
they were colourless in the remaining two anatomotypes. Rhizomorphs
were observed only in BM03M3 and 6MM2. The transition between a

plectenchyma and a pseudoparenchyma in BM03M3 was different from the
plectenchymatous mantle in PD10 and 6MM2. The cell diameter in outer
layer was more variable and greater on average in PD10 than in 6MM2 and
BM03M3. A matrix was present in BM03M3 outer mantle layer, in PD10 in
deeper mantle layers and it was absent in 6MM2. Hyphae from outermost
layer were variably shaped in BM03M3 and PD10 and they were mostly
cylindrical in 6MM2. Staghorn-like hyphae were observed only in PD10.
Ramifications included polytomies with up to three branches in BM03M3
and 6MM2; they could ramify in three to many branches in PD10. Clusters
of emanating hyphae were only observed in 6MM2’s outer mantle layer.
Simple septa were frequently curved in 6MM2, either straight or curved
in PD10 and straight in BM03M3. Hyphal ends in emanating hyphae were
thin-walled in 6MM2 and either thick or thin-walled in both, BM03M3 and
PD10. Emanating elements similar to awl-shaped cystidia were lacking in
PD10; they were scattered in BM03M3 and often in clusters in 6MM2.
BM03M3 anatomotype was particularly close to the unidentified
sample Y62M1 (Urban et al. 2003, Agerer and Rambold 2004–2011). Both
are yellowish and hydrophilic. Shared anatomical features between the
two anatomotypes included the transition between a dense plectenchyma
of thick-walled ramified hyphae and a pseudoparenchyma in the outer
layer (type E/C/H, Agerer 2006). This mantle layer organization was also
observed in Sebacinoid ectomycorrhiza M2 (Selosse et al. 2002b). Remaining
shared features between BM03M3 and sample Y62M1 (Urban et al. 2003)
included: the superficial, loose hyphal net with variably shaped, thick-
walled hyphae with protrusions and inflations, the emanating hyphae
originating from the net and the plectenchymatous deeper layers. The main
differences between BM03M3 and sample Y62M1 included: the yellowish
cell walls in BM03M3, the rhizomorphs lacking in Y62M1, the greater size
(3.5–7 versus 2.2–3.5 µm) and the different shape of hyphae in Y62M1
superficial net, the location of polytomies, either in the net (BM03M3) or
the emanating hyphae (Y62M1) and the variable diameter of emanating
hyphae only in Y62M1 (1–4 versus 2.8–3.2 µm). Thick-walled, straight or
wavy emanating hyphae with polytomies in Y62M1 (Urban et al. 2003)
were classified by Agerer and Rambold (2004–2011) as awl-shaped (type A)
or ramified (type E) cystidia. Shorter emanating hyphae in BM03M3 were
similar to bent awl-shaped cystidia; the frequent simple septa and the lack
of anatomical differences with longer emanating hyphae suggest that they
are a transition between emanating hyphae and cystidia.
PD10 anatomotype shared the plectenchymatous mantle with squarrose
branched hyphae and the lack of rhizomorphs observed in most Sebacinales
EcM. The matrix previously observed in Sebacinales anatomotypes was
superficial; in PD10, it was covered by a matrix-free outer layer. Superficial
hyphal net with variably shaped hyphae as in PD10 was also reported on
Sebacina incrustans + Picea abies (Urban et al. 2003), sample Y62M1 (Urban
et al. 2003), Quercirhiza dendrohyphidiomorpha (Azul et al. 2006) and Pinirhiza
multifurcata (Wei and Agerer 2011). No structures similar to PD10 staghorn-
like hyphae (thick-walled, often swollen hyphae with numerous finger-
like outgrowths and ramifications including polytomies with numerous
branches) have been previously described on Sebacinales EcM. Schematic
representations of cystidia with finger-like outgrowths (type E and I) were
reported by Agerer (1991). The term hyphae is preferred for PD10 outermost
mantle hyphal cells because these structures were bearing a variable
number of outgrowths, with variable length and sometimes septa. PD10
variably shaped hyphae are called staghorn-like hyphae for the similarity
with staghorn hyphae on Pseudotsuga menziesii (Mirb.) Franco + Byssoporia
(Poria) terrestris var. lilacinorosea M.J. Larsen & Zak EcM anatomotype (Zak
and Larsen 1978, Agerer and Rambold 2004–2011). Differences between B.
terrestris and PD10 staghorn-like hyphae included the thin walls, and the
lack of inflations and conspicuous polytomies in the first anatomotype.
6MM2 anatomotype shared features of Sebacina incrustans + Picea abies
(sample Y129M5) (Urban et al. 2003, Agerer and Rambold 2004–2011) and
Sebacinaceae sp. + Betula papyrifera Marshall (Twieg and Durall 2009). A
matrix was lacking and the thick-walled plectenchymatous outer mantle
layer was covered by numerous thick-walled emanating elements in these
three anatomotypes. Rhizomorphs were lacking in Sebacina incrustans + Picea
abies (L.) H. Karst. (sample Y129M5) (Urban et al. 2003) and Sebacinaceae sp.
+ Betula papyrifera (Twieg and Durall 2009). Emanating elements in Sebacina
incrustans + Picea abies (Urban et al. 2003, Agerer and Rambold 2004–2011)
and Sebacinaceae sp. + Betula papyrifera (Twieg and Durall 2009) were
interpreted as awl-shaped cystidia and emanating hyphae. 6MM2 shorter
emanating hyphae were similar to awl-shaped cystidia (Agerer 2006). The
thinner wall at cell distal end and the frequent septa suggest they are a
transitional growing stage of emanating hyphae. Despite similarities with
Sebacina incrustans anatomotype, the presence of rhizomorphs and the
absence of cystidia indicate that 6MM2 belongs to a different species.
5. Conclusions
This study confirmed that morpho-anatomical features are important
information, together with ultrastructure and DNA sequences to confirm
the EcM status of Sebacinales species. This first study of tropical Sebacinales
EcM anatomotypes corroborated the earlier conclusion (Agerer 2006) that
Sebacinales anatomotypes share general morpho-anatomical features.
Specimens BM03M3, PD10 and 6MM2 could be clearly distinguished

from each other. These results are in agreement with the view that EcM
morpho-anatomical features are useful for an integrative taxonomy of this
diverse fungal group for which the study of fruitbodies is difficult (Wei
and Agerer 2011). Further, combined morpho-anatomical and molecular-
phylogenetic studies are necessary to define affiliations of the three P.
dipterocarpacea Sebacinales EcM anatomotypes to species already known
to science, or to hint at still undetected new species.
This study demonstrated the need to expand Sebacinales anatomotype
morpho-anatomical studies. Comparisons with previously described
anatomotypes demonstrated that BM03M3, PD10 and 6MM2 are new
Sebacinales EcM anatomotypes to science. Yellowish cell walls in
BM03M3 were observed for the first time in Sebacinales EcM. Infrequent
rhizomorphs were already described from the Chinese anatomotype
Pinirhiza nondextrinoidea (Wei and Agerer 2011), but their characteristics
(rhizomorph type A/B, hyphae imbedded in a matrix) were different from
the type B rhizomorphs in BM03M3 and 6MM2. Rhizomorph types can
differ in Sebacinales and the capacity to form rhizomorphs in this fungal
group is more widespread amongst species than previously assumed
(comp. Agerer 2006). These results are not only important for the taxonomy
of Sebacinales EcM but also for the understanding of Sebacinales ecology.
BM03M3 and 6MM2 EcM clearly belong to the medium distance exploration
type (Agerer 2001). PD10 staghorn-like hyphae were observed for the
first time in Sebacinales. This new feature in Sebacinales anatomotypes
demonstrates the importance of tropical studies for a more comprehensive
taxonomy of EcM.
Ackowledgements
The Instituto Venezolano de Investigaciones Científicas and University
of Tübingen provided support including lab facilities. I am particularly
grateful to R Bauer for his help in ultrastructural analyses, and M. Weiß
and I. Kottke for facilitating molecular and light microscope work at an
early stage of this project. Thanks to R. Agerer, anonymous reviewers for
helpful comments on the manuscript and Krista L. McGuire and Caitlyn
Gillikin for improving the language.
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CHAPTER
6
Abundance, Distribution, and
Function of Pisolithus albus
and other Ectomycorrhizal
Fungi of Ultramafic Soils in
New Caledonia
Philippe Jourand,1,* Fabian Carriconde,2
Marc Ducousso,3 Clarisse Majorel,1 Laure Hannibal,1
Yves Prin3 and Michel Lebrun4
1. Introduction
Ultramafic soils, also known as “serpentine soils” in literature, are a
weathered product from ultramafic bedrock that covers less than 1% of the
earth’s surface (Coleman and Jove 1992). These soils are characterized by
high concentrations of iron oxides (up to 85% w/w), unbalanced calcium-
1
IRD, UR040 LSTM, Centre IRD, BPA5, Promenade Roger Laroque, 98848 Nouméa Cedex,
Nouvelle-Calédonie.
2
Institut Agronomique néo-Calédonien (IAC), Axe 2 ‘Diversités biologique et fonctionnelle
des écosystèmes terrestres’, Nouméa IRD research centre, BP18239, 98857 Nouméa, Nouvelle-
Calédonie.
3
CIRAD, UMR LSTM, TA A-82 ⁄ J Campus International de Baillarguet, 34398 Montpellier
Cedex 5 France.
4
Université Montpellier 2, UMR28 LSTM, TA A-82 ⁄ J Campus International deBaillarguet,
34398 Montpellier Cedex 5, France.
*Corresponding author: philippe.jourand@ird.fr
Ectomycorrhizal Fungi of Ultramafic Soils in New Caledonia 101
to-magnesium ratio (up to 1/30 that consequently may influence both

Mg and Ca plant nutrition), and the presence of various heavy metals at
high concentrations, such as chromium, cobalt, manganese and nickel,
all of which are mostly toxic for many plants (Brooks 1987). They are
also extremely deficient in elements that are essential for plant nutrition,
including nitrogen, phosphorus and potassium (Brooks 1987, Chiarucci
and Baker 2007). Previous studies have shown that ultramafic soils are
characterized by a high biological diversity of plants as described in Proctor
(2003) and micro organisms that use various mechanisms to cope with the
extreme edaphic conditions, in particular adaptation to toxic heavy metals
(Brady et al. 2005, Kazakou et al. 2008, Rajkumar et al. 2009). Recently, major
data about the ecological traits of ultramafic soils have been reviewed to
propose these soils as a model system in ecology and conservation,mostly
because of their high plant diversity (Harrisson and Rajakaruna 2011).
In ultramafic ecosystems, it is well known that most plants tolerant to
these extreme soils are involved in mycorrhizal associations, which may
greatly enhance plant nutrition (such as P assimilation) and reduce metal
toxicity on plants (Alexander et al. 2007, Smith and Read 2008). Studies
carried out on ectomycorrhizal (ECM) fungal communities in ultramafic
soils showed a high diversity of fungal species developing ECM symbioses
with plants growing on these substrates (Moser et al. 2005, Urban et al.
2008). In addition, it was recently demonstrated that ultramafic soils do not
limit, and can even promote, the ectomycorrhizal fungal diversity (Moser
et al. 2009, Branco and Ree 2010, Branco 2010). However, the comparison of
ECM fungal diversity between serpentine and non-serpentine soils showed
differences within the fungal population structure (Brealey et al. 2006),
sometimes with the presence of unique species (Moser et al. 2005). Moreover,
studies about physiological behaviour such as metal tolerance within a same
fungal species present on both serpentine and non-serpentine soils have
suggested adaptive evolution, raising questions about the adaptation and
evolution of fungal species on these soils (Gonçalves et al. 2007, 2009).
Here, we have presented a review carried out on ECM fungi collected
from ultramafic soils in New Caledonia, which is a tropical archipelago
located in the South Pacific Ocean (Fig. 1). In New Caledonia, these soils
cover one-third of the main island due to geological evolution (Fig. 1). As
a result of the presence of such ultramafic outcrops, numerous endemic
ecosystems have developed (Jaffré 1992), making the main island a
biodiversity hot spot (Myers et al. 2000). In the first section, we have
summarized results from existing studies and new results of ECM fungal
diversity found on these extreme soils. In the second section, we have
gathered data about the ECM Pisolithus albus (Cooke and Massee) isolated
from ultramafic soils in New Caledonia: diversity, metal-tolerance and
symbiotic interactions with its host plant are presented.
Fig. 1. Geographical map of the New Caledonian archipelago in the South Pacific Ocean with
location of ultramafic massifs (in grey). Data from Perrier et al. (2006a).
2. Plant ECM Status and Fungal Diversity in Ultramafic Soils

of New Caledonia
Considering the vascular plants:fungi ratio of 1:6 as reported by Hawksworth
(1991, 2001) and the number of vascular plants of 3,371 species identified in
New Caledonia (T. Jaffré, personal communication), we could hypothesize
that at least 20,000 fungal species inhabit the archipelago. Referring to
the available literature and herbarium data, the mycologists Horak and
Mouchacca listed about 420 Ascomycota and Basidiomycota taxa in New
Caledonia (Horak and Mouchacca 1998, Mouchacca 1998, Mouchacca and
Horak 1998), which would indicate that approximately 2% of the species
have been inventoried.
The studies undertaken by Perrier, though preliminary, are to date the
only ones that have characterized the ECM status of some New Caledonian
plant species and the related ECM fungal diversity (Perrier 2005, Perrier
et al. 2006a,b). In New Caledonia, two main plant formations are basically
distinguished on ultramafic rocks: sclerophyllous scrubland formations,
called “maquis” or “maquis minier”, and rain forest formations (Jaffré and
L’Huillier 2010). According to the type of soil, the altitude, and the floristic
composition, many groups are further identified. The plant formations
studied by Perrier were located on the ultramafic Koniambo Massif and

correspond to four distinct vegetation groups (Fig. 2): a maquis with
emerging Araucaria trees, a lingo-herbaceous maquis, a Tristaniopsis spp.
maquis and a rain forest dominated by Nothofagus balansae with patches
of N. codonandra. Investigation of the root systems of 19 species revealed
that two Tristaniopsis species, T. calobuxus and T. guillainii, are involved in
ECM symbioses. These species belong to the Myrtaceae (Leptospermoideae
group), a well-known plant family frequently found to be associated with
ECM fungi (Smith and Read 2008, Wang and Qiu 2006). Nothofagus balansae
and N. codonandra roots were also characterized by the presence of a fungal
mantle and a Hartig net. Another New Caledonian species, N. aequilateralis,
has also been previously shown to be able to develop ECM associations
(McCoy 1991). Nothofagus are indirectly, by the presence of putative ECM
fungal fruit bodies, and/or directly, by investigation of the root system,
defined in other regions of the world (i.e., Australia, New Zealand, Papua
New Guinea and South America) as ECM trees (Horak and Wood 1990,
Garnica et al. 2003, Tedersoo et al. 2008, Dickie et al. 2010), and subsequently
recognized as an important ECM genus in the Southern Hemisphere (Smith
and Read 2008).
Fig. 2. Repartition of the four distinct plant formations (sites 1 to 4) on the topographic sequence
studied by Perrier et al. (2006) at the Koniambo Massif. Most abundant and potential species
for restoration purpose within each vegetation type are shown. Plants with ECM structures
or ECM-like-structures on their root systems are indicated by an asterisk or two asterisks,
respectively. The distance in meters (m) from the valley to the plateau and the altitude are
given in abscissa and ordinate, respectively. Modified data from Perrier et al. (2006a).
Surprisingly, Perrier et al. (2006a) observed ECM-like structures on

Cyperaceae roots as Costularia arundinacea, with the presence of a fungal
mantle but the absence of a Hartig net. Such observation has already
been done on two other Cyperaceae species belonging to the genus
Carex (Harrington and Mitchell 2002). However, the colonization of C.
arundinacea roots was only observed on sites 3 and 4, dominated by T.
guillainii and N. balansae respectively (Fig. 2). The vicinity of both ECM
plants may thus explain the colonization of C. arundinacea root systems.
Further investigations led to the identification of other plant species as ECM
(Table 1) (Amir and Ducousso 2010, F. Carriconde personal communication).
Regarding the Myrtaceae, four additional Tristaniopsis species, two
Melaleuca species and the monospecific and endemic genus Arillastrum
are involved in such associations (Table 1). The Fabaceae Acacia spirorbis
has also been identified as ECM (Ducousso et al. 2012). Finally, in New
Caledonia, the ECM status has been characterized for only 13 plant species
among Fabaceae, Myrtaceae and Nothofagaceae (Table 1). Giving the large
representation of the Myrtaceae family, especially the Leptospermoidae
group, and the Fabaceae family in New Caledonia (Morat et al. 2012), we
Table 1. Plant families and species in New Caledonia characterized as ECM. The biogeographical
native status is given according to Jaffré et al. (2001) (N: native, i.e., species for which their
natural distribution area extend beyond the boundaries of New Caledonia; E: endemic species;
EE: endemic genus). The types of soils on which species are encountered are also indicated (C:
calcareous; UM: ultramafic soils; VS: volcano-sedimentary). For species known to be present
on more than one type of soil, the predominant types are highlighted in bold. Modified data
from Amir and Ducousso (2010).
Family Species Biogeographical status Type soil

Fabaceae Acacia spirorbis N C, VS, UM
Arillastrum gummiferum EE UM
Melaleuca pancheri E UM
Melaleuca quinquenervia N C, VS, UM
Myrtaceae
Tristaniopsis calobuxus E UM, VS

Tristaniopsis glauca E UM
Tristaniopsis guillainii E UM
Tristaniopsis macphersonii E UM
Tristaniopsis ninndoensis E VS
Tristaniopsis vieillardii E UM
Nothofagus aequilateralis E UM
Nothofagaceae
Nothofagus balansae E UM
Nothofagus codonandra E UM
could expect a large number of species to be involved in such mutualistic

interaction. This clear lack of knowledge is furthermore well-illustrated by
the fact that the ECM status remains unknown for two Nothofagus species
in New Caledonia: N. baumanniae and N. discoidea.
The aboveground and belowground fungal diversity has been, to some
extent, investigated on the topographic sequence at the Koniambo Massif
(Fig. 2) by collecting sporocarps, ectomycorrhizal root tips and hyphal mats
in the soil (Table 2, Perrier 2005). Molecular identification has been carried
out by sequencing the nuclear ribosomal DNA (rDNA) internal transcribed
spacer (ITS), a widely used marker in mycology and recently defined as the
reference region for fungal DNA barcoding (Schoch et al. 2012). Twenty-
nine sporocarps, 11 ECM root tips and 7 hyphae collected from soil cores
were successfully sequenced (Table 2, Perrier 2005). Comparison of the
generated ITS sequences to the international available database GenBank
using the BLAST algorithm (Altschul et al. 1990) showed the presence
of several genera (Table 2). Interestingly, out of the total of 47 samples,
45 presented a percentage of similarity less than 97% (Table 2), a value
commonly used to differentiate ECM species (e.g., Tedersoo et al. 2003,
Izzo et al. 2005, Smith et al. 2007). Two sporocarps, K66C and KC03C, had
a percentage of similarity >97% with samples from Australia and New
Zealand, respectively. Overall, these results suggest there is a diverse and
unique ECM fungal assemblage at these study sites and possibly across
New Caledonia at a regional scale.
Indeed, the description in the last few years of new putative ECM
species, such as the impressive Podoserpula miranda (Fig. 3), thought to be
associated with Arillastrum gummiferum in the South of New Caledonia
(Ducousso et al. 2009), or the chanterelle, Cantharellus garnieri (Fig. 3)
collected under distinct potential host trees in different localities and type
of soils (Ducousso et al. 2004), strengthened the idea of the high fungal
diversity in the archipelago. Regarding the abundance of the different
fungal genera at Koniambo’s sites, samples belonging to the Cortinarius
genus were largely represented. Indeed, out of the 29 sporocarps, 11 ECM
root tips and 7 hyphal mats collected, 11 (~38%), 6 (~55%), and 5 (~71%)
were assigned to this genus. The large belowground representation of
Cortinarius has already been highlighted in Nothofagus forests in Australia
and New Zealand (Tedersoo et al. 2008, Dickie et al. 2010). Co-evolution
between Cortinariaceae and Nothofagus in Australia has been suggested
(Bougher et al. 1994), and could thus be one of the main driving forces
that may have led to the diversification of this fungal group in the Pacific
region.However, the limited sampling size of Perrier’s study (in total only 47
samples), and particularly the very restricted number of studies undergone
to date on fungal diversity, do not allow us to draw any conclusions on
the diversity level and the structure of this diversity on the archipelago.
Table 2. Sporocarps, ECM root tips and hyphae samples collected in the Tristaniopsis spp. maquis (site 3) and the rain forest dominated by Nothofagus
106
balansae (site 4), located on the topographic sequence at the Koniambo Massif and genotyped by sequencing of the ITS region. The host plant (putative),
the morphospecies when available, the ITS sequence length, the closest BLAST match and the related information are presented. ITS sequence data
generated by Perrier (2005) were recently analyzed.
GenBank Sequence
Plant Host plant Bases Best match GenBank
Sample reference Sample type Morphospecies accesion lenght Closest species BLAST match ‡ % Similarity
formation (putative) † matched accesion number
number (bp)
K66C Sporophore 3 Tristaniopsis guillainii Pisolithus sp FJ656011 527 Pisolithus sp 520/526 99% AF270787
K02C Sporophore 4 Nothofagus balansae Boletus sp FJ656001 609 Boletus sp 384/449 86% EU569234
K05C Sporophore 4 Nothofagus balansae nd FJ656002 551 Cortinarius subgemmeus* 498/561 89% JX000354
K06C Sporophore 4 Nothofagus balansae - FJ656003 605 Phellodon sp 544/597 91% GU222318
K09C Sporophore 4 Nothofagus balansae nd FJ656004 547 Austrogautieria macrospora* 438/504 87% GQ981492
K10C Sporophore 4 Nothofagus balansae nd FJ656005 670 Tricholoma imbricatum 628/668 94% AY573537
K10C Sporophore 4 Nothofagus balansae nd FJ656005 670 Tricholoma imbricatum 626/668 94% AY573537
K12C Sporophore 4 Nothofagus balansae nd FJ656006 572 Cortinarius austrovenetus 534/573 93% GQ890318
K14C Sporophore 4 Nothofagus balansae Inocybe sp FJ656007 506 Dermocybe largofulgens* 483/504 96% GU233324
K16C Sporophore 4 Nothofagus balansae nd FJ656008 706 Lactarius scrobiculatus 660/719 92% EU597079
K18C Sporophore 4 Nothofagus balansae Lactaroides FJ656009 598 Russula zonaria * 526/569 92% DQ421990
KC02C Sporophore 4 Nothofagus balansae nd FJ656012 436 Cortinarius lividus 390/433 90% AF539734
KC05C Sporophore 4 Nothofagus balansae nd FJ656014 580 Inocybe aeruginascens* 491/569 86% GU949591
KC08C Sporophore 4 Nothofagus balansae nd FJ656016 670 Lactarius olympianus 624/684 91% EF685079
KC11C Sporophore 4 Nothofagus balansae nd FJ656018 609 Russula sp 577/608 95% GU222292
KC12C Sporophore 4 Nothofagus balansae nd FJ656019 518 Cortinarius flammuloides 460/540 85% AF539716
KC16C Sporophore 4 Nothofagus balansae nd FJ656020 574 Cortinarius multiformis 518/589 88% AF389135
KC17C Sporophore 4 Nothofagus balansae nd FJ656021 574 Leratiomyces ceres 394/411 96% HQ604750
KC19C Sporophore 4 Nothofagus balansae nd FJ656022 583 Cortinarius singularis * 508/584 87% JQ287672
KC22C Sporophore 4 Nothofagus balansae nd FJ656023 528 Austrogautieria macrospora* 442/503 88% GQ981492
KD37C Sporophore 4 Nothofagus codonandra nd FJ656038 582 Cortinarius eutactus* 559/582 96% JX000366
KC23C Sporophore 4 Tristaniopsis guillainii nd FJ656024 414 Cortinarius elaiops* 241/262 92% JX000369
K01C Sporophore 4 Nothofagus balansae nd FJ656000 701 Tricholoma ustale 632/713 89% AF458435
K22C Sporophore 4 Nothofagus balansae nd FJ656010 582 Inocybe aeruginascens* 491/569 86% GU949591
KC03C Sporophore 4 Nothofagus balansae nd FJ656013 612 Russula sp 593/613 97% GU222292
KC06C Sporophore 4 Nothofagus balansae nd FJ656015 436 Phaeocollybia redheadii 394/411 96% JN102541
KC10C Sporophore 4 Nothofagus balansae nd FJ656017 447 Cortinarius aff. austrosanguineus 427/454 94% GQ890317
KD36C Sporophore 4 Nothofagus codonandra nd FJ656037 585 Cortinarius elaiops* 554/586 95% JX000369
KD42C Sporophore 4 Nothofagus codonandra nd FJ656039 485 Tricholoma ustale 402/455 88% AF458435
KE01-2M ECM 3 Tristaniopsis guillainii - FJ656040 457 Piloderma sp 400/450 89% JQ711951
KE02M ECM 3 Tristaniopsis guillainii - FJ656041 511 Cortinarius vernicifer* 387/449 86% JX000370
KE04M ECM 3 Tristaniopsis guillainii - FJ656042 438 Piloderma sp 383/430 89% JQ711951
KD10M ECM 4 Nothofagus balansae - FJ656025 552 Oidiodendron chlamydosporicum 477/519 92% AF062789
KE06M ECM 4 Nothofagus balansae - FJ656043 584 Cortinarius amoenus 544/590 92% AF389160
KE12-1M ECM 4 Nothofagus balansae - FJ656045 620 Tricholoma ustale 557/640 87% AF458435
KD18M ECM 4 Nothofagus codonandra nd FJ656026 474 Cortinarius calyptratus* 425/476 89% EU525980
f g yp
KD29-2M ECM 4 Nothofagus codonandra - FJ656033 608 Cortinarius elaiops* 558/606 92% JX000369
KD31''-2M ECM 4 Nothofagus codonandra - FJ656034 570 Cortinarius elaiops* 518/600 86% JX000369
KD31'M ECM 4 Nothofagus codonandra - FJ656035 682 Tomentellopsis submollis 642/684 94% JQ711898
KD36-2M ECM 4 Nothofagus codonandra - FJ656036 584 Cortinarius singularis* 509/584 87% JQ287672
KD19_1S Hyphae 3 Tristaniopsis guillainii - FJ656027 698 Lycoperdon sp 682/726 94% JX029934
KD19_2S Hyphae 3 Tristaniopsis guillainii nd FJ656028 592 Cortinarius eutactus* 560/597 94% HQ533023
KD19_9S Hyphae 3 Tristaniopsis guillainii - FJ656029 410 Cortinarius sp 350/402 87% JQ287690
KD20_5S Hyphae 3 Tristaniopsis guillainii - FJ656031 585 Cortinarius sp 543/594 91% JN942302
KD20_6S Hyphae 3 Tristaniopsis guillainii nd FJ656032 615 Cortinarius sp 552/621 89% JN942302
KE12_2S Hyphae 4 Nothofagus balansae - FJ656046 608 Tricholoma ustale 554/629 88% AF458435
KE18_2S Hyphae 4 Nothofagus codonandra - FJ656047 540 Cortinarius subgemmeus* 477/560 85% JX000354
† ECM root tips were sampled by tracing the roots from the tree trunks.
‡ Voucher specimens are indicated by an asterisk.
Ectomycorrhizal Fungi of Ultramafic Soils in New Caledonia
107
Fig. 3. (A) Podoserpula miranda and (B) Cantharellus garnieri. Photos by courtesy from Ducousso
Marc, CIRAD.
Although preliminary analysis of the ECM diversity has been achieved, a

thorough description of ECM and fungal communities and the interaction
with host-plants in New Caledonia should be carried out. In order to really
investigate such fungal diversity and better understand the mechanisms
involved, molecular ecology studies on ECM communities by sequencing
sporocarps and ectomycorrhizas using the classical Sanger approach,
complemented by the use of next generation sequencing on soil cores,
should be undertaken.
3. Pisolithus albus from Ultramafic Soils in New Caledonia:

Diversity and Physiological Response to Nickel
Pisolithus albus (Cooke and Massee) is a fungal species belonging to Pisolithus
Alb. and Schwein known to be one of the major ectomycorrhizal Boletale
distributed on a worldwide scale that forms ectomycorrhizal symbioses
with a broad range of angiosperm and gymnosperm tree species (Marx
1977, Martin et al. 2002). Pisolithus is also regarded as an early colonizer
that persists on sites subject to edaphic stresses (Anderson et al. 1998).
In New Caledonia, P. Albus fruit bodies are very abundant. The species
also develops ectomycorrhizal associations with many endemic plants
belonging to various genera of the Myrtaceae such as Melaleuca, Tristaniopsis
and Sannantha, and one Mimosaceae, i.e., Acacia spirorbis (Perrier 2005). In
New Caledonia most of the plants able to form ECM with P. albus dominate
specific zones in their respective ecosystem: for example, Tristaniopsis genus
colonizes specific zones of the ultramafic ecosystem at an altitude from 400
to 900 meters (L’Huillier et al. 2010). Altogether, the abundance of P. albus
and its ability to develop ECM symbioses with endemic plants that colonize
specific ecosystems in New Caledonia has led to the study of the genetic
diversity of P. albus in New Caledonia.
3.1 Diversity of Pisolithus albus and their symbioses in New

Caledonia
Isolates of ectomycorrhizal P. albus were sampled from both ultramafic

and non-ultramafic soils in New Caledonia in order to investigate
the relationships between (i) genetic diversity and (ii) the edaphic
constraintssuch as the deficiency of major nutrient elements (N, K and
P), the unbalanced Ca/Mg ratio and the presence of heavy metals at high
concentrations (Jourand et al. 2010a). Fruiting body description, spore
morphology (Fig. 4) and phylogenetic analysis based on internal transcribed
spacer (ITS) rDNA (as previously reported by Martin et al. 2002) sequences
confirmed that all isolates belong to P. albus and are closely related to
other Australasian specimens (Fig. 5). In addition, the ecology of P. albus
isolated from New Caledonia confirmed the dominant association with
endemic plants belonging to genera of the Myrtaceae family (e.g., Melaleuca,
Sannantha, Tristaniopsis) or the Fabaceae family (e.g., Acacia).
Altogether, the ecological and molecular data of P. albus isolated
in New Caledonia were in agreement with the phylogeography of the
ectomycorrhizal Pisolithus genus inferred from rDNA-ITS sequences,
suggesting that (1) evolutionary lineages within Pisolithus are related to
the biogeographical origin of their plant hosts (Martin et al. 2002) and (2)
a long-distance dispersal event of ectomycorrhizal fungi from Australia
Fig. 4. Pisolithus albus from New Caledonia. A: Pisolithus albus MD07-117 from the Koniambo
massif; B: Pisolithus albus MD07-228 from the Ouen-Toro, Noumea; C: cross section of Pisolithus
albus MD07-166 from Pindjen water-fall and D: globose spores (8.77 to 9.62 µm) of Pisolithus
albus MD06-379 from Poum, erected spines (1.2 µm) are clearly visible. From Jourand et al.
(2010a).
might explain the introduction of Pisolithus species in the South Pacific

zone (Moyersoen et al. 2003). Interestingly, the use of other molecular
tools such as ITS-restriction fragment length polymorphism (Fig. 6A)
and amplified fragment length polymorphism markers (AFLP) (Fig. 6B),
showed the existence of one genotype within P. albus grouping isolates from
ultramafic soils (Jourand et al. 2010a). Such results raised the question of
the presence a fungal ecotype on ultramafic soils, as described for plants
found on these soils (Harrison and Rajakaruna 2011). They also contribute
to the hypothesis of a link between the phylogenetic population structure
and the ecological adaptation due to the particular mineral constraints,
P. aurantioscabrosus, SE Asia
41
Pisolithus sp.10, Australia
95
87 Pisolithus sp.8 and P. microcarpus,
55
Australia, S America
72
NC
40
52 NC
32
100 51
P. albus
44 NC New Caledonia,
23 65 NC Australia, New-Zealand,
SE Asia, W Africa
NC
68
13
100 Pisolithus sp.1, E Africa
63 98
P. marmoratus, Australia
37
Pisolithus sp.3 and P. sp.4, Europe
57
46
76
P. tinctorius, N America, Europe
100
Pisolithus sp.5, SE Asia

98
Fig. 5. Phylogenetic synthetic relationships among representative Pisolithus sp. from New
Caledonia collection sites and worldwide reference isolates. The phylogeny is based on
the analysis of the rDNA ITS1, 5.8S and ITS2 sequences. Tree shown is a 50% Majority rule
consensus of the most parsimonious trees (Tree Length = 3733) obtained with PAUP4 (see
Materials & Methods). Values indicated at tree nodes are percentage values of 1000 bootstrap
replicates under MP criterion using fast stepwise addition (only values >50% are shown).
The tree was rooted with Suillus luteus ITS sequences. Significant bootstrap frequencies are
indicated. Abbreviations: S America: South America; SE Asia: South East Asia; W Africa: West
Africa, E Africa: East Africa.
in particular ultramafism, as observed in ectomycorrhizal communities

from other ultramafic soils (Urban et al. 2008). To further investigate this
hypothesis, considering that nickel is one of the most toxic and bioavailable
metal found at high concentrations in these soils (Echevarria et al. 2006),
P. albus molecular and physiological responses to nickel were assessed in
a further study.
A) P. albus ITS RFLP profiles
B) P. albus AFLP profile relationship
Fig. 6. A) Representative patterns of ITS restriction fragment length polymorphism (RFLP)

profiles of Pisolithus albus isolates from both ultramafic and volcano sedimentary soils compared
to both undigested amplified ITS and 100 pb DNA Ladder (Promega). Arrows highlight
major differences between profiles. B) Genetic relationship within P. albus isolates from New
Caledonia according to AFLP analysis. Bootstrap consensus UPGMA tree obtained for 882
AFLP scored fragments obtained with the 9 selective primers pairs on the 27 P. albus isolates
(100 replicates). Data from Jourand et al. (2010a).
3.2 Tolerance and adaptation to nickel of Pisolithus albus from New

Caledonia
In ultramafic soils, nickel (Ni) is one the most bioavailable and phytotoxic
element: nickel content may reach up to 10 g/kg in ultramafic soils when
compared with the average 50 mg/kg in cultivated soils (Wenzel and
Jockwer 1999, Echevarria et al. 2006). This mineral element is a crucial
selecting factor for plant survival on ultramafic soils: to grow on such
high concentrations of nickel as found in serpentine environments (often
coinciding with high concentrations of other heavy metals), plants had to
develop major adaptations that include exclusion of the absorption of the
toxic metal by the roots and/or metal hyperaccumulation with internal
complexation and compartmentation (Kazakou et al. 2008). In addition,
ECM symbioses might contribute to limit the metal accessibility and uptake
by the plant (Colpaert et al. 2011).
3.2.1 Pisolithus albus nickel content and in vitro tolerance

In the previous study, the nickel concentration in fruiting body tissues
of Pisolithus albus isolates from New Caledonia was assessed, as well as
the in vitro nickel tolerance of cultivated mycelia from isolates collected
from soil type (ultramafic vs non-ultramafic) where P. albus were collected
(Jourand et al. 2010a). In fruiting bodies of P. albus from ultramafic soils,
the nickel concentration reached an average of 5.7µg/g of dried tissue. In
contrast, tissue of carpophores of isolates collected from non-ultramafic soils
contained 2.5 times less nickel. In addition, P. albus mycelia from ultramafic
soils included isolates with high variations of in vitro nickel-tolerance, with
both nickel-tolerant isolates (with an average that half the maximal effective
concentration of Ni that reduced fungal growth by 50% was 575 mM) and
nickel-sensitive isolates (average Ni EC50 37 mM). In contrast, all isolates
from non-ultramafic soils were found to be nickel-sensitive (average Ni
EC50 at 32 mM).
Within Pisolithus spp., previous studies have showed that some isolates
were able to tolerate high concentrations of nickel. For example isolates of
Pisolithus tinctorius were found to tolerate nickel with a Ni EC50 ranging
from 126 to 170 mM (Tam 1995). Aggangan et al. (1998) also described one
isolate of P. tinctorius from ultramafic soils in New Caledonia able to grow
on nickel from 20 to 200 µM. More recently, Blaudez et al. (2000) and Ray et
al. (2005) reported isolates of P. tinctorius that are able to grow on medium
with nickel concentrations ranging from 17 to 350 µM. The mycelia from
P. albus isolates from New Caledonian ultramafic soils displayed both
in vitro nickel-sensitive and nickel-tolerant phenotypes. In addition, the
nickel-tolerant isolates presented a noteworthy tolerance to Ni with an
average Ni EC50 two to three times higher than the Ni EC50 already reported
for other Pisolithus spp. mentioned above. To explain the high variability in
nickel-tolerance observations, it was first hypothesized that such variations
could be correlated to high real fluctuations of bioavailable nickel content
in ultramafic soils, which is assessed as the DTPA-Ni fraction according to
Echevarria et al. (2006). Perrier et al. (2006a) reported that the nickel-DTPA
concentrations in ultramafic soils varied in a range from 17 to 980 µmol/
kg. Assuming that the average nickel-DTPA concentration does not reflect
real fluctuations of bioavailable nickel in ultramafic soils, and considering
the range of nickel-DTPA concentrations in ultramafic soils reported by
Perrier et al. (2006a,b), it is not surprising to find isolates of P. albus with high
variations in nickel tolerance from the same ultramafic site. Similar variations
in metal-tolerant fungal populations in correlation to metal-soil content have
already been reported. For instance, in Suilloid fungi, populations displayed
zinc tolerance relative to zinc concentrations in polluted soils, suggesting
an evolutionary adaptation of fungi to the soil environment (Colpaert et
al. 2004). More recently, evidence of adaptation to nickel was provided in
isolates of Cenococcumgeophilum from ultramafic soils in Portugal and the
USA (Gonçalves et al. 2009). No clear relationship between the phenotypic
physiological response to nickel and the population genetic differentiation
observed within P. albus from soils could be established as the nickel-tolerant
isolates from ultramafic soils did not cluster in a homogeneous group. It
was thus tempting to speculate that the capacity of some P. albus to tolerate
high nickel concentrations reflects the expression of an adaptive response
to high concentrations of bioavailable nickel in soils as suggested for other
fungi in response to high heavy metal levels (Hartley et al. 1997, Colpaert
et al. 2004, Gonçalves et al. 2009). However, if New Caledonian population
of P. albus seems to be structured into one ecotype, nickel tolerance alone
might not be a sufficient feature to explain such results. Thus, the ultramafic
constraint should be considered as a whole, even if each factor (N, P, K
contents, Ca/Mg imbalance, heavy metal presence) is studied separately,
as suggested by Kazakou et al. (2008).
3.2.2 Pisolithus albus transcriptomic response to nickel

In another study on nickel-tolerant Pisolithus albus isolated from ultramafic
soils in New Caledonia, the comparison of the transcriptomes of a nickel-
tolerant isolate in the presence and absence of nickel was monitored by
using pyrosequencing and quantitative polymerase chain reaction (qPCR)
approaches in order to identify genes involved in the specific molecular
response to nickel and to quantify their expression (Majorel et al. 2012).
As a result of the experiment, two non-normalized cDNA libraries were
obtained from one nickel-tolerant P. albus isolate grown in the presence and
absence of nickel. A total of 19,518 genes could be obtained through the de

novo assembly of the sequence reads from the two non-normalized cDNA
libraries. The expression of 30% of these genes was regulated by nickel.
Further analysis identified 4,211 genes (21%) that were up-regulated by
nickel and 1763 genes (9%) that were down-regulated by nickel. The global
statistical distribution of these 19,518 genes is presented on a scatter plot
in Fig. 7A. The genes, for which expression was induced most markedly
by nickel, encoded products that were putatively involved in a variety of
biological functions, such as the modification of cellular components (53%)
and the regulation of biological processes (27%) and molecular functions
(20%) (Fig. 7B). Compared to most previous studies conducted on ECM
samples isolated from soils polluted with heavy metals as a result of human
activities (Jacob et al. 2004, Muller et al. 2007, Ruytinx et al. 2011), this study
was the first repository of its kind. These results clearly suggested a positive
transcriptomic response of the fungus to nickel-rich environments, which
may contribute to the tolerance of the fungus to the extreme conditions as
found in New Caledonia. The analysis of the results based on gene ontology
(GO) analysis and functional genetic tools also suggests the role of these
genes as putative adaptive mechanisms of nickel tolerance in P. albus. The
majority of genes up-regulated by nickel belonged to the GO category
‘cellular component’. Information on the annotations of these genes is
valuable for the further investigation of gene functions, cellular structures
and biological processes that might be involved in the tolerance of fungi
to nickel via extracellular and intracellular mechanisms, as suggested by
Bellion et al. (2006).
In the second step of the experiment, ten genes that were analyzed as
the most nickel-induced in pyrosequencing were characterized by qPCR
analysis in both nickel-tolerant and nickel-sensitive P. albus isolates from
ultramafic soils. Among them, six genes were expressed exclusively in nickel-
tolerant isolates as well as in ECM samples in situ. In addition, in the nickel-
tolerant isolates, the presence of nickel increased their level of expression by
between one- and nine-fold (Fig. 8). Their functional classification showed
that these genes encoded for putative proteins involved either in chitin
cell wall rearrangements as GPI-anchor-like protein and class III chitinase,
or biological regulations as vacuolar protein sorting and APC amino acid
permease, suggesting a possible role of fungi in metal immobilization and
consequently in reducing metal toxicity when in symbiosis with plants. In
previous studies involving fungi, many genes involved in the response to
stress induced by heavy metals were found to encode proteins that function
as metal transporters or metal-binding proteins (Jacob et al. 2004, Bellion
et al. 2006, Bolchi et al. 2011). However, in Majorel et al. (2012), most of the
genes overexpressed in the presence of nickel did not encode proteins that
are generally involved in metal-stress responses. This suggested that the
A) Gene expression level repartition in Ni-tolerant Pisolithus albus
B) Gene functional GO terms assignment and distribution in Ni-tolerant P. albus
Fig. 7. A) Scatter plot presenting gene expression levels in Pisolithus albus Ni-tolerant
ecotypefree-living mycelium grown without or with Ni at 250 µM. The expression levels of
genes were normalized using a scale of 0 to 10,000. Each circle in the plot represents expression
of one gene. B) Functional GO terms assignment and distribution of total sequences of two
transcriptomes of Ni-tolerant P. albus with (+250 µM) and without nickel, among Gene
Ontology (GO) biological process, molecular function and cellular component. From Majorel
et al. (2012).
80
Gene N° 1 (GPI- anchor-like) Gene N° 2 (predicted protein)
1.2x
70 4
60 1x
1.5x
mRNA accumulation
mRNA accumulation
3
Arbitrary units
Arbitrary units
50
40 2.2x
2
30
1.2x
20 1.4x 1
10 ND ND
ND ND
0 0
MD06 -337 MD09 -001 MD09 -045 MD09 -063 MD09 -078 MD06-337 MD09 -001 MD09-045 MD09 -063 MD09 -078
Ni-tolerant Ni-sensitive Ni-tolerant Ni-sensitive
Gene N° 5 (vacuolar protein sorting) Gene N° 7 (class III chitinase)

5 1,4
3.6x
mRNA accumulation
4
mRNA accumulation
1x
(Arbitrary units)
Arbitrary units
1 2.5x
3
2 0,6
1x
1x
1 1.4x
ND ND 0,2 ND ND
0 0
MD06 -337 MD09- 001 MD09 -045 MD09 -063 MD09 -078 MD06 -337 MD09 -001 MD09 -045 MD09-063 MD09 -078
Gene N°6 (S - adenosylmethionine transferase) Gene N°9 (APC amino acid permease)
30
1.6x 9x
25 400
mRNA accumulation
mRNA accumulation
(Arbitrary units)
Arbitrary units
20 300
1.3x
15 60
10 40
1x
5 1.2x 20 1.3x
ND ND ND ND
0 0
MD06 -337 MD09 -001 MD09 -045 MD09 -063 MD09 -078 MD06 -337 MD09 -001 MD09-045 MD09-063 MD09-078
Fig. 8. Comparison of mRNA accumulation profiles for six selected Ni up-regulated genes in
five P. albus isolates from ultramafic soil in presence of nickel 50 µM (black columns) and in
absence of nickel (grey columns). Three nickel-tolerant isolates (MD06-337, MD09-045, and
MD07-001) and two nickel-sensitive isolates (MD09-078 and MD09-063) were compared.
Transcript accumulation was quantified by qPCR using 2-ΔΔCT method with normalization to
two reference genes, GAPDH and EF4α, and is expressed as arbitrary units. The data indicate
mean values ± S.D. values, calculated from three technical replicates with triplicate biological
samples. The fold induction by nickel is presented above the black columns in italics. ND:
mRNA non-detected (Ct values >37). From Majorel et al. (2012).
mechanisms that underlie the nickel tolerance in P. albus from ultramafic

soils might differ from those of other fungi. In particular, that might
reflect a long-term adaption to nickel in natural environment, in contrast
to short-term adaptation on metal contaminated soils. Among the genes
in which expression was remarkably induced in presence of nickel, and
exclusively expressed in nickel-tolerant, it was interesting to identify genes
that encode chitinase-like and glycosylphosphatidylinositol (GPI) cell-wall
structural proteins that are involved in extracellular processes and encode
putative cell-wall proteins. Recently, it was suggested that modifications
of structural elements of the cell wall, such as the rearrangement of chitin
and biosynthesis of glucan- or galactosamine-containing polymers, might
play a key role in modulating the integrity of the cell wall and its capacity
to immobilize heavy metals. In this way, such modifications could confer
tolerance to metals and affect the ability of fungi to survive in stressful
environments (Meharg 2003, Bellion et al. 2006, Fuchs and Mylonakis
2009).
Altogether, these results evidenced a strong and specific transcriptomic
response to nickel of ultramafic-adapted P. albus both in vitro and in situ.
This led the authors to hypothesize that the presence of both nickel-tolerant
and nickel-sensitive fungal phenotypes in ultramafic soils might reflect
environment-dependent phenotypic responses to variations in the effective
concentrations of nickel in heterogeneous ultramafic habitats (Majorel et
al. 2012).
3.3 Role of ECM symbiose between nickel-tolerant Pisolithus albus

and its host plant Eucalyptus globulus exposed at toxic nickel
concentrations
As ECM symbioses are known to play a major role in the fitness of plants
in the presence of heavy metals (Jentschke and Godbold 2000), experiments
were carried out to analyse the symbiotic interactions between P. albus
and one of its host plants in the presence of nickel. Ectomycorrhizal
Pisolithus albus isolated in nickel-rich ultramafic soils from New Caledonia
and showing in vitro adaptive nickel tolerance were inoculated to
Eucalyptus globulus Labill used as a Myrtaceae plant-host model to study
ectomycorrhizal symbiosis. Plants were then exposed to a nickel dose-
response experiment with increased nickel treatments up to 60 mg/kg soil
as maximum extractable nickel content found in ultramafic soils (Perrier
et al. 2006a). Results showed that plants inoculated with ultramafic ECM
P. albus were able to tolerate high and toxic concentrations of Ni (up to
60 mg/kg) while uninoculated controls were not (Fig. 9). At the highest
nickel concentration tested, root growths were more than 20-fold higher
and shoot growths more than 30-fold higher in ECM plants compared with
Fig. 9. Eucalyptus globulus seedlings after 12-weeks growth. A and A’ mycorrhizal; B and B’:
non-mycorrhizal (controls). A and B: no nickel added; A’ and B’ seedlings treated with Ni.
From Jourand et al. (2010b).
control plants. Ergosterol was also measured in roots as it is a major sterol

in fungi and is a good indicator of the level of mycorrhizal colonization of
roots (Martin et al. 1990). Without nickel, roots had a mean level of 19.7%
ectomycorrhization. At low nickel concentrations (0.6 and 6 mg/kg), the
level of root ECM colonization varied from 15.6 to 27.8%. At high and toxic
nickel concentrations (30 and 60 mg/kg), the level of root colonization was
significantly reduced to around 9%, but confirmed the presence of viable
ECM. At the highest nickel concentration tested, the improved growth in
ECM plants was also associated witha 2.4-fold reduction in root nickel
concentration but a massive 60-fold reduction in transfer of nickel from root
to shoots, while for all other major plant nutrient elements analyzed, i.e., N,
P, K, Ca and Mg, no significant differences in concentration were noted in
either shoots or roots in response to nickel treatments or fungal treatment.
To determine whether nickel tolerance was related to the release of metal
binding compounds, exudates from roots were analyzed. The two principal
chemical components of the exudate solution were non-protein thiols and
oxalate. Control plants excreted significantly more thiols and oxalate than
plants developing ECM symbiose with P. albus, with the increase being more
evident at higher nickel concentrations: control plants released 5-fold more

thiols at 30 and 60 mg/kg of nickel, and 12- and 8-fold more oxalate at 30
and 60 mg/kg nickel, respectively. All these results confirmed that the nickel
tolerance of the ECM has a substantial beneficial effect on the plant host.
Ultramafic ECM isolates produced significant increases in growth in both the
absence and low concentrations of nickel (from 0.6 to 6 mg/kg). Plant root
surface was greatly increased, and the high level of mycorrhizal colonization
is consistent with previous data on the interaction between Pisolithus
and Eucalyptus (Martin et al. 1990, Brundrett et al. 1996). At low nickel
concentrations, the increase in both shoot and root biomass observed in ECM
plants compared with non-inoculated plants is probably a consequence of
better mineral nutrition (Marschner 1995, Finlay 2004). However, at toxic
levels of nickel the contribution of the ECM symbiosis with ultramafic
P. albus to host nickel tolerance was more substantial. Interestingly, P. albus
isolates could withstand in vitro high nickel concentrations but accumulated
very little nickel in its tissue (Jourand et al. 2010b). The lower nickel uptake
by mycorrhizal plants could not be explained by increased release of metal-
complexing chelates since these were 5- to 12-fold lower in mycorrhizal
plants at high nickel concentrations. It was proposed that the fungal sheath
covering the plant roots acts as an effective barrier to limit transfer of nickel
from soil into the root tissue.
4. Conclusions
Overall, the observations about ECM diversity found on ultramafic soils
in New Caledonia raise very compelling questions about the evolutionary
processes involved in fungal diversification in New Caledonia and at a
regional scale. The focus on ECM Pisolithus albus isolated from soils in New
Caledonia highlighted the identification of an ultramafic nickel-tolerant
ecotype as reported in Jourand et al. (2010a), showing specific and adaptive
molecular response to this metal (Majorel et al. 2012), and having a key role
in plant host adaptation to toxic nickel concentrations as found in these soils
(Jourand et al. 2010b). Together, these results constitute an important step in
evaluating the potential of ECM symbioses for plant adaptation to ultramafic
soils containing high concentrations of heavy metals, which is a prerequisite
for their use in strategies for ecological restoration of mine sites as suggested
by Reddell et al. (1999), Perrier et al. (2006a) and, more recently, Khosla and
Reddy (2008). Further characterization of ECM fungal communities in New
Caledonia would increase knowledge about fungal diversity and identify
fungal species that might be relevant for plant inoculation purpose and
their direct implications in restoration strategies.
Acknowledgements
Most of these studies were supported by (i) the GIP CNRT “Nickel and its
Environment” [grant number GIPCNRT98] and (ii) the ANR ECCO2005
and BIODIV2007 research programs entitled “Niko” and “Ultrabio”
respectively. The authors wish to thank Pr R. Reid (University of Adelaide,
South Australia), Dr T. Jaffré (IRD, Nouméa, New Caledonia), S. Santoni
(INRA, Montpellier, France), M.E. Soupe, J. Riss, C. Richert for their
respective contributions and/orsuggestions. They are also thankful Mr.
Pierrick Gailhbaud and Mr Antoine Leveau of Koniambo Nickel Society
(KNS), Vavouto, Koné, New Caledonia. The authors thank the anonymous
referees for their valuable comments on this study, and Krista L. McGuire
and Caitlyn Gillikin for improving the language.
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CHAPTER
7
Diversity and Function of
Ectomycorrhiza between
Scleroderma and Afzelia
Species in Burkina Faso
(West Africa)
Kadidia Bibata Sanon,1,* Amadou Mustapha Bâ2 and
Robin Duponnois3
1. Introduction
Most forest trees draw water and nutrients from the soil through fungi
associated with their root systems. This symbiotic association, called
mycorrhizas, contributes to water and mineral nutrition, and root protection.
In return, the fungus receives photosynthetic products necessary for its
growth and development (Smith and Read 2008).
1
Institut de l’Environnement et de Recherches Agricoles, Département Productions Forestières
(INERA/DPF), Laboratoire de Microbiologie Forestière, BP 7047 Ouagadougou 03, Burkina
Faso.
2
Laboratoire Commun de Microbiologie (UCAD-IRD-ISRA) BP 1386 Bel Air, Dakar,
Sénégal.
3
Laboratoire Ecologie & Environnement (Unité associée au CNRST, URAC 32). Faculté des
Sciences Semalia. Université Cadi Ayyad. Marrakech. Maroc.
*Corresponding author: sbkady@gmail.com
Diversity and Function of Scleroderma in Burkina Faso 127
Two main types of mycorrhizal association have been described

following their morphological characteristics: the endomycorrhizas and
ectomycorrhizas. The endomycorrhizas are widespread and associated with
approximately 80% of terrestrial plants (Fortin et al. 2008). They are found
in cultivated plants and most tropical trees. Ectomycorrhizas (ECM), are
frequent and widespread in forests and woodlands of temperate and boreal
regions (Alexander 2006). However, a minority of tropical trees, ecologically
and economically important such as Dipterocarpaceae, Fagaceae, Myrtaceae,
Phyllanthaceae and Caesalpinioideae form EcM. In tropical Africa, surveys
of mycorrhizal symbiosis and its recognition in the technical itineraries
of agricultural production and production of reforestation plants remain
limited (Bâ et al. 2011, Bâ et al. 2012). In Burkina Faso, the first studies on
mycorrhizal symbiosis dates back to 1989 with Afzelia africana Sm and
Pisolithus tinctorius (Pers.) Coker. & Couch. (Sary 1989). A few years later,
works have been carried out on both types of symbiosis. The studies on
endomycorrhizal symbiosis include among others: (i) The diversity of
Glomales associated with some Australian Acacia introduced in Burkina
Faso (Bâ et al. 1996a); (ii) The effect of inoculation with mycorrhizal fungal
strains on growth, nutrition and water stress resistance of some wild or
domesticated fruit-tree species in tropical Africa [Ziziphus mauritiana Lam.,
Balanites aegyptiaca (L.) Delile, Parkia biglobosa (Jacq.) R. Br. (Guissou et al.
1998, Guissou et al. 2001, Bâ et al. 2001)]; (iii) The response to inoculation of
Shea tree (Vitellaria paradoxa Kotchy) and Faidherbia albida (Delile) A. Chev.
with or without fertilizers or rock phosphate (Bâ and Guissou 1996, Bâ et
al. 1996b, Dianda et al. 2009).
Burkina Faso is a dry tropical country with 400 mm rainfall in the
north to 1100 mm in the south-west. EcM species are very scarce, confined
to the south and south-western woodlands and gallery forests. Pioneering
EcM investigations (Sanon et al. 1997, Sanon 1999) addressed trees species
and putative fungal partner. These works have allowed isolating and
making pure cultures of Scleroderma species. Scleroderma species appear to
be good candidates for controlled mycorrhization due to their relatively
easy cultivation and their early stage, and have been the subject of detailed
studies (Sanon et al. 1997, Bâ et al. 1999, 2002, Sanon et al. 2002, Sanon et
al. 2009a, b).
2. Partners of Ectomycorrhizal Symbiosis in Burkina Faso

2.1 Some features of the climate and vegetation in Burkina Faso
Burkina Faso is a landlocked country located in the heart of West Africa

between latitudes N 009° 20’ and 15° 05’, longitudes E 002° 03’ to W 5°
20’. It is a relatively flat country especially in its northern part and covers
274,000 km2.
The climate is characterized by two strongly contrasted seasons: a dry

season and a rainy season. The dry season varies from 8 to 9 months in the
North and 5–6 months in the Southwestern part. The rainy season extends
from May to October in the South, Southwest and June-July to September
in the north. Precipitations are irregularly distributed in space and time.
The average annual rainfall decreases from Southwest (1100 mm) to the
north (less than 400 mm) (Fig. 1). Rainfall is concentrated during the single
rainy season with a maximum in August. Since the drought of 1973–1974,
the original isohyets lost between 100 and 200 mm (Kessler and Geerling
1994).
Based on rainfall distribution, the country is divided into three climatic
regions (Laclavère 1993) (Fig. 1). The Sudanian zone which occupies the
entire south of the country with about 6 months of rainfall (>900 mm), the
Sudano-Sahelian zone, located in the center, accounts for half of the country
Fig. 1. Climatic regions of Burkina Faso and localization of prospected areas. Data from Sanon
et al. (2009a).
with 4–5 months of rainfall between 900 and 600 mm, the Sahelian zone in
the north with about 2–3 months of rain and an annual rainfall less than
600 mm. According to Boussim (2010), two phytogeograpical zones can be
distinguished: the Soudanian zone extending from N 009°20 to N 13° making
2/3 of the land area, and the Sahelian zone (N 13° to 15°) that represent one
third of the country. The vegetation is characterized by the predominance of
mixed woody and herbaceous formations patchily distributed mostly in the
northern part (steppes, savannas, woodlands). The natural vegetation covers
60% of the country (FAO 1987). Gallery forests (1%) and woodlands (1%)
occur in the southern part. Tiger bush (1%) and steppe (4%) are common
in the North, whilst wooded savannas and shrublands (53%) can be found
in the rest the country.
Ectomycorrhizal-rich vegetations are located in the wetter southern
areas. They are mostly Caesalpinioid- and Phyllanthioid-dominated
woodlands of the Sudanian zone in the Southwestern, Southern and Eastern
parts of the country (Fig. 1). In many such woodlands, Anogeissus leiocarpus
(DC.) Guill. & Perr. dominates with other species such as Pterocarpus
erinaceus Poir., Burkea africana Hook., Afzelia africana Sm., Albizia chevalieri
Harms. and mainly Isoberlinia doka Craib. & Stapf., I. tomentosa (Harms.)
Craib. & Stapf. and Detarium microcarpum Guill. & Perr. (Thombiano et al.
2012). The presence of Isoberlinia doka as monospecific planting or associated
with Isoberlinia tomentosa also characterizes this area. Gallery forests, mainly
those of Kou and Mouhoun Reserve Forests are exclusively dominated by
EcM tree species such as Berlinia grandiflora (Vahl) Hutch. & Dalziel.
2.2 Ectomycorrhizal trees species with their myco-symbionts
Different tropical plant families known to form EcM symbiosis have been
reported (Ducousso et al. 2008, Bâ et al. 2011, Bechem and Alexander 2012).
Compared to these data, an inventory was first performed in the field in
the Sudanian zone. It allowed identifying seven ectomycorrhizal species
sorted into three families or subfamily and five genera (Table 1).
Assessment of mycorrhizal status of these species was performed on the
roots of young plants, mature trees and natural regeneration. Arbuscular
mycorrhizal were not observed on roots of Isoberlinia spp. Some studies have
reported the presence of arbuscular mycorrhizal fungi on A. africana roots
(Thoen and Ducousso 1989), however we did not find this type of symbiosis
in our samples. For B. grandiflora, the presence or not of AM has not been
evaluated. Sites containing these EcM species were identified mainly in the
south-west and to a lesser extent in the south and east of the country (Fig.
1). These sites can be grouped as shown in Table 2.
Sites contained U. guineensis and B. grandiflora (3 sites in Southwest),
and the site of A. africana in Southwest are located in gallery forests. Apart
Table 1. Ectomycorrhizal tree species so far in Burkina Faso.
Family or Subfamily Genera Species Symbiotic statuta

Caesalpinioideae Afzelia A.africana ECM
Berlinia B. grandiflora ECM
Isoberlinia I. doka ECM
Isoberlinia I. tomentosa ECM
Dipterocarpaceae Monotes M. kerstingii ECM & AM
Phyllanthaceae Uapaca U. guineensis ECM & AM

Uapaca U. somon ECM & AM
Sanon et al. (1997). a ECM : Ectomycorrhiza ; MA ; Arbuscular Mycorrhiza.
Table 2. The sites prospected with the dominant host plants.
Regions Sites number Dominant host plants

South-west 3 U. guineensis and B. grandiflora
2 Isoberlinia spp.
1 A. africana
1 I. dalziellii and M. kerstingii
1 Isoberlinia spp. and M. kerstingii
1 M. kerstingii
1 U. somon and Isoberlinia spp.
South 2 I. doka
1 A. africana
1 I. doka and A. africana
East 2 I. doka
1 A. africana
1 I. doka and A. africana
from the site of Isoberlinia spp. and M. kerstingii in Southwest which were
identified in 2000 and explored, all other sites were surveyed in 1994, 1995,
2000, 2005 and 2006. Sites in South and East were surveyed in 2000 and fungi
frequently encountered were Scleroderma species. Except gallery forests, the
other trees species are found in woodlands and savannas. Surveys were
conducted during the rainy season, the period of fungi fruiting, particularly
in June, July and August. All putative ECM fungi fruitbodies near these
trees were harvested. They were cleaned, photographed and described
morphologically. They were transported to the laboratory for isolation in
pure culture and/or dried in an oven at 50°C and stored in herbarium of
the Laboratory of Microbiology INERA/DPF.
The sampled specimens are sorted into 18 genera (Fig. 2) and 8 orders
(Aphyllophorales, Agaricales, Boletales, Cantharellales, Gautierales,
Hymenogastrales, Russulales and Sclerodermatales) and identified
according to the herbarium of the Microbiology Laboratory of IRD Senegal
Fig. 2. Genera of ectomycorrhizal fungi identified by their morphology. (From Sanon et al.
1997, Sanon 1999, Sanon et al. 2012, in press).
(formerly ORSTOM), Ducousso (personal communication) and Verbeken

(personal communication). The most represented genera are Russula,
Amanita, Boletelus, Leccinum and Scleroderma.
From the above mentioned EcM taxa, isolation and pure culture were
made from some Scleroderma species as highlighted in Sanon et al. (1997) and
Sanon (1999). The mycorrhizal status of these isolates was confirmed in vitro
with certain host plants (Afzelia africana, A. quanzensis Welw., Isoberlina doka,
I. tomentosa, Brachystegia speciformis). An analysis of the genetic diversity of
different species of Scleroderma and testing for controlled mycorrhization in
nurseries were then carried out (Bâ et al. 1999, 2002, Sanon et al. 2009a).
3. Scleroderma species
Scleroderma belongs to Gasteromycete and to the Sclerodermataceae family.
This genus is widespread in tropical and temperate zone and is a symbiont
of many families of temperate and tropical plants of economic importance
such as Pinaceae, Myrtaceae, Fagaceae, Dipterocarpaceae, Gnetaceae and
subfamily Caesalpinioideae (Munyanziza and Kuyper 1995, Sims et al.
1997, Bechem and Alexander 2012). The genus Scleroderma encompasses
more than 25 accepted species (Sims et al. 1995, Sims et al. 1997, Guzman et
al. 2004). However, many species remain unidentified especially in tropical
Africa and Asia (Sanon et al. 1997, David and David 1998, Sims et al. 1999).
A revised key to the species of the genus Scleroderma was developed by
Sims et al. (1995). This key is based primarily on the characteristics of the
peridium and spores. Thus, three morphotypes or sections are accepted
with the genus Scleroderma depending on the morphology of their spores:
Section Aculeatispora with spiny or warty spores; section Sclerangium with
sub-reticulate spores and the section Scleroderma with spores totally covered
with reticulum (Sims et al. 1995).
3.1 Morphological diversity of Scleroderma
Scleroderma sporocarps occur most frequently at the beginning of the rainy

season (June-early July) and are almost absent when the rains are well
established and abundant. The fruiting bodies collected from 1994 to 1997
and 2000 have been described morphologically. They are sub-spherical or
spherical and contracted to the base to form a pseudo-stem structure (Fig. 3).
The outer surface of the peridium can be smooth or scaly. Based on colour
and ornamentation of spores and peridium, six morphological species have
been identified belonging to the sections Aculeatispora and Scleroderma, and
a new group not yet described in the literature (Table 3, Fig. 4).
Fig. 3. Sporocarps of S. verrucosum.
3.4 Genetic Diversity of Scleroderma
Inter- and intraspecies differences in the ability to stimulate host plant

growth were identified among ectomycorrhizal fungi in different studies
performed in the laboratory, nursery and in the field (Malajczuk et al.
1990, Lei et al. 1990, Le Tacon et al. 1992, Thomson et al. 1994, Bâ et al.
Table 3. Characteristics of sporocarps and basidiospores of Scleroderma species.
Species Sporocarps Spores diameter Spores Sections

color (µm) ornamentation
Scleroderma Brown-black 7–9 reticulated Scleroderma
dictyosporum Pat.
Scleroderma Brown 5–9 warty Aculeatispora
verrucosum Pers. 1–2 µm
Scleroderma sp1 Pinkish-brown 9–13 Spiny Aculeatispora
2–3 µm
Scleroderma sp2 Brown 5–7 - New
Scleroderma sp3 Brown 5–10 Very small spines Aculeatispora
Scleroderma sp4 Yellowish 7–9 - New

Sanon et al. (2009a).
Fig. 4. Spores of Scleroderma sp2 without ornamentations (G. 100x, photonic microscope).
1999, 2002, Diédhiou et al. 2005). To attest whether the increased growth
of host plants resulted from inoculated symbionts, it was essential to
assess the persistence of these symbionts. In the absence of fructification,
study of the persistence of mycosymbionts implied that it was possible to
identify them from the mycorrhizas formed in situ. However, tracing the
identity of a fungal species from the EcM root tips was quite tricky, due
to poorly variable morphological and structural characteristics (Agerer
1987, Gardes et al. 1991, Sanon 1999, Sanon et al. 2002). To overcome these
difficulties, molecular biology techniques are increasingly used to enable
the identification of ectomycorrhizal fungi from fruiting bodies, cultured
mycelia and mycorrhizas (Gardes et al. 1991, Liang et al. 2004, Matsushita
et al. 2005, Ruiz-Diez et al. 2006, Sica et al. 2007). Also, although the
morphological characteristics allowed for the identification of a relatively
large number of species of the genus Scleroderma, molecular tools provided
more detail in the identification of ectomycorrhizal fungi (Hansen et al. 2002,
Rivière et al. 2007, Tedersoo et al. 2007). Various studies have shown that in
vitro DNA amplification (PCR) followed by the analysis of Polymorphism
Restriction Fragment Length (RFLP), or sequencing the internal transcribed
spacer (ITS) and inter-genic spacer (IGS) are among the most important
tools for analyzing inter and intra-specific fungal symbionts (Liang et al.
2004, Matsushita et al. 2005, Ruiz-Diez et al. 2006, Sica et al. 2007). The
ribosomal DNA spacers (ITS and IGS) are known to be inter-and intra-
specific variable and used as markers to distinguish multiple species or
isolates of ectomycorrhizal fungi (Karen et al. 1997, Peter et al. 2001, Horton
2002, Gomes et al. 2002).
To assess the inter- and intraspecific diversity of Scleroderma harvested
in Burkina Faso, the ITS and IGS1 regions were amplified and digested with
two restriction enzymes HinfI and MboI. DNA was extracted from isolates
and fruiting bodies using the methods described by Grube et al. (1995)
and Martin et al. (1997) or the DNeasy kit according to the manufacturer’s
instructions (Qiagen, France). The universal primers and ITS1/ITS4
and CNL12/5SA were used to amplify ITS and IGS1, respectively. The
amplification conditions, digestion and electrophoresis are described by
Among the six morphological species identified (Table 3), the analysis of
length polymorphism restriction fragments revealed eight ribotypes noted,
A, B, C, D, E, F, G and H with 1-3 ribotypes within each morphological
species (Table 4). Two ribotypes were identified within the morphotype S.
dictyosporum (A, B), three for S. verrucosum (C, D, E), one for Scleroderma
sp1 (F) and two for Scleroderma sp2 (G, H). Some ribotypes were more
represented than others; this was the case of ribotype A of S. dictyosporum,
C of S. verrucosum and H of Scleroderma sp2. However, no relation was
established between the species origin (host plant) and ribotypes observed.
RFLP profiles of Scleroderma sp3 and Scleroderma sp4 were identical to those
of ribotype D of S. verrucosum and A of S. dictyosporum respectively (Sanon
et al. 2009a). This suggested that these two species are rather ecotypes of
S. verrucosum and S. dictyosporum, respectively. Thus, the six morphospecies
identified should be grouped into four species: S. dictyosporum, S. verrucosum,
Scleroderma sp1 and Scleroderma sp2.
Except ribotype G of Scleroderma sp2, ITS of at least two ribotypes of
each species have been sequenced. ITS sequences were obtained from 9
fruiting bodies and 16 isolates in culture. They were submitted to GenBank
database and similar sequences were identified using the algorithm Blastn.
Table 4. Size of restriction fragments (in base pairs) of the ITS and IGS1 regions digested with
MboI and HinfI. The fruiting bodies are noted “SD, SV, SP1, SP2, SP3 or SP4” and isolates “IR”.
Fragments less than 50 bp are not shown on the table. Data from Sanon et al. (2009a).
Species ITS IGS1 Ribotypes

HinfI MboI HinfI MboI
S. dictyosporum
(4IR et 4SD) 174, 127, 113, 208, 159, 135, 119 143, 135, 128 228, 153, A
87, 60 86
(1IR et 1SD) 287, 240 218, 198, 167 160, 151, 82 280, 240 B
S. verrucosum
(5IR et 10SV) 311, 246, 93 171, 162, 153, 119 182, 167, 119 238, 151, C
(2IR) 285, 146, 110, 66 254, 146, 124, 182, 147, 63 54
98, 77 228, 178 D
(2SV) 264, 114, 104, 94 208, 159, 135, 92 182, 139, 119 238, 210 E
Scleroderma sp1
(all 4 samples) 322, 149, 110, 66 241, 153, 140, 119, 286, 143 228, 196, F
102 54
Scleroderma sp2
(1SP2 et 2IR) 331, 294, 87 284, 227, 91 232, 143, 88 228, 153 G
(10SP2 et 2IR) 206, 186, 99 152, 137, 113, 106 173, 139, 124 231, 209 H
The sequences of Scleroderma from Burkina showed low similarity with

other Scleroderma in GenBank (89% maximum similarity, Sanon et al. 2009a).
Seven genotypes were identified for all 25 samples analyzed and sequences
were deposited in GenBank (Table 5).
The phylogenetic tree (Neighbor-joining method, MEGA software)
(Kumar et al. 2004) from these sequences can distinguish three different
branches (Fig. 5). Branch 1 summarizes the morphological species
S. verrucosum and Scleroderma sp1, branch 2 summarizes the morphotypes
of S. dictyosporum and the third branch includes the morphotype Scleroderma
sp2. For each branch, the ribotypes were supported by high bootstrap
values, suggesting that the ribotypes identified within each morphological
species can be considered as different phylogenetic species as for Pisolithus
(Martin et al. 2002). Thus, samples identified as S. verrucosum include
three species, those identified as S. dictyosporum include two species
and one species sequenced for Scleroderma sp2 (Fig. 5). Scleroderma sp1
is closer to S. verrucosum than any other species, from which it differs by
a few deletions and insertions at ITS2 (sequence alignment matrix, data
not shown), thus belonging to the S. verrucosum morphotype (species 4).
Both morphological species Scleroderma sp3 (IR252) and Scleroderma sp4
(IR408) were supported by high bootstrap values (100%) with species 2 of
S. verrucosum and S. dictyosporum morphotypes, respectively (Fig. 5). This
confirms the results obtained by PCR/RFLP and shows that Scleroderma
sp3 (IR252) and Scleroderma sp4 (IR408) are respectively morphotypes of
S. verrucosum and S. dictyosporum. Scleroderma sp2 was genetically distant
Table 5. Sporocarps and isolates of Scleroderma sequenced with their GenBank accession
numbers. Isolates are noted “IR” and sporocarps “SD, SV, SP1, SP2, SP3 or SP4”. Data from
Species/Ribotypes/Voucher number GenBank accession number

S. dictyosporum
Ribotype A
IR109 FJ840442
IR250 FJ840444
IR412 FJ84044
IR602 FJ840447
IR408 FJ840445
ORS7731 FJ840448
Ribotype B
IR215 FJ840443
SD-4901 FJ840449
S. verrucosum
Ribotype C
IR256 FJ840454
IR114 FJ840451
IR110 FJ840450
IR118 FJ840452
SV1803 FJ840458
SV2802 FJ840460
SV5602 FJ840461
Ribotype D
IR500 FJ840455
IR600 FJ840456
IR252 FJ840453
Ribotype E
SV1801 FJ840457
SV1804 FJ840459
Scleroderma sp1
Ribotype F
IR406 FJ840462
IR410 FJ840463
Scleroderma sp2
Ribotype H
SSP2-1806 FJ840464
SSP2-2803 FJ840465
SSP2-3901 FJ840466
from other species analyzed (52% bootstrap value) but was grouped with
the genus Scleroderma. The morphology of spores of this species suggested
that it might be a new species specific to Africa. However, further studies
are needed on a larger number of samples and also the sequencing of the
second ribotype of this species.
Fig. 5. Neighbor joining phylogenetic tree of Scleroderma species based on ITS sequences.
Rhizopogon occidentalis is used as outgroup. Numerical values on the branches are the bootstrap
values of 1000 replications. Data from Sanon et al. (2009a).
4. Controlled Mycorrhization of Afzelia africana

The inoculation of a host plant with a well-defined ectomycorrhizal fungus
strain under controlled conditions and the evaluation of the effect of the
fungus on host growth is called “controlled mycorrhization.” It allows the
selection of fungi compatible and efficient for the production of seedlings

for reforestation. The criteria for the selection of the best strains include the
ability to form mycorrhizas with seedlings, the beneficial effect on growth of
inoculated plants and the mineral content (especially phosphorus) of various
organs of the plants. Also, the length of the extramatrical network developed
in the soil by the mycosymbionts, or the proportion of metabolically active
hyphae in the soil, associated with other parameters, have been proposed
by some authors to characterize the efficacy of the fungus (Jones et al. 1990,
Hamel et al. 1990). This technique is widely used in temperate regions
(France, Spain, USA, Canada) (Le Tacon et al. 1992). However, there are few
studies on tropical trees, especially in Africa. Nevertheless, in recent years,
there is a renewed interest in the study of controlled mycorrhization of some
local species. In Burkina Faso, works on controlled mycorrhization were
carried out mainly on the genus Scleroderma and the host plant A. africana,
one of the most important ectomycorrhizal species in Burkina Faso, and A.
quanzensis, a species of East Africa (Bâ et al. 1999, 2002).
4.1 Inoculum production
The solid mycelial inoculum was used in two experiments on A. africana and
A. quanzensis in the nursery. The inoculum was produced on a solid substrate
composed of peat and vermiculite according to the method described by
Duponnois and Garbaye (1991).
4.2 Fungal inoculation
Controlled mycorrhization of A. africana and A. quanzensis was carried out

on a sandy soil from Sangalkam (Senegal) which was free of ectomycorrhizal
propagules and displayed the following physico-chemical characteristics:
clay (5.4%), silt (5.8%), sand (88.8%), pH in water (6.03), 0.39% total C, total
N (0.027%), total MO (0.68%), C/N 14, P total (41.92%), available P, Bray
1 (2.15%). The substrate was sieved (2 mm) and mixed with the inoculum
(10: 1, v/v). The mixture was dispensed into polyethylene bags of 2 liters
and watered with tap water. Two germinated seeds were planted at 2 seeds
per bag. Ten days after planting, the number of seedlings was reduced to
one per bag.
For A. africana, two provenances (Nazinga, Burkina Faso (AaBF) and
Diatock, Senegal (AaSN)) were inoculated with two isolates of S. dictyosporum
(IR109 and IR408), one isolate of S. verrucosum (IR406) and one isolate of
Thelephora sp (ORSXM002) (Bâ and Thoen 1990, Sanon 1999, Diédhiou et
al. 2004) or not inoculated. The experiment was factorial (2 provenances x
(4 fungal strains + 1 control). Each treatment was replicated 10 times and
the treatments were arranged into randomized blocks in a screen house

under the climatic conditions of Dakar (average daily temperature 25–30°
C, photoperiod approximately 12 h). Watering was done once a day with
tap water. The experiment duration was 4 months (Bâ et al. 1999).
For A. quanzensis, a species from East Africa, one provenance was
inoculated with S. dictyosporum (IR408) and Thelephora sp. (ORSXM002).
The experiment design was single randomization with 3 treatments and
10 replicates per treatment. The experimental conditions were the same as
above and the duration of the test was also 4 months (Bâ et al. 2002).
4.3 Growth and nutrition of two provenances of A. africana
The two provenances of A. africana differed in their response to inoculation

with the four fungal strains. Mycorrhization rate varied in roughly the
same proportions for the two provenances, 32% to 69% for AaBF and 22%
to 64% for AaSN (Table 6). For the Burkina Faso provenance (AaBF), the
highest rates of mycorrhization were obtained with S. dictyosporum, IR109
(69%) and IR408 (64%). Thelephora sp., ORSXM002 formed mycorrhiza with
AaBF with medium rate of 46%. S. verrucosum IR406 has a lower rate of 32%.
Isolates IR408 and IR406 gave the lowest rates of mycorrhization (22% and
32%) with the provenance AaSN (Bâ et al. 1999).
Growth (total dry weight) of AaSN was not significantly improved by
inoculation despite mycorrhization rate of 58% and 64% with IR109 and
ORSXM002 respectively, while that of AaBF was improved by inoculation
with IR408 and ORSXM002 (Table 6). The increased growth observed for
this provenance could be attributed to the development of the root system as
reflected by the total biomass of inoculated plants. These results confirmed
those obtained by Bâ (1990) on the positive effect of inoculation with
ORSXM002 on growth of A. africana (from Bayottes) due to an increase in
root biomass. They were also consistent with those obtained by Diédhiou
et al. (2005) from a Senegalese A. africana inoculated for 7 months in the
nursery with different mycorrhizal fungi with IR408 and ORSXM002.
The mycorrhizal dependency of the two provenances of A. africana was
relatively low 0.15% to 32% compared to the values obtained with tropical
species introduced in West Africa such as the hybrid Eucalyptus urophylla
x E. kirtonia and Acacia mangium (Garbaye et al. 1988, Duponnois and Bâ
1999). However, the comparison was difficult because of different culture
conditions. A mycorrhizal dependency of at most 30% was also obtained
with another provenance of A. africana inoculated with different isolates of
Scleroderma, Pisolithus and Thelephora after 7 months of culture (Diédhiou
et al. 2005).
Table 6. Effect of inoculation with different isolates of ectomycorrhizal fungi on growth parameters and nutrition of two provenances of A. africana
140
seedlings. MD, Mycorrhizal dependency. Data from Bâ et al. (1999).
Provenances of A. africana Height (cm) Roots dry Total dry Colonization MD (%) N (%) P (%) K (%)
and fungal isolates weight (g) weight (g) (%)
AaBF
IR109 41.97 b 2.83 abc 7.81 ab 69.00 a 21.25 ab 1.96 d 0.11 bc 1.41 cde
IR406 44.96 ab 2.53 bc 7.73 ab 32.00 abc 20.43 ab 2.33 ab 0.09 c 2.12 a
IR408 47.78 ab 3.29 ab 9.00 a 64.00 a 31.66 a 1.94 d 0.14 a 1.66 bc
ORSXM002 42.75 b 3.98 a 9.10 a 46.00 ab 32.24 a 1.85 d 0.10 bc 1.45 cd
Control 38.98 b 1.76 c 6.15 b 0.00 c - 2.02 cd 0.10 bc 1.02 e
AaSN
IR109 46.85 ab 2.07 bc 6.34 b 58.00 ab 0.15 b 2.03 cd 0.13 ab 1.55 c
IR406 47.80 ab 2.71 abc 6.95 ab 38.00 ab 8.92 ab 2.28 abc 0.10 bc 2.05 ab
IR408 52.40 a 2.17 bc 7.03 ab 22.00 bc 9.95 ab 2.54 abc 0.11 bc 2.11 a
ORSXM002 52.78 a 2.92 abc 8.18 ab 64.00a 22.61 ab 2.08 bcd 0.12 abc 1.80 abc
Control 52.60 a 1;73 c 6.33 b 0.00 c - 1.90 d 0.10 bc 1.13 de
Values followed by the same letter within columns are not significantly different using ANOVA (SAS, Bonferroni test, P<0.05).
One major effect of mycorrhization is generally to improve the

phosphorus nutrition of host plants. Here, however, when comparing
the control treatments and those inoculated, the increased P, N, Mg and
Ca contents of plants inoculated with some strains was not related to
the stimulation of plant growth in terms of biomass produced, except
for P content of AaBF inoculated with IR408 (Table 6). Only the leaf K
concentration was higher in inoculated plants compared to controls but
unrelated to the stimulation of plant growth (Bâ et al. 1999). This suggested
that these minerals were not limiting factors for the growth of these two
A. africana provenances. Thus, the high rate of mycorrhization do not
necessarily translate into a high concentration of minerals in leaves of
inoculated plants, there was no clear link between the level of colonization
of inoculated plants and the potential benefit for plants. In conclusion, two
fungal species, S. dictyosporum IR408 and Thelephora sp. ORSXM002 seemed
to be the most suitable species to improve seedling growth of A. africana
in nursery.
4.4 Growth and nutrition of A. quanzensis
Based on the results of the previous study, the two promising isolates
identified were tested on A. quanzensis (Bâ et al. 2002). A. quanzensis
responded differently to inoculation depending on the isolate. Thelephora
sp. ORSXM002 efficiently colonized the root system of inoculated plants
and stimulated root biomass compared to S. dictyosporum IR408 (Table 7).
The mycorrhization rate obtained with ORSXM002 (66.40%) was similar
to that obtained with the Senegalese A. africana provenance (64%) (Bâ et al.
1999). However, for IR408, this rate was very low (19.40%). Stimulation of
root biomass by ORSXM002 was not reflected in the total biomass of plants.
Conversely, this strain had stimulated the total biomass of A. africana in
the same conditions. Therefore, mycorrhizal dependency of A. quanzensis
inoculated with these two species does not exceed 16%, which was quite
Table 7. Effect of inoculation with IR408 and ORSXM002 on growth parameters and nutrition
of A. quanzensis seedlings. MD, Mycorrhizal dependency. Data from Bâ et al. (2002).
A. quanzensis/ Height Shoot dry Root dry Colonization MD N (%) P (%) K (%)
Isolates (cm) weight (g) weight (g) (%) (%)
inoculated
IR408 69.86 b 8.84 a 2.26 a 19.40 b 3.50 a 2.09 a 0.13 a 1.63 b
ORSXM002 68.22 ab 9.14 a 3.60 b 66.40 a 16.00 a 2.14 a 0.14 a 1.56 b
Control 64.28 a 8.80 a 1.91 a 0.00 c - 2.15 a 0.12 a 0.81 a
Values followed by the same letter within columns are not significantly different using ANOVA
(SAS, Bonferroni test, P<0.05).
low compared to A. africana (Bâ et al. 1999). These data suggested that the
different origin of strains and host plant may influence the response to
inoculation with A. quanzensis compared to A. africana. As in the case of A.
africana, only the K content of the shoot was greater in plants inoculated
with both strains (Table 7).
5. Conclusion
This work allowed the identification of ectomycorrhizal fungi associated
with seven host plants in Southwest, South and East of Burkina Faso (A.
africana, B. grandiflora, I. doka, I. dalzeillii, M. kerstingii, U. guineensis, U. somon).
All harvested fungi belonged to genera already identified as ectomycorrhizal
and some were identified at the species level. An unexpected diversity of
ectomycorrhizal fungi (about 78 morphological species) could be detected
despite the geo-climatic conditions of the regions surveyed and phenological
fluctuation of the fungi from one year to another in low humidity areas.
Our inventory revealed the dominance of species of the genera Russula,
Boletelus, Leccinum, Amanita and Scleroderma. For the latter, ITS sequencing of
ribotypes showed that they belonged to at least seven phylogenetic species:
4 species for S. verrucosum morphotype, 2 for S. dictyosporum and a single
ribotype sequenced for Scleroderma sp2. The particular morphology of
spores of Scleroderma sp2 and its position on the phylogenetic tree compared
with other species of Scleroderma suggested a new species unique to Africa,
especially in West Africa.
We tested the effectiveness of four fungal isolates, S. dictyosporum,
IR109 and IR408, S. verrucosum, IR406 and Thelephora sp, ORSXM002, on
the growth and mineral nutrition on two provenances of A. africana in
the nursery. We have shown that the inoculation induced a wide range of
plant growth stimulation (0.17% to 48% of biomass increase) and improved
mineral nutrition, especially K. The use of two provenances of A. africana
has highlighted the strong influence of the genetic background of the host
plant on the beneficial effect of mycorrhization. Mycorrhizal dependency
indicated that the Senegalese origin was less dependent on mycorrhiza
for growth than that of Burkina Faso. The use of fungal strains effective to
improve A. africana plant growth in controlled mycorrhization programs
can be oriented towards S. dictyosporum IR408 and Thelephora sp. ORSXM002
for both tree provenances. These two isolates tested on A. quanzensis have
shown a limited significant effect compared to A. africana suggesting that
the origin of the host plant in relation to the fungal isolates tested may
influence the response to inoculation.
However, these strains should also be tested under other soil conditions
because our experiment was conducted in a sandy soil unrepresentative
of A. africana forests in West Africa. Any selection of strain must therefore
be supplemented by tests under various conditions and respecting local
conditions of plantations.
Acknowledgements
Krista McGuire and Jean Garbaye for improving the language.
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CHAPTER
8
The Physiology of
Scleroderma sinnamariense
Mont. (Sclerodermaceae),
an Ectomycorrhizal Fungus
Associated with Gnetum spp.
(Gnetaceae)
Eneke Esoeyang Tambe Bechem
1. Introduction
Scleroderma sinnamariense Mont. is a bright yellow ectomycorrhizal fungus
with a pantropical distribution (Guzman and Ovrebo 2000) which was
originally described in French Guiana. This fungus is a gasteromycete and
produces macroscopic sporocarps called “puffballs” (Fig. 1). These yellowish
leathery, rounded structures become dry and crack at maturity, releasing
dry powdery basidiospores which are dispersed by wind. The basidiomes
are not eaten in Cameroon by humans but it is possible that they are eaten
by some animals including small mammals, birds and insects. They are,
however, eaten by humans in Asia. S. sinnamariense is commonly recorded
Department of Botany and Plant Physiology, Faculty of Science, University of Buea, P. O. Box
63 Buea, South West Region, Republic of Cameroon.
Email: tamenekeso@yahoo.co.uk
Fig. 1. Sporocarps of S. sinnamariense. Bar is 2.5 mm.

in SE Asia (Sims et al. 1995) where it is reported to form ectomycorrhizas

with members of the Dipterocarpaceae and Gnetaceae (Watling 1994, Cadiz
and Florido 2001). It has also been recorded in Cameroon (Bechem and
Alexander 2009, 2012a, Onguene and Kuyper 2001, Ingleby 1999) where it
forms ectomycorrhiza with Gnetum spp. The ectomycorrhizal status of this
fungus is proven, but unlike other species of the genus Scleroderma, such
as S. citrinum Pers., S. aurantium Viall.: Pers., S. vulgare Horn., which have
also been shown to occur as saprotrophs on rotting wood (Jeffries 1999),
S. sinnamariense has been demonstrated to form typical ECM (Bechem and
Alexander 2009). There are no records to show that this species is capable
of a free-living saprotrophic existence. Richter and Bruhn (1989) suggested
that the genus was wholly mycorrhizal and not partly saprotrophic. This
suggestion was based on data collected from enzyme tests.
The difficulty encountered with the growth of this fungus in vitro as well
as its relatively slow growth rate are some of the reasons why few studies
exist on its physiology as well as on its fungus-host interaction.
2. Taxonomy and Ecology

S. sinnamariense is a variable species. It belongs to the Sclerodermaceae
which also contains Pisolithus, a genus that has been widely studied. This
species is believed to have been described using several different names
such as S. aureum Massee from New Guinea, S. chrysastrum Martin from
Panama, S. luteum Lloyd from Malaya and S. pantherinum Matt. from Africa
(Guzman 1970). Demoulin and Dring (1971) described two related species,
S. congolense and S. schmitzii from the Congo basin in Africa which also
The Physiology of Scleroderma sinnamariense Mont. 149
have a yellow endoperidium. However, their spores and basidiomata are

smaller than those of S. sinnamariense. A molecular phylogenetic analysis
(Fig. 2) carried out by Bechem and Alexander (2012a), placed a sequence
from S. sinnamariense associated with Gnetum spp. in Cameroon in the
same group as that obtained from a basidiome collected beneath G. gnemon
in Malaysia. Another group included a sequence from S. sinnamariense
associated with Gnetum spp. as well as that obtained from a culture isolated
from a basidiome growing with Hopea odorata in Malaysia. From the said
phylogenetic analysis (Fig. 2), it was concluded that S. sinnamariense forming
EM with Gnetum spp. in Cameroon was not distinct and therefore was
the same as that forming EM with dipterocarps and Gnetum in Asia. This
conclusion was however based on a single “non-Gnetum’ sequence.
Fig. 2. Molecular phylogenetic tree-diagram of single linkage analysis of RFLP patterns from
digestion of the ITS region of rDNA amplified from EM root tips from Gnetum, mycelium,
and EM fruiting bodies. Numbers at nodes indicate bootstrap indices over 50% obtained after
1,000 replicates. Data from Bechem and Alexander (2012a).
These authors also observed variations within the ITS sequences

obtained from yellow mycorrhizas and yellow basidiomes, and even
between yellow mycorrhizas from different provenances in Cameroon.
S. sinnamariense isolated from Gnetum spp. in Cameroon could not form EM
with Hopea odorata from Malaysia (Bechem and Alexander 2009), in spite of
the fact that they were not quite distinct based on molecular studies.
Generally, Scleroderma has a wide and inconsistent distribution of
species. Tropical species such as S. sinnamariense are able to withstand
high temperatures and can grow at 30°C in axenic cultures. This species
has been recorded principally in forest habitats in association with Gnetum
spp. (Bechem and Alexander 2009, 2012a, Cadiz and Florido 2001), Shorea
sp. and Hopea sp. (Lee et al. 1997). This fungus like most basidiomycetes
produces extensive extramatrical mycelia (Fig. 3), which may aggregate into
rhizomorphs (Bechem and Alexander 2012a), used in channeling nutrients
and water to and from the host (Hasselquist et al. 2010).
Fig. 3. Yellow Gnetum EM root tip formed with S. sinnamariense, showing extensive extramatrical
mycelia. Bar is 0.3 mm.
3. Growth in Culture
Just like most mycobionts, S. sinnamariense can be isolated from EM root tips
but recovery is often less than 20% and sometimes can be as low as 5% or
less of the number of plated tips (Molina and Palmer 1982). The low success
rates could be attributed to many factors including the inability of fungi to
grow on the media used, insufficient surface sterilization, and requirement
of special nutrients. The mycobiont can also be isolated from spores and
sporocarps. Several media types have been used for the isolation of EM
fungi from root tips. These include modified Melin-Norkran (MMN) agar
(Marx 1969), Hagems agar (Modess 1941), Pachlewski agar (Pachlewski
and Pachlewska 1974), modified woody plant medium, potato dextrose
agar, etc.
S. sinnamariense was successfully isolated from Gnetum EM root tips
on MMN agar containing benomyl (1 ug/ml), chlortetracycline (30 ug/ml)
and streptomycin (10 ug/ml) (Bechem 2004). More than 350 individual EM
root tips were plated with a fungal symbiont recovery rate of 38%. This was
obtained after surface sterilization using 6% Cl2 for 4 min. In the said study,
only one fungus type was isolated from the yellow morphotype unlike other
studies such as Zak and Marx (1964), who isolated two or more fungi from
the same pine EM roots.
Scleroderma sinnamarense was a yellow culture with prominent hyphae
which had numerous clamp connections. The arrangement of hyphae in
culture was similar to that observed on the mycorrhiza tip. Growth rate
of the fungus was very slow in comparison to some isolates of Pisolithus
sp., an observation similar to that of Sims et al. (1999). Growth rarely
exceeded 35–40 mm in ten weeks, giving a growth rate of 0.5–0.57 mm d–1.
This growth rate was much slower than that observed on the same fungus
species studied by Sims et al. (1999) where growth rate was as high as 1.5
mm d–1 on some isolates. Growth was slower on Malt extract agar (MEA),
Potato dextrose agar (PDA), and on Hagem’s agar, in comparison to MMN
and Pachlewski agar (Fig. 4) (Bechem 2004).
S. sinnamariense species possessed colonies with matted mycelial growth
which sometimes had a floccose periphery, with an irregular to occasionally
feathery outline. Cultures were bright yellow in color, with the reverse
showing the same coloration throughout. Observation of this fungal isolate
was quite similar to that of Sims et al. (1999). Color was impermanent, with
cultures occasionally changing to white after prolonged storage. The reverse
side of these white cultures was whitish in the extremities but yellowish in
the centre. The fungus exuded a yellowish-green pigment and sometimes
produced faint greenish droplets into the agar. Putra et al. (2011) isolated
two known coloring constituents, methyl 4, 4’-dimethoxyvulpinate and
Fig. 4. Dry weight yields (mg) of five isolates of S. sinnamariense following growth for 30 days
on liquid nutrient media, modified Melin Norkran (MMN) and Pachlewski (PACH). Vertical
bars represent standard errors of mean. Each point is a mean of three replicates.
4, 4’-dimethoxyvulpinic acid, from the fruiting body of this fungus. The

second compound was shown to have inhibitory effects on the growth of
some pathogenic microbes. This fungus could tolerate temperatures of up
to 30°C.
4. Nitrogen Nutrition
Nitrogen is present in soil in diverse forms such as ammonium, nitrate,
proteins, peptides and amino acids. Studies using different fungal species
have shown that EM fungi have the ability to take up and transfer N to the
partner plant. Studies on the use of different N sources by Scleroderma spp.
are lacking but some fungi species such as Hebeloma spp. and Paxillus species
have been widely studied in this respect (Abuzinadah and Read 1986).
S. sinnamariense was studied to evaluate its ability to use different N
sources for growth (Bechem 2012a). This fungus produced measurable
biomass (Table 1) following growth on MMN liquid broth containing a
range of nitrogen sources including ammonium, nitrate, glutamic acid,
arginine, alanine, glycine, peptone, and bovine serum albumin (BSA)
(Bechem 2012a).
This Scleroderma produced greater biomass on ammonium-N than
that produced on nitrate-N. Growth in ammonium was accompanied by
a fall in pH of the medium, while an increase in pH was observed with
Table 1. Dry weight yields (mg) after 30 days of growth of Scleroderma sinnamariense on liquid
MMN media containing different nitrogen sources.
N source S. sinnamariense
Mean ± s.e.m
Calcium nitrate 4.2 ± 0.1
Ammonium sulphate 7.2 ± 1.1
Glutamic acid 8.8 ± 1.2
Arginine 12.9 ± 1.9
Alanine 11.2 ± 0.6
Glycine 2.8 ± 0.5
Bovine Serum Albumin 3.5 ± 0.2
Peptone 16.7 ± 1.4
No Nitrogen 2.5 ± 0.1
Values are means ± standard errors of mean. Number of replicates (n) = 3. Least significant
difference at 95 % (one-way comparison) was 3.324.
growth in nitrate. This inability to use nitrate had been observed in other
ectomycorrhizal fungi species (Rangel-Castro et al. 2002, Sawyer et al.
2003).
The fungus grew best on peptone-N, producing a biomass which was
more than twice (131.9%) the yield observed on ammonium-N. Growth
on BSA was poor, when compared to ammonium. However, utilization of
this N source was better at very low pH (3.0). This low pH optimum for
proteolytic activity suggested that broad spectrum acid proteases were
involved in the catalytic process.
Glycine could not provide the N needed for growth by the fungus. Its
utilization was therefore limited. But arginine, alanine and glutamic acid
were good sources of nitrogen for the fungus. Its growth on these N sources
was higher than that observed in ammonium. It is possible to conclude from
this observation that free amino acid pools in soil as well as those released
directly by the action of fungal proteases might help to supplement the
supply of mineral N.
Ammonium has always been thought to be the N source for the growth
of microorganisms. The results of the study carried out by Bechem (2012a)
showed that organic N sources like peptone, arginine, and alanine were
better for the growth of Scleroderma sinnamariense.
In Cameroon where this fungus was collected, the pH of the soils is
mostly acidic and rarely exceeds 4.5. It is therefore possible that under such
conditions the activity of the proteinases necessary for protein uptake and
utilization would be optimal, enabling the endophyte to access organic N
from the rhizosphere and bulk soil. The phytobiont can therefore access
these N sources via the mycobiont.
5. Carbon Nutrition
There has been no published study on the growth of Scleroderma spp. on
different carbon sources, except for the study by Bechem (2012b). She
evaluated the ability of S. sinnamariense to grow on different C sources in
the presence of either ammonium-N or peptone-N. The C sources used in
the study included; glucose, sucrose, soluble starch, cellulose, cellulose
plus ‘starter’ glucose. She observed that glucose was the best carbon source
for this fungus and that growth on glucose was significantly greater when
peptone was the N source. Growth on the other C sources was relatively
poor, implying that the fungus could hardly utilize soluble starch, cellulose,
and cellulose with ‘starter’ glucose in peptone or ammonium-N. The ability
of the fungus to use sucrose only in ammonium-N might have been due to
changes in pH which resulted following ammonium uptake. Hampp and
Schaeffer (1994) had observed that sucrose hydrolysis was significant at
pH levels below 4.0. In the study by Bechem (2012b) the media were not
buffered and uptake of ammonium would have caused acidification of the
medium. The drop in pH may have favored the hydrolysis of sucrose to
its constituent glucose and fructose, which might have been used by the
fungus for growth. In this study, ‘starter’ glucose caused adaptive growth
of the fungus in cellulose in the presence of ammonium and peptone-N.
There is a possibility that the limited growth of Scleroderma on sucrose
and cellulose could restrict the fungus to the root of the host plant due to
unavailability of nutrients.
6. Phosphorus Nutrition
Some researchers had suggested that the release of organic acids from EM
fungi may solubilize recalcitrant mineral phosphates (Leyval and Berthelin,
1986). Others believed that before insoluble organic P can be broken down
by fungal phosphatases and the resulting phosphate absorbed by the roots,
they would first have to be solubilized (Lapeyrie et al. 1991).
In the experiment reported by Bechem (2011), the ability of
S. sinnamariense to use calcium tetra hydrogen di-orthophosphate CaH4(PO4)2,
hydroxyapatite Ca5(PO4)3OH, calcium phytate C6H6Ca6O24P6 and amorphous
iron phosphate FePO4 as P sources for growth was evaluated. The effect
of the internal P status of the inoculum, the medium N source and the
presence/absence of buffer on growth was also determined.
In the said study, P-sufficient S. sinnamariense showed growth on all P
sources with peptone as sole N source except on FePO4 in unbuffered media.
The source of medium P and the presence of buffer had significant effects
(P< 0.001) on colony diameter. Maximum growth of 40 mm was observed
on FePO4 in buffered conditions. Growth of fungus in buffered medium
was superior to that observed in unbuffered medium. The difference was

greatest for FePO4. The interaction between the P source and buffer had a
significant effect (P<0.001) on growth.
Fungal growth on phytate in unbuffered medium was comparable to
that on CaH4(PO4)2 and hydroxyapatite but better than that on FePO4. The
fungus grew on all P sources with ammonium as the N source, but growth
on CaH4(PO4)2 and hydroxyapatite was better than growth on FePO4.
P-sufficient inoculum grew better in comparison to P-starved inoculum
on CaH4(PO4)2 and hydroxyapatite but not on FePO4..
P-starved fungus demonstrated an ability to solubilize hydroxyapatite
and CaH4(PO4)2 which was superior to that shown by P-sufficient fungus.
This difference was greatest for hydroxyapatite. Solubilization of CaH4(PO4)2
by P-sufficient fungus was comparable to solubilization of hydroxyapatite.
This was probably due to proton expulsion. The resulting pH change in the
medium might have led to the observed solubilization. This phenomenon
is confirmed by studies which showed that during ammonium transport
by the hyphae, there is H+ secretion into the medium (Jennings 1997).
In an earlier assay, this author had observed that growth of Scleroderma
on peptone was followed by fall in the pH of medium. However, the
resulting decrease in pH was not significant enough to bring about
solubilization of hydroxyapatite and CaH4(PO4)2 in peptone-N as observed
on ammonium-N. Nonetheless, the fact that Scleroderma grew on these P
sources implied that there was some solubilization to permit growth. It is
probable that an unknown proportion of the insoluble P provided must
have dissolved, thus supporting growth without any apparent indication
of solubilization. It is unfortunate that other similar studies such as Van
Leerdam et al. (2001) did not indicate whether growth was recorded in the
absence of solubilization or not.
The inability of Scleroderma to solubilize some of the P sources assayed
does not necessarily imply that they would not be able to solubilize these P
sources under natural conditions, with the help of some bacteria. Villegas
and Fortin (2002) showed that the extramatrical mycelium of Glomus
intraradices could solubilize sparingly soluble calcium phosphate in vitro
only when grown in association with Pseudomonas aeroginosa and P. putida.
These bacteria were also unable to solubilize this P source without the
help of the fungus. There is therefore a possibility that in association with
some mycorrhiza helping bacteria, many more P sources could be accessed
in situ.
It was also interesting to note that solubilization of a P source did not
necessarily mean better growth on that P source. Bechem (2011) noted that
within the same P source, in situations where solubilization occurred, the
fungal growth diameter was inferior to that observed in the absence of
solubilization. A probable reason for such an observation may be that the
fall in pH which is thought to have assisted in the solubilization process

demonstrated adverse effects on the fungal culture, causing a reduction in
growth in comparison to the situation where solubilization did not occur.
Alternatively, the observed solubilization of the P sources led to an increase
in the amount of available P in the growth medium. The attained levels
may eventually become toxic to the culture.
Cairney and Smith (1992) found that inoculum P status does influence
hyphal absorption of soluble phosphate. The internal P status of the fungal
inoculum had an effect on the solubilization of hydroxyapatite but not of
CaH4(PO4)2. Van Leerdam et al. (2001) did not observe any effect of P status
of inoculum on solubilization in their study. Although they concluded
that solubilization activities were not dependent on internal phosphorus
concentrations, the author of this chapter believes from her assay that this
may vary with P source and with the fungal species.
The fact that Scleroderma could solubilize hydroxyapatite and calcium
tetrahydrogen di-orthophosphate in vitro does not necessarily imply that
its plant partner can utilize these P sources in situ. It would be necessary to
demonstrate that solubilization of a P source leads to uptake and eventual
translocation to the host plant. Such a demonstration would confirm that
the host plant can utilize these P sources via their fungal partners.
Scleroderma exhibited cell-bound phosphatase activity. The greatest
activity was shown by P-starved mycelium assayed in medium lacking
inorganic P. It was apparent that the absence of N from the assay medium,
just like the presence of P, suppressed activity.
Extracellular phosphatase activity was less than cell-bound activity by
a factor of 100. Again the greatest activity was shown by P-starved mycelia
assayed in medium lacking inorganic P. The absence of P from the assay
medium appeared to not affect extracellular activity to the same extent as
cell-bound activity.
Cell-bound activity was almost completely suppressed by the
presence of P in the assay medium but there was no effect of P on the
low levels of extracellular phosphatase detected. The results showed that
levels of p-nitrophenol phosphatase (pNPPase) activity of some species
of ectomycorrhizal fungi grown in pure culture are partially influenced
by the nitrogen and phosphorus source as well as internal P status of the
inocula and assay medium. Cell-bound phosphatase activity of Scleroderma
was greatly influenced by the presence of inorganic P and peptone-N in
the inoculum which was demonstrated in the absence of inorganic P and
presence of peptone-N in assay solution.
This study showed that a source of nitrogen in the assay solution is
vital for the activation of cell-bound acid phosphatase activity. Overall,
activity was slightly higher when a phosphorus source was absent in
the assay medium. This observation goes to confirm findings by others
that the production of acid phosphatase is enhanced in the absence of P

in ectomycorrhiza fungi (Kropp 1990, Antibus et al. 1992, Tibbett et al.
1998). The highest expression of both cell-bound and extracellular acid
phosphatase activity was observed following growth with peptone as the
sole N source but in the absence of P. This indicated that inorganic P is
an important regulator of both cell-bound and extracellular phosphatase
activity. Such activity was also dependent on the nitrogen source. Growth
was not proportional to acid phosphatase production. From such an
observation it is possible that P may not be the only limiting factor of mycelia
growth in this experiment.
The ability to restrict secretion when a product is plentiful and to
increase production where product is limited suggests that ectomycorrhizal
fungi have an economic regulation of phosphatase production brought into
harmony by environmental P concentration and therefore aimed at external
(soil) substrates (Tibbett et al. 1998a).
With phosphorus being one of the least available plant nutrients found
in soil, phosphatases are believed to be very important in their uptake.
Phosphatases produced by plants and microbes are presumed to convert
organic P into available inorganic P, which is then absorbed by plants.
Therefore the observations from this experiment would shed some light into
the possible mechanisms by which these fungi in mycorrhizal associations
facilitate their host plants with P uptake in a P-limiting environment.
In this study, cell-bound phosphatase was the most active enzyme, an
observation similar to that of McElhinney and Mitchell (1993). This was
probably because cell-bound phosphatases are thought to be most important
in cleavage and procurement of P (Calleja et al. 1980). Nonetheless, in the
study carried out by Tibbett et al. (1998a) it was observed that extracellular
phosphatases accounted for the largest fraction of pNPPase. They put
forward an argument that intimated that contact between substrate
and enzyme was not the only factor required for enzyme activity. They
emphasised the fact that proper alignment of both substrate and enzyme
was also vital for substrate to be transformed to product.
Acid phosphatases are thought to be vital in the uptake and eventual
transfer of phosphate to the plant host. The fact that S. sinnamariense could
express acid phosphatase activity in pure culture does not necessarily mean
that it could access unavailable P sources when in symbiosis. There is a
possibility that the phosphatases produced in symbiosis might be slightly
different from those produced in pure culture. But some researchers such as
Gianinazzi-Pearson and Gianinazzi (1989) showed that acid phosphatases
produced by ectomycorrhizal hyphae were the same as those produced by
the fungus in pure culture. Since phosphorus acquisition is one of the most
important functions of the ectomycorrhizal symbiosis, acid phosphatase
activity is of considerable interest in selecting strains for inoculation

experiments.
In view of the association of Gnetum plants with organic soils of low
phosphorus status, the demonstration that Scleroderma sinnamariense (the
ectomycorrhizal endophyte of Gnetum spp. in Cameroon) can produce
high acid phosphatase activity in organic environments with low P may
be of considerable importance in the establishment and growth of Gnetum
plants in tropical soil communities. In such an environment, absorption
of phosphorus would be enhanced by the mycorrhizal system. The
phosphatase released by the mycorrhizal fungal mantle and attached
mycelium ramifying through the soil would probably catalyze the hydrolysis
of complex phosphorus compounds found in the organic layer into more
readily absorbed forms. Organic acids produced by the fungus may lead
to the solubilization of insoluble P sources, which would be taken up by
the fungus and eventually translocated to the plant host. This improved
P nutritional status would make the plant more fit so as to better compete
for other limiting factors in its environment.
The results from Bechem (2011) also indicated that the nitrogen and
phosphorus status of the mycorrhizosphere is an important determinant
of acid phosphatase activity. For there to be any meaningful and concrete
conclusion, more of such studies are needed on tropical mycorrhizal fungi
species.
7. Ectomycorrhiza Formation
Scleroderma spp. have been reported generally to form white mycorrhizas
from clamped hyphae (Jeffries 1999). S. sinnamariense, however, has been
reported to form bright yellow mycorrhizas with Gnetum spp. (Bechem
and Alexander 2012a, Onguene and Kuyper 2001, Ingleby 1999). The
same fungus was reported to form dark brown EM with Shorea leprosula
Miq. but the hyphae in the rhizomorphs linking the sporocarps to the EM
were said to be bright yellow (Lee et al. 1997). This fungus seems to have a
narrow host range and has been associated only with Gnetum, Parashorea,
and Shorea. EM synthesis with rooted Gnetum cuttings under controlled
conditions was possible with the use of spores, soil, and mycorrhizal tips
as inoculum (Bechem and Alexander 2009).
8. Growth Responses and Nutritional Benefits to the Host

Plant
Species of Scleroderma have been used to improve on the early growth of
some tree species and growth responses have been positive both in screen
house and field trials (Jeffries 1999, Castellano 1996, Bâ et al. 2002). Most
of the studies reported used species other than S. sinnamariense except for
Dodd et al. (1996) who used this fungus to inoculate seedlings of Parashorea
malanonan. Height increments of over 22% were observed on inoculated
seedlings when compared to uninoculated plants.
Bechem and Alexander (2012b) carried out an experiment in which they
evaluated the effects of mycorrhization and the addition of P (30mg/kg and
300mg/kg) on the growth of rooted Gnetum cuttings. It was noted that the
number of new EM tips decreased with an increase in P level implying that
the rate of colonization decreased with increasing P concentrations. This
finding demonstrated that the level of fertilization that can be applied to
Gnetum plants in farms and plantations is of critical importance and the
type of fertilizer must be carefully chosen so as to allow for the continuous
development and survival of the mycorrhizas. Ectomycorrhization led to an
increase in shoot, root and total plant dry weights, root: shoot ratio, and shoot
elongation as well as the total number of new leaves. Ectomycorrhization
of Gnetum in this study therefore led to improved access to added P. Non-
ectomycorrhizal plants in the said study responded to phosphorus addition
by increased root and total plant dry weights, shoot elongation and total
number of new leaves. An increase in foliar P concentration was also
demonstrated, thus suggesting that P availability may have been one of the
limiting factors under these experimental conditions. In a previous study,
Lee and Alexander (1994) had demonstrated that non-mycorrhizal Hopea
odorata responded to P addition by showing an increase in shoot dry weight
and P concentration. They also observed that ectomycorrhizal colonization
increased shoot P concentration and dry weight by about 100% as compared
to those of uncolonized plants growing on P amended soil.
Both P addition and EM colonization led to an increase in total
foliar P. Mycorrhizal colonization increased foliar N concentrations at
all P concentrations. Foliar N concentrations of mycorrhizal plants were
higher when P was added, but this was not the case for non-mycorrhizal
plants. There was a linear relationship between percent ectomycorrhizal
colonization and total foliar N at low P concentrations but this was significant
(P< 0.05) only in absence of added P. However, there was no evidence of an
association between total foliar P and percent colonization.
The relationship between ectomycorrhizal colonization and total foliar
N at low P concentration in the study by Bechem and Alexander (2012b)
gives an indication of the functional relationship between EM colonization
and nutrient (N, P) acquisition by plant species of the tropical rain forests.
Although ectomycorrhization and P addition had significant effects on
total foliar P in the said study, the analysis of data did not show any linear
relationship between percent colonization and foliar P. This observation
was inconsistent with that of Moyersoen et al. (1998) in which a clear
relationship between P uptake and mycorrhizal colonization was observed

in Tetraberlinia moreliana, an ectomycorrhizal tree species from the lowland
tropical forests of Cameroon.
The linear relationship between total foliar N and ectomycorrhizal
colonization at low P concentration suggested that Gnetum would be
partially dependent on EM for the uptake of N in limiting situations.
Nonetheless, N is more available for uptake by trees in most lowland
tropical rain forests (Vitousek and Sandford 1986, Martinelli et al. 1999). It is
probable that in situations where there is a seasonal fluctuation of nutrients,
mycorrhizally-assisted N capture would be important (Hodge 2003). EM
colonization increased total foliar P at both low and high P availability,
an observation similar to that of Moyersoen et al. (1998) in which EM
colonization was observed to increase P uptake of T. moreliana at both low
and high P availability.
The increase in shoot elongation observed on mycorrhizal plants was
probably not due to improved P nutrition only. This was because there was
a response at all P concentration. It was therefore possible that there were
additional factors brought about by EM colonization besides improved P
nutrition.
This experiment showed that Gnetum plants do respond positively to
ectomycorrhizal colonization and that the response may not be attributed
solely to increased uptake of nutrients including phosphorus and nitrogen. It
also demonstrated that mycorrhization and fertilization would be necessary
for the establishment of Gnetum farms and plantations.
9. Conclusion
From the findings of this researcher and her collaborators, there is further
evidence that some tropical ectomycorrhiza fungi are able to use amino acids
and some proteins as a sole N source. The stimulatory effect of peptone
was interesting, although the implication of this observation in nature is
to be determined. There was evidence that the fungus was able to utilize
organic P and N. It expressed both extracellular and cell-bound phosphatase
activity, with activity being higher in the absence of inorganic P. There is
therefore a possibility that the fungus would be able to access some organic
P sources in nature when inorganic P is limiting, an ability which would
be beneficial to the host plant. It is conclusive that ectomycorrhizas are an
integral part of Gnetum’s physiology. Any useful and effective conservation
strategy therefore would have to incorporate the diversity and biology of
the fungal partner.
Acknowledgements
I appreciate the assistance from colleagues at the Department of Botany and
Plant Physiology, University of Buea for their support. I am indebted to Dr
Egbe Enow Andrew for reading this manuscript. I thank the anonymous
referees for their valuable comments on this study, and Krista L. McGuire
and Caitlyn Gillikin for improving the language.
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CHAPTER
9
Alleviation of Salt Stress by
Scleroderma bermudense in
Coccoloba uvifera Seedlings in
the French West Indies
Amadou Mustapha Bâ,1,2,3,* Raymond Avril,1
Eric Bandou,1 Seynabou Sène,2 Robin Duponnois,3 Régis
Courtecuisse,4 Samba Sylla2 and Abdala Diédhiou2
1. Introduction
More than 800 million ha of land throughout the world is affected by
salt levels. The soil types most affected by excess salt are found in 30% of
Australian soils (Flowers and Colmer 2008). Excess salt in soils can have
two origins: (i) natural causes, i.e., weathering of parent rocks, deposition
of oceanic salts carried in wind and rain (10 mg/kg of NaCl would deposit
1
Laboratoire de Biologie et Physiologie Végétales (LBPV), Faculté des Sciences Exactes et
Naturelles, Université des Antilles et de la Guyane, BP. 592, 97159, Pointe-à-Pitre, Guadeloupe,
France.
2
Bel Air, BP. 1386, CP. 18524 Dakar, Sénégal.
3
Laboratoire des Symbioses Tropicales et Méditerranéennes (LSTM), UMR113, Campus de
Baillarguet, A10/J, 34398 Montpellier, Cedex 5, France.
4
Département de Mycologie et de Botanique, Université de Lille, BP. 83, F-59006 Lille cedex,
France.
*Corresponding author: amadou.ba@ird.fr, amadou.ba@univ-ag.fr
Alleviation of Salt Stress by Scleroderma bermudense by Coccoloba uvifera 165
10 kg/ha of salt for each 100 mm of rainfall per year); (ii) human causes,
i.e., excess amounts of fertilizers, irrigation with brackish water (20% of
irrigated lands in the world are affected), and (iii) increased sea level caused
by global climate changes. Subsequently, two main groups of salted soils
exist: saline soils (soluble salts) and alkaline sodic soils (adsorbed sodium).
Saline soils affect plant growth without changing the physical properties
of soil. Soil is classified as saline when electrical conductivity ECe is ≥
4 dS m–1 (~40 mM or 2.4‰ NaCl) and generates an osmotic pressure of
0.2 MPa. Saline soils cover 397 million ha worldwide (FAO 2005). Sodic
soils affect plant growth and soil structure by saturating the adsorption
complex by Na+, which causes soil structure to evolve to an unstable state
of dispersion. This can be overcome through the flocculant power of Ca++
from gypsum (CaSO42H2O). Soil is classified as sodic when Exchangeable
Sodium Percentage (ESP) is ≥ 15% of cation exchange capacity. Sodic soils
cover 434 million ha worldwide (FAO 2005).
Plants respond to salinity stress at two levels: (i) osmotic stress (rapid
response) due to the increase in external osmotic pressure (reduction of
shoot growth rate); this is the osmotic or water-deficit effect of salinity;
(ii) Salt-specific effect (slow response) due to the accumulation of toxic
ions (Na+, Cl–) in leaves (decrease of shoot growth rate only for sensitive
plant). This is the toxic effect of salinity (Munns 2005). The osmotic stress
not only has an immediate effect on growth, but also has a greater effect
on growth rates than the ionic stress. To overcome salt-stress problems, it
is possible to select salt-tolerance plants, to use biological processes such
as mycorrhizal interactions, or to desalinate soil by leaching excessive
salts (Parida and Das 2005). The desalinisation of soils is not economically
viable for sustainable agriculture. Although the results are somewhat
variable, salt tolerant plants develop mechanisms such as osmotic and ionic
homeostasis. Osmotic homeostasis consists of osmotic adjustment through
the active accumulation of compatible solutes (e.g., K+, sucrose, proline,
glycine betaine) in the cytosol to maintain turgor of cells (e.g., stomatal
opening) that would otherwise dehydrate. Ionic homeostasis consists of
Na+ exclusion by roots through transporters (e.g., type SOS1) to ensure that
Na+ does not accumulate to toxic concentrations in leaves, or Na+ and Cl–
compartmentation through transporters (e.g., type NHX) in the vacuoles
to avoid toxic concentrations within the cytoplasm.
The majority of terrestrial plants form symbioses with mycorrhizal fungi
(Smith and Read 2008). There is considerable evidence that mycorrhizal
fungi can improve plant growth and nutrition in soils subject to a range
of saline stress (Giri and Mukerji 2004, Parida and Das 2005, Bandou et al.
2006, Bois et al. 2006). Mycorrhiza can help moderately salt-tolerant plants
prevent Na+ and Cl– translocation to shoot and leaf tissues, and improved
salt tolerance after mycorrhizal colonization may also result from more
efficient P uptake by mycorrhizal plants in P-deficient soils, leading to

increased growth and the subsequent dilution of toxic ions effects (Juniper
and Abbott 1993, Giri and Mukerji 2004). In this respect, ectomycorrhizal
(ECM) fungi contribute to render trees more resistant to salt stress than
non-ECM plants. The identification of salt-tolerant ECM fungi could help
enhance tree survival and growth in salted soils (Bois et al. 2006, Chen et
al. 2001, Tang et al. 2009). Many studies have been conducted to test salt
tolerance of ECM fungi in axenic cultures and little is known about the
salt tolerance of the ECM association (Kernaghan et al. 2002). Chen et al.
(2001) screened eighteen isolates of Pisolithus for resistance to NaCl during
growth in axenic cultures and found that three of them are broadly resistant
to salinity and may represent useful ECM inoculants for the outplanting
of compatible, salt-resistant host trees at saline sites. Levels of mannitol,
glycerol, and trehalose, and some other metabolites, such as the proline
amino acid, were observed to increase in fungi under osmotice stress (Shen
et al. 1999). Bois et al. (2006) tested the effects of three selected ECM fungi
for their in vitro tolerance to excess NaCl, on the growth of Picea glauca and
Pinus banksiana seedlings over a gradient of NaCl concentration. Although
the different ECM fungi altered the physiological response of the host in
different ways (e.g., organic osmolyte accumulation, water content, Na+
accumulation and allocation), inoculation with salt-stress tolerant ECM
fungi increased growth and reduced the negative effects of excess NaCl (Bois
et al. 2006). Paxillus involutus was salt tolerant, showing biomass increases
in media containing up to 500 mM NaCl after 4 wk of growth (Langenfeld-
Heyser et al. 2007). Whole salt-sensitive poplar hybrid Populus x canescens
performance was positively affected by inoculation with P. involutus because
total biomass was greater and its leaves accumulated less Na+ than non-
mycorrhizal plants (Langenfeld-Heyser et al. 2007).
Coastal vegetation usually consists of low numbers of plant species that
are specifically adapted to environments that experience frequent stress
such as intermittent salinity, drought, intense insolation, instability of sand,
low fertility, etc. In this context, Coccoloba uvifera (L.) L. (Polygonaceae), also
named seagrape, often grows in pure stands within well-drained sandy
soils that are slightly to moderately alkaline (Parrota 1994). It is considered
a drought-hardy and non-halophyte woody plant relatively tolerant to
salt. This tree is naturally associated with ECM fungi and one of them,
Scleroderma bermudense, alleviates saline stress and stimulates growth and
improves nutrition of seagrape seedlings (Bandou et al. 2006). Here, we
examined (i) how the salinity affected the ECM communities associated
with three species of Coccoloba (C. uvifera, C. swartzii and C. pubescens) along
a salinity gradient in Guadeloupe and Martinique (Lesser Antilles); (ii) the
ability of ECM fungi to grow in pure culture and to alleviate salt stress in
seagrape seedlings over a range of NaCl levels.
2. Distribution of Coccoloba uvifera, C. swartzii and

C. pubescens, and their Putative Ectomycorrhizal Fungi
along a Salinity Gradient
The genus Coccoloba belongs to the family of Polygonaceae. This plant
family has two ECM genera, Polygonum and Coccoloba, including 300 and
400 species, respectively (Howard 1988). Coccoloba includes several species
that predominate in South America, the largest number of species being
in Brazil. Of the Coccoloba species found in the Caribbean, ten species
have been identified, including six species in Guadeloupe and Martinique
(Table 1). Of these species of Coccoloba, three have been particularly
encountered: C. uvifera, C. pubescens and C. swartzii. C. uvifera is the most
known and widespread species in the Caribbean basin. It is an important
tree for edible fruits, ornamental plantings, and coastal windbreak along
Caribbean beaches and roadsides. Seagrape is the dominant tree species in
beach canopies. It is encountered in rocky, calcareous and volcanic sandy
soils which are alkaline and poor in terms of P, N and K. C. pubescens, also
named grandleaf (diameter 2.5–45 cm) seagrape, is a tree distributed in the
xero-mesophytic forests in Guadeloupe and Martinique (Lesser Antilles).
C. swartzii is a tree encountered in the degraded xerophytic forests in
Guadeloupe and Martinique. C. pubescens and C. swartzii are often found
together in Martinique. Given the natural distribution of the three species
of Coccoloba along a salinity gradient, it could be inferred that C. uvifera is
more adapted to salinity than the other two Coccoloba species (Fig. 1). In
fact, only mature trees of C. uvifera with abundant seedling recruitment
were well developed within areas far from the sea where salt levels were
low (0–2‰), whereas mature trees near the sea (2–15‰) were shunted
and seedlings were almost absent (Fig. 1). We observed that mature trees
of seagrape adjacent to the sea were smaller than mature trees growing far
from the sea. Measurements were not made to estimate their difference
in terms of sizes. Nevertheless, we noted that the number of mycorrhizal
and non-mycorrhizal root tips was nearly three times higher on mature
trees growing in salted soil at 0–2‰ than those at 2–15‰, suggesting a
negative impact of salinity on root development of mature trees of C. uvifera.
Naturally regenerating seedlings under the canopy of mature trees were
high in low salinity levels, whereas very little occurred under the mature
trees growing near the sea. This suggests a negative impact of salinity on
naturally regenerating C. uvifera seedlings. Salinity is considered to be one
of the most significant environmental factors limiting plant germination,
growth, and productivity (Flowers and Colmer 2008). Salinity reduces the
availability of water and increases toxic ion concentrations in soils. This
could affect imbibition, germination, and root elongation. However, the
Table 1. Diversity of Coccoloba species in the Caribbean basin (nd= not determined).
168
Species Synonymous Distribution Country Habitat Reference

Coccolobaascendens Uviferaascendens Fairly Guadeloupe, Martinique, South America, Basaltic hills and Fournet (2002)
common Trinidad, Dominica, St. Lucia, Grenade forests
C. caravellae none rare Endemic to Martinique nd Fournet (2002)
C. coronata C. caribaea nd Central America, Trinidad and Tobago, South nd Howard (1988)
America, Grenadines, Grenade
C. diversifolia C. laurifolia nd Florida, Mexico, Central America, Greater nd Howard (1988)
Antilles, Antigua
C. dussii none Fairly Guadeloupe, Trinidad, St. Lucia, St. Vincent, nd Fournet (2002)
common Grenade Howard (1988)
C. krugii Uviferakrugii rare St. Martin, Bahamas, Greater Antilles, Anguilla, nd Fournet (2002)
Guadeloupe, St. Christophe, St. Kitts
C. pubescens C. grandifolia, Fairly Guadeloupe, Desirade, Marie-Galante, Coastal Fournet (2002)
C. antiguensis, common Martinique, Hispaniola, Porto Rico, Barbuda, mesophytic, Howard (1988)
C. bonfilsiana Antigua, Nevis, Montserrat, Dominica, St. Lucia, Basaltic or
Barbados calcareous hills
C. swartzii C. diversifolia, Common Bahamas, Greater Antilles, Central America, Basaltic hills. Fournet (2002)
C. barbadensis, South America, Antigua, Saba, St. Eustache, Degraded Howard (1988)
C. punctata, St. Christophe, St. Kitts, Nieves, Montserrat, xerophytic forest.
Coccolobensisdiversifolia, Guadeloupe, Dominica, Marie-Galante,
C. neglecta Martinique, St. Lucia, St. Vincent, Grenadines,
Grenade, Barbados
C. uvifera Polygonumuvifera Very Bahamas, Greater Antilles, Mexico, Central Sandy coast and Fournet (2002)
common America, South America, Anguilla, Antigua, Saba, sometimes rocky Howard (1988)
St. Eustache, St. Bartelemy, St. Christophe, St.
Kitts, Nieves, Montserrat, Guadeloupe, Dominica,

Marie-Galante, Martinique, St. Lucia, St. Vincent,
Grenadines, Grenade, Barbados
C. venosa C. nivea Quite rare St. Martin, St. Bartelemy, Guadeloupe, Les Coastal dry sand, Fournet (2002)
Saintes, Marie-Galante, Martinique, Hispaniola, tuff or basalt. Howard (1988)
Porto Rico, Trinidad and Tobago, Venezuela, Calcareous ills
Antigua, Saba, St. Eustache, Montserrat, in Pterocarpus
Dominica, St. Lucia, Grenadines, Grenade, forest behind the
Barbados mangrove swamp
Fig. 1. Distribution of three Coccoloba species (Coccoloba uvifera, C. swartzii and C. pubescens) and
their putative ectomycorrhizal fungi along a salinity gradient in Martinique and Guadeloupe
(Lesser Antilles).
way in which salinity exerts its influence on these vital processes through
an osmotic effect or a specific ion toxicity is still not resolved (Katembe et
al. 1998).
Table 2 shows the diversity of putative ECM fungi associated with
different Coccoloba species in the Greater and Lesser Antilles. Some of them
were common with those we collected in Martinique and Guadeloupe. We
collected 19 putative ECM fungi, of which 6 were found under C. uvifera
and 13 under C. pubescens and C. swartzii along a gradient of salinity
(Fig. 1) (Bessard et al. 2008). The 11 epigeous (Russula, Lactarius, Fistulinella,
Scleroderma, Boletelus, Clavaria, Clavulina, Coltricia, Limacella, Ramaria and
Thelephora) and 2 hypogeous (Austrogautieria and Melanogaster) fungal
species were collected under C. pubescens and C. swartzii in areas without
salt, whereas 8 different fungal species were collected in C. uvifera in salted
sandy soils (0–2‰ of salinity) (Fig. 1). Diversity of ECM fungi increased
when the salinity was low. It could be structured by salinity, host preference,
and/or charcteristics of soils along the salinity gradient. Also, abundance
of sporocarps fruiting from under mature trees and seedlings in C. uvifera
coastal forest were assessed over the salinity gradient (Fig. 2). Six species
of sporocarps were identified in low salinity levels (0–2‰) and only one
of them (S. bermudense) fructified in high salinity levels (2–15‰). Thus,
sporocarps of S. bermudense were more abundant in high salt levels than
Table 2. Putative ectomycorrhizal fungi associated with different Coccoloba species in the Greater and Lesser Antilles.
170
Fungal family Fungal species Host plant Country Reference

Amanitaceae Amanitacystidiosa C. uvifera Anguilla, Guana (Virgin Islands), Miller et al. 2000
PuertoRico
A.antillana C. diversifolia, C. pubescens Martinique, Trinidad Pegler 1983
A .arenicola C. uvifera PuertoRico, Guadeloupe Miller et al. 2000,
Bandou 2005
A .microspora C. uvifera PuertoRico Miller et al. 2000
A. craseoderma C. pubescens Martinique Pegler 1983
Boletaceae Austrogautieria sp. C. pubescens, C. swartzii Martinique Bessard et al. 2008
Boletusroborculus Coccoloba sp. Porto Rico Miller et al. 2000
Boleteluscubensis C. pubescens, C. swartzii Martinique Bessard et al. 2008
Fistulinellagloeocarpa C. diversifolia Martinique Pegler 1983, Bessard et
al. 2008
Limacella sp. C. pubescens, C. swartzii Martinique Pegler 1983, Bessard et
al. 2008
Melanogaster sp. C. pubescens, C. swartzii Martinique Bessard et al. 2008
Xerocomushypoxanthus C. uvifera Martinique Pegler 1983
X. coccolobae C. uvifera, C. pubescens, Martinique Pegler 1983
C. swartzii
X.guadelupae Coccoloba sp. Martinique, Guadeloupe, Dominica Pegler 1983
X.cuneipes C. uvifera Martinique Pegler 1983

Sclerodermabermudense PuertoRico, Guadeloupe, Martinique Guzman et al. 2004,
Bandou et al. 2006
S. stellatum Coccoloba sp., Cuba, Puerto Rico Kreisel 1971
C. uvifera
Scleroderma sp. C. swartzii, C. pubescens Martinique Bessard et al. 2008
Cantharellaceae Cantharelluscinnabarinus C. uvifera Martinique, Guadeloupe, PuertoRico, Pegler 1983, Bandou
Cuba 2005
Cortinariaceae Inocybe littoralis C. uvifera Martinique, Guadeloupe Pegler 1983, Bandou
2005
I.xerophytica C. uvifera Guadeloupe Pegler 1983, Bandou
2005
Gomphaceae Ramariacyanocephala C. swartzii, C. pubescens Martinique Bessard et al. 2008
Hymenochaetaceae Coltricia sp. C. swartzii, C. pubescens Martinique Bessard et al. 2008
Inocybaceae Inocybe littoralis C. uvifera Martinique, Guadeloupe Pegler 1983, Bandou
2005, Bessard et al. 2008
I. xerophytica C. uvifera Martinique, Guadeloupe Pegler 1983, Bandou
2005, Bessard et al. 2008
Russulaceae Lactariuscoccolobae C. uvifera Anguilla, Guana (Virgin Islands), Miller et al. 2000
Puerto Rico
L. caribaeus C. pubescens, C. diversifolia Martinique Pegler 1983
L. castaneibadius C. diversifolia, C. swartzii, Martinique Pegler 1983, Bessard et
C. pubescens al. 2008
L. hygrophoroides C. pubescens Martinique Pegler 1983
L. murinipes Coccoloba sp. Martinique Pegler 1983
L. nebulosus C. pubescens, C. diversifolia Martinique Pegler 1983
Russulacremeolilacina C. diversifolia, C. uvifera Martinique, Guadeloupe Pegler 1983, Bandou
2005
R. brevipes C. swartzii Martinique Pegler 1983
R. littoralis C. uvifera Martinique, Virgin Islands, PuertoRico
R. martinica C. diversifolia Martinique Pegler 1983
R. metachromaticae C. uvifera Martinique Pegler 1983
Thelephoraceae Thelephora sp. C. swartzii, C. pubescens Martinique Bessard et al. 2008
70
60
Number of sporocarps
Salted soil (2-15‰)
50 Salted soil (0-2‰)
40
30
20
10
Fig. 2. Number of fungal species described by examining epigeous sporocarps collected in an

ECM sampling plot within a period of 8 months in a C. uvifera coastal forest in Guadeloupe.
in low salt levels. Only Scleroderma bermudense fructified at salinity levels

above 15‰. This suggests that fruiting of S. bermudense could tolerate
salinity stress. The productivity of S. bermudense was greater than of any
other species of ECM fungi, which permitted it to occupy soils with high salt
levels in coastal forests (Fig. 2). It is well known that fungi require localized
outbreaks of rain to provide the soil with sufficient water for them to develop
and fructify. Their fruiting depends on rainfall distribution and intensity.
The lack of available soil water due to salinity could have a detrimental
effect on the fruiting of all the fungal species except S. bermudense. The
species accumulation curves for sporocarps in both salt levels tended
to level off, indicating that most ECM fungal taxa were detected in our
sampling effort (Fig. 3).
3. In vitro Selection of ECM Fungi for use in Saline Soils

Among the sporocarps collected from under the three Coccoloba species,
only 3 of them (S. bermudense isolate GUA 09.06.06, Scleroderma sp. isolate
CLMART07.157 and Melanogaster sp. isolate CLMART06.004) were
successfully isolated and cultivated in pure culture. Two fungal species
(Scleroderma sp. and Melanogaster sp.) were from a non-salt-affected soil, and
one (S. bermudense) was from salt-affected soil along the salinity gradient
(Fig. 1). S. bermudense grew better in pure culture than Melanogaster sp. and
Scleroderma sp. whatever the levels of NaCl (Fig. 4). S. bermudense showed
significant peaks in radial growth at two concentrations of NaCl (100–200
mM). Other fungi showed an apparent general decline in radial growth with
4
Number of species
0
1 15 29 43 57 71 85 99 113127141155169183
Number of sporocarps
Fig. 3. Species accumulation curve for sporocarps in a C. uvifera coastal forest in Guadeloupe.
Dotted lines indicate 95% CI.
9 f S. bermudense
f Melanogaster sp.
8
Scleroderma sp.
7 e
e
Radial growth (cm)
6 e
5
4 d cd
3 bc
ab
2 a a
ab ab a a
1
0
0 100 200 300 500
NaCl (mM)
Fig. 4. Radial growth of S. bermudense, Melanogaster sp. and Scleroderma sp. in the presence of
NaCl at a range of concentrations in axenic culture for 1 mon (Two-ways ANOVA, P<5%).
increasing NaCl concentration, although the decline was not significant with
incresasing NaCl up to 100 mM. Similar results were found for biomass
yield for all parameters (Fig. 5). Selection of the salt concentration ranges
used in the present study was based on field measurements of soil salinity
(Bandou 2005, Bandou et al. 2006). Our data indicated that S. bermudense
were broadly resistant to NaCl at concentrations encountered in the saline
soils of littoral forests. This fungus could also require Na+ for growth as
their radial growth and biomass yield increased at two concentrations
(100–200 mM) relative to the zero NaCl treatment. Chen et al. (2001) also
found that some isolates of Pisolithus showed clear peaks in biomass yield
at concentrations in the range of 25–100 mM NaCl. Much of the information
on fungal salt tolerance comes from studies on aquatic fungi (Clipson
and Jennings 1992). While some marine fungi require Na+ for growth i.e.
one fungal species growsat 12.8 M NaCl in the Great Salt Lake, terrestrial
fungi appear to have less of a requirement. The observed peaks could also
reflect responses to lowered external water potential generated by NaCl
in the medium. This hypothesis is supported by similar peaks observed in
experiments in which the growth of ECM fungi was measured at different
0.25
S. bermudense
Melanogaster sp.
h
0.2 Scleroderma sp.
gh
Dry mass (g)
0.15
f
f f
0.1
e
de bcd
cde cd
0.05 abc
ab
a a a
0
0 100 200 300 500
NaCl (mM)
Fig. 5. Biomass yield of S. bermudense, Melanogaster sp. and Scleroderma sp. in the presence of
external water potentials through application of the organic osmotica such

as polyethylene glycol (Jennings 1995).
It is well known that ECM fungi tolerate high osmotic potential and toxic
metal ions by combining compartmentalization of toxic ions and production
of polyols that acts as nontoxic osmoregulators (Clipson and Jennings 1992,
Chen et al. 2001, Bois et al. 2006). Such an ability may explain the observed
growth of ECM fungi under conditions of high external osmotic stress,
and may in part explain the resistance to salt that we have identified in
S. bermudense. Interestingly, S. bermudense produced more fungal biomass
and accumulated more Na+ than Melanogaster sp. and Scleroderma sp. under
salt stress, showing that S. bermudense was more tolerant to high NaCl
concentrations (Figs. 5 and 6). Also, compatible osmolytes (e.g., proline)
accumulated in the cytoplasm of S. bermudense to counter excess ions in
the vacuole (Fig. 7). The results of this study showed that S. bermudense
accumulated more Na+ than the other ECM fungi, which means a physical
barrier for plant development supported by S. bermudense was stronger
than those supported by the other fungi. S. bermudense has been selected
to alleviate salt stress in seagrape seedlings.
9000
S. bermudense
8000 e
Melanogaster sp. de
Scleroderma sp. de
7000 de de
d d
6000
Na (ppm)
5000
c
4000 bc
3000
ab
2000 a
a
1000
a a a
0
0 100 200 300 500
NaCl (mM)
Fig. 6. Na contents of S. bermudense, Melanogaster sp. and Scleroderma sp. in the presence of
1200
S. bermudense
Proline concentrations (μg g-1 DM)

Melanogaster sp. c c c
1000
Scleroderma sp.
800 b
a
600 a a
a a a
a a
400 a a a
200
0
0 100 200 300 500
NaCl (mM)
Fig. 7. Proline contents of S. bermudense, Melanogaster sp. and Scleroderma sp. in the presence of
4. Scleroderma bermudense Alleviates Salt Stress in

Seagrape Seedlings
The beneficial effects of S. bermudense on seagrape seedlings occurred not
only during NaCl stress but also under non-stressed conditions, suggesting
that the ECM effect in improving plant growth was not a specific process
induced by saline stress (Table 3). These results recall observations on the
beneficial effect of AM fungi on plant growth under salt stress conditions
(Al-Karaki et al. 2001, Yano-Melo et al. 2003, Giri and Mukerji 2004, Tian
et al. 2004). Although ECM colonization by S. bermudense was reduced
with increasing NaCl levels, ECM dependency of seagrape seedlings was
increased. This suggests that the activity of the ECM symbiosis between
seagrape seedlings and S. bermudense was strengthened under saline stress
conditions once the symbiosis was established.
In our study, ECM plants showed considerably reduced Na and Cl
uptake into roots as compared to non-inoculated plants (Figs. 8 and 9).
Moreover, ECM inoculation of plants lowered Na and Cl translocation to
shoot and leaf tissues. The marked differences in Na and Cl concentrations
between ECM and non-ECM plant tissues suggest that the mechanisms of
enhanced tolerance to salt stress in ECM plants were related to exclusion of
Table 3. Effects of inoculation with Scleroderma bermudense and NaCl levels on growth variables
and ectomycorrhizal (ECM) colonization in seedlings of Coccoloba uvifera at 3 mon (p<1%)
(Bandou et al. 2006).
ECM status NaCl levels Height Total biomass EM EM colonization

(mM) (cm) (g) dependency (%) (%)
Non-inoculated 0 6.2 b 1.4 b - 0.0 a
200 5.0 a 0.7 a - 0.0 a
350 5.5 a 0.6 a - 0.0 a
500 5.1 a 0.6 a - 0.0 a
Inoculated 0 7.2 d 3.1 f 55.8 a 89.4 d
200 6.8 cd 2.6 e 71.7 c 66.6 c
350 7.0 d 2.3 d 75.2 c 46.3 b
500 6.2 bc 1.9 c 69.5 c 41.6 b
0.16
Stem
g
0.14 Leaf fg g
fg fg
0.12 Root f
0.1
e
P (%)
e
0.08 de e
de
bc cd
0.06
bc bc bc
bc bc
0.04 ab ab ab
ab ab
a
0.02
0
0 200 350 500 0 200 350 500
Controls Inoculated
NaCl (mM)
Fig. 8. Changes in phosphorus (P) concentrations in the leaves, shoots, and roots of C. uvifera
seedlings in response to NaCl levels and inoculation with the ectomycorrhizal (ECM) fungus
S. bermudense at 3 mon (Bandou et al. 2006).
Na and Cl by the ECM plants growing in the NaCl treatments. One of the
main mechanisms for enhanced salinity tolerance in ECM plant has also
been proposed to be the exclusion of Na and Cl (Giri and Mukerji 2004,
Tian et al. 2004).
ECM plants had higher concentrations of P than non-inoculated plants
particularly under salt stress conditions. This could be due to the extended
network of hyphae beyond the depletion zone around roots that acquire
more P and therefore suppress the adverse effect of salinity stress. These
results suggest that S. bermudense can alleviate the deleterious effects of NaCl
stress on plants by mechanisms which may also be related to improvement
of Puptake (Fig. 8).
If Na and Cl are sequestered into plant vacuoles, K and organic solutes
(e.g., proline) should accumulate in the cytoplasm and organelles to balance
the osmotic pressure of intravacuolar ions (Munns 2002). Na and Cl were
not accumulated under salt stress and found at low concentrations in ECM
seedlings of C. uvifera when compared to controls (Figs. 9 and 10).
It was also noteworthy that K concentrations were higher in leaves of
ECM seagrape seedlings than in controls at all salinity levels. S. bermudense
seemed to protect seedlings by increasing the K/Na ratio to maintain
higher turgor under salt stress (Figs. 11 and 12). It is well known that
high concentrations of K and low concentrations of Na can play a major
role in salt stress tolerance by regulating stomatal openings and osmotic
potential in the vacuoles (Zhu 2003, Munns 2005). Some studies indicated
that plants subjected to increased salinity accumulated less K because
Na uptake competed with the uptake of K, leading to decreased K levels
(Larcher 1995, Parida and Das 2005). Reductions in Na and Cl uptake and
2.5
i
i Stem
2 Leaf
i
Root
h
1.5 h
Na (%)
1
g
efg fg d-g fg fg
efg
0.5 a-e a-e b-f
b-f c-f
abc abc a-d a-d
ab a
0
0 200 350 500 0 200 350 500
Controls Inoculated
NaCl (mM)
Fig. 9. Changes in sodium (Na) concentrations in the leaves, shoots, and roots of C. uvifera
5 i
Stem
i
Leaf
4 i
Root
Cl (%)
h
3
h h
h gh gh
2 fg
d-f ef
c-f b-e b-e
1 a-e
abc abc b-e b-e
abc ab ab a
0
0 200 350 500 0 200 350 500
Controls Inoculated
NaCl (mM)
Fig. 10. Changes in chloride (Cl) concentrations in the leaves, shoots, and roots of C. uvifera
2,5
Stem
Leaf j i ij
2
Root
h
1,5
K (%)
efg
fg g efg
1 efg d-g efg c-g efg
c-g fg
a-d a-e abc b-g b-g
a ab a-d
b-f
0,5
0
0 200 350 500 0 200 350 500
Controls Inoculated
NaCl (mM)
Fig. 11. Changes in potassium (K) concentrations in the leaves, shoots, and roots of C. uvifera
24
22 Stem a
20 Leaf
18
Root
K/Na (%) 16
14
12
ab
10
c
8
6
d
d de
4 de def
de de de de de
def e
2 def def def def e e
f f f
0
0 200 350 500 0 200 350 500
Controls Inoculated
NaCl (mM)
Fig. 12. Changes in ration K/Na concentrations in the leaves, shoots, and roots of C. uvifera
enhanced P and K nutrition may be significant in helping the ECM plants

to grow under saline conditions (Table 4). There were significant negative
correlations between Na and Cl concentrations in the leaves, shoots, and
roots, and production of biomass (Table 4). The exclusion of Na and Cl
could be the main mechanism whereby mycorrhiza protected seagrape
seedlings against salinity.
The contribution of ECM fungi to plant growth in stressed soil is not
limited to the improvement in mineral nutrition but also to the changes
in the morphological characters of the leaves and the water status of
plants (Augé 2001). Measurements of minimal leaf water potentials are
considered informative on evapotranspiration to assess the water status of
plants (Larcher 1995). S. bermudense improved the water status of seagrape
Table 4. Correlation coefficients (r) between K, P, Na, Cl, and proline, and total biomass
production (Bandou et al. 2006).
a a b
Inoculated Non-inoculated Inoculated plus non-inoculated
K 0,65* 0.50 0.91*
P 0.85* 0.25 0.55*
Na –0.70* –0.94* –0.74*
Cl –0.71* –0.91* –0.79*
Proline 0.95* –0.93* 0.94*
a b
* Significant (p<1%); 10 df; 22 df
seedlings despite their higher evaporative leaf surface (Table 5). Also, it is
noteworthy that leaf proline content was higher in leaves of ECM seagrape
seedlings than in the controls at all salinity levels (Fig. 13). S. bermudense
seemed to also protect seagrape seedlings by increasing the proline content
to maintain higher turgor under salt stress.
Table 5. Effects of inoculation with Scleroderma bermudense and NaCl levels on the number of
leaves, leaf area, and minimal leaf water potential in seedlings of Coccoloba uvifera at 3 mon
(p<1%) (Bandou et al. 2006).
ECM status NaCl levels Number of Leaf area (cm2) Minimal leaf water
(mM) leaves potential (-bars)
Non-inoculated 0 3.60 d 33.7 b 9.2 e
200 2.90 c 18.7 a 13.4 bc
350 2.30 b 15.6 a 14.0 b
500 1.90 a 15.7 a 15.0 a
Inoculated 0 4.10 e 80.9 e 5.9 f
200 4.20 e 76.4 d 11.1 d
350 3.80 de 68.9 d 12.2 cd
500 3.60 d 56.05 c 15.50 a
900 d
Leaf proline content (mmol g DW-1)
800
700
600
c
500
400
300 b
200
a a a
100 a a
0
0 200 350 500 0 200 350 500
Controls Inoculated
NaCl (mM)
Fig. 13. Changes in proline concentrations in the leaves of C. uvifera seedlings in response
to NaCl levels and inoculation with the ectomycorrhizal (ECM) fungus S. bermudense at
3 mon.
5. Conclusion
Salinity had a negative impact on growth of mature trees and naturally
regenerating seedlings of C. uvifera. Salinity could contribute to the
structuration of the three Coccoloba species as well as their associated ECM
fungi. The data from the present study also suggest that S. bermudense possess
considerable resistance to salinity. Also, seagrape tolerance to salt stress was
considerably enhanced by S. bermudense. Reductions in Na and Cl uptake
and enhanced P nutrition may be significant in helping the ECM plants
to grow under saline conditions. Furthermore, the higher accumulation
of K in the leaves of ECM plants could render ECM plants more tolerant
to osmotic stress induced by exposure to salt. The overall improved water
status of seagrape seedlings related to ECM inoculation with S. bermudense
may also be a contributing factor. Further studies should be done to
elucidate the physiological mechanisms (e.g., stomatal conductance, water
use efficiency, transpiration, photosynthetic rate, osmotic adjustment) by
which S. bermudense improved the water status of seagrape seedlings. Our
results should be taken cautiously as the experiment was a greenhouse study
with 3 month-old seedlings. From an ecological point of view, the ECM
symbiosis may be an important factor for the establishment and survival
of seagrape stands under saline stress along the beaches of the Caribbean.
Moreover, transplanting ECM-inoculated seagrape to such degraded sites
not only benefits the individual plant but, more importantly, may result in
the development of ornamental plantings and coastal windbreaks along
Caribbean beaches and roadsides.
Acknowledgements
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441–445.
CHAPTER
10
The Contribution of
Ectomycorrhizal Fungal
Feedbacks to the Maintenance
of Tropical Monodominant
Rain Forests
Krista L. McGuire
1. Introduction
Tropical monodominant forests have been classically defined as areas of
forest where one tree species comprises >60% of the stems or basal area of
canopy trees (Torti et al. 2001). These forests occur throughout all tropical
regions (Fig. 1) and have been noted as interesting deviations from the
more extensive high-diversity rain forests (Leigh et al. 2004). A number of
mechanisms have been suggested to explain their existence and persistence
among the mosaics of highly diverse forest (Connell and Lowman 1989,
Hart 1990, Torti and Coley 1999), however, a single explanation for tropical
monodominance does not suffice (Torti et al. 2001, Peh et al. 2011). It
is now widely accepted that multiple mechanisms must be operating
simultaneously that lead to feedbacks, which enable a monodominant tree
species to persist and maintain site dominance (McGuire 2008).
Columbia University, Barnard College Department of Biology, USA.

Email: kmcguire@barnard.edu
Fig. 1. Map illustrating examples of monodominant and co-dominant forests with the tree
species or genus listed and the tree family in parentheses. This diagram is not an exhaustive
list of monodominant species, but it illustrates how monodominant forests are found in all
tropical regions.
One attribute that many monodominant trees have in common is

that they form ectomycorrhizal (ECM) associations. Most tropical tree
species form associations with arbuscular mycorrhizal (AM) fungi, but
there is a strong correlation between the dominance of tropical trees in
lowland rain forests and ECM associations (Torti et al. 2001). This pattern
is particularly interesting when considering how few tree species actually
form ECM associations. A recent synthesis found that nearly 75% of all
angiosperm plant species form AM associations, whereas only about 2%
form ECM associations (Brundrett 2009). Nonetheless, the majority of
tropical monodominant trees form ECM associations, suggesting that this
trait somehow contributes to the success of monodominant trees. However,
being ECM does not guarantee that a tree will become locally dominant, as
there are numerous examples of less abundant tree species that form ECM
associations, as well as monodominant trees that are AM. For this reason,
Torti et al. (2001) developed a list of traits that most monodominant trees
The Contribution of Ectomycorrhizal Fungal Feedbacks 187
share and suggested that there must be interactions among these traits that
together promote positive feedbacks leading to monodominance.
Community feedback models clearly demonstrate that negative plant-
soil feedbacks lead to plant coexistence and positive feedbacks lead to
dominance (Bever et al. 2010, Bever et al. 2012). These models account for
individual plant responses to differing soil microbes and then scale these
interactions to community-level patterns of plant diversity. The negative
plant-soil feedbacks are predicted to originate from plant-pathogen
interactions and the positive feedbacks are derived from plant-symbiont
interactions. These theoretical models have been empirically supported by a
number of studies, although the majority of the experimental work has been
done as pot studies and herbaceous plant manipulations. Very few tropical
studies have been performed and little is known about the functioning of
plant-soil feedbacks in undisturbed systems. Tropical ECM monodominant
forests are ideal model systems for exploring these feedbacks, as they offer a
natural diversity gradient that has not been significantly altered by human
manipulation. Since the outcome of undisturbed forest dynamics in these
systems is that of single species dominance, it can be inferred that positive
plant-soil feedbacks have outweighed negative feedbacks in these systems
(Bever et al. 2010).
For the current review, I will be focusing on a subset of tropical
monodominant tree species: those that form ectomycorrhizal (ECM)
associations in lowland, aseasonal rain forests, and are not associated
with extreme edaphic conditions or periodic disturbances. The reason for
focusing on ECM monodominant forests is that the mechanisms operating
to maintain monodominance likely include ECM-mediated feedbacks that
are unique to these systems, as the ecology, evolution, and physiology of
ECM fungi are different than for fungi that form arbuscular mycorrhizal
(AM) associations, the dominant mycorrhizal type in most diverse tropical
forests. As such, there are several attributes of ECM fungi that may confer
a competitive advantage to their host trees, particularly when growing in
nutrient-poor soils (Smith and Read 2008). For example, ECM fungi can
grow much larger than AM fungi due to the septate nature of their cells.
When ECM hyphae are broken, they can begin growing again from the last
septation, enabling mycelia to grow tens and sometimes hundreds of meters
in diameter. By contrast, AM fungi have coenocytic hyphae, so following
cellular damage they must resume growth from the root-fungal interface.
Consequently, AM fungi are not thought to grow mycelia that are more than
several centimeters in diameter. This discrepancy in size places ECM fungi
at an advantage in terms of nutrient foraging area and space occupancy.
In fact, where ECM fungi occur, they can comprise up to 90% of the fungal
biomass (Wallander et al. 2001). Also, the recent evolutionary divergence
of multiple ECM fungal lineages from decomposer fungi has implications
for functional differences in organic resource acquisition capabilities of AM

and ECM fungi, as many ECM fungi possess and express genes that are
involved in the degradation of organic polymers (Abuzinadah and Read
1986, Floudas et al. 2012). These advantages are realized for ECM trees in
terms of growth, nutrient uptake, drought tolerance, pathogen resistance,
and productivity.
This review explores empirical studies and hypothetical mechanisms
that explicitly incorporate the unique biology of ECM fungi and focuses
on a particular ECM monodominant system from Guyana, South America.
Finally, these various mechanisms are synthesized into a feedback
framework (Fig. 2) that integrates three non-mutually exclusive positive
mechanisms derived from the ECM symbiosis that may contribute to
monodominance: 1) ECM fungal mutualists preferentially benefit the ECM
host tree, but donot benefit heterospecific trees, 2) positive feedbacks from
ECM fungal mutualists are stronger than negative feedbacks from other soil
microorganisms, and 3) ECM fungal mutualists suppress positive plant-soil
feedbacks for heterospecific trees.
βA CB
CA DB
Fig. 2. Incorporation of monodominant and mixed forest trees into theoretical soil-feedback
models (adapted from Bever et al. 2010, 2012). The direction and strength of the plant-soil
feedbacks should correlate with the relative abundance of the tree species. The net pairwise
effect can be quantified by evaluating ((αA + βB) – (αB + βA)).
2. Dicymbe corymbosa as a Case Study

2.1 Study site
The ECM monodominant system used as a case study for exploring

these feedback mechanisms is located in Guyana, South America (Fig.
3; 5°4’ N, 59°58, 700 m a.s.l.). The forest in this region is intact, aseasonal
rain forest receiving approximately 4000 mm mean annual precipitation.
There are extensive stands of ECM, monodominant Dicymbe corymbosa
(Caesalpiniaceae) Spruce ex Benth. in the interior forming mosaics with
higher diversity, mixed forest in which D. corymbosa is not found. Dicymbe
corymbosa is an emergent tree species reaching 30–40 m and reaches
dominance levels of 70–90 percent where it occurs (Henkel 2003, McGuire
2008). This monodominant system was first described by Richards
(Richards 1952) and has been intensively studied by teams of ecologists and
mycologists since the early 2000s. Apart from the Gilbertiodendrondewevreii
monodominant forest in the Congo (Hart 1990, Torti et al. 2001), it has
become one of the most intensively studied ECM, monodominant forests
in the tropics.
Fig. 3. Map showing the ECM monodominant Dicymbe corymbosa forest and mixed forest
sites in Guyana.
2.2 Positive ECM-mediated feedbacks
Field experiments have revealed several positive feedbacks that are

seemingly conveyed by the ECM mutualism in D. corymbosa, but do not
benefit heterospecific trees. One of the major feedbacks is that of seedling
facilitation near to conspecific adult trees in the monodominant forest
(McGuire 2007a,b). That is, seedlings grow and survive better when planted
near other D. corymbosa trees compared to when they are planted near
heterospecific trees. To date, there have been two mechanisms explaining
the positive distance/density-dependence of D. corymbosa seedling
establishment. The first mechanism is that common mycorrhizal networks
facilitate seedling growth and survival. This mechanism was supported by a
field experiment using nylon mesh designed to exclude or allow the mycelial
network to connect to the seedling roots. After one year, seedlings with
access to the ECM network had significantly greater growth and survival
than seedlings excluded from the network (McGuire 2007a). However, all
seedlings had nearly 100% ECM root colonization, so the seedling responses
were not due to limited ECM colonization, but rather access to the common
mycorrhizal network. Mycorrhizal networks have been shown to influence
plant community dynamics and seedling establishment in temperate
ECM systems (Newman 1988, Simard and Durall 2004, Simard 2009), and
detailed tracer studies using autoradiography have revealed that nutrient
translocation can occur from plant to plant via these connections (Brownlee
et al. 1983, Finlay and Read 1986, Wu et al. 2012). Translocation of P and C
via mycorrhizal networks has been demonstrated in pine seedlings, and
implies that there is the potential that monodominant ECM adult trees
may support conspecific seedlings by similar pathways. The mechanism of
photosynthate transfer would be particularly advantageous for seedlings
in the light-limited understory of the tropical rain forest, as access to light
is often the most limiting resource for seedling survival (Chazdon 1988).
This positive feedback would be limited to the ECM monodominant tree
in an otherwise AM rain forest community, and, therefore,fits within the
plant-soil feedback framework to explain tropical ECM monodominance.
A second mechanism by which positive distance/density-dependence
occurs is enhanced recruitment near to conspecific adults due to greater ECM
inoculum density. This mechanism was supported in another experiment in
which D. corymbosa seeds were planted in both the monodominant and the
mixed forest, along with four other non-ECM tree species commonly found
in both forests (McGuire 2007b). In the mixed forest, D. corymbosa seedlings
had significantly lower mycorrhizal colonization after one year compared
to seedlings planted in the monodominant forest. The reduced colonization
was reflected in the lower survival of seedlings planted in mixed forest.
Responses of the non-ECM seedlings varied, but the magnitude of survival
in the monodominant forest relative to the mixed forest was much smaller
than the advantage gained by D. corymbosa seedlings (Fig. 4). It was possible
that these mixed tree species were benefitting from lower pathogen and
seed predator densities, since the overall density of their conspecific adult
trees was lower in the monodominant forest, although these mechanisms
have not been explicitly tested. However, two seed density treatments were
implemented for all tree species to test the prevalence of seed predation in
both forest types, and there were no notable differences in seed predation
rates in either forest type for any of the density treatments. Thus, it appeared
that seedling recruitment, rather than seed survival, was more reflective
of canopy diversity patterns, and that ECM inoculum density was a major
factor driving these patterns for D. corymbosa. The reduced availability of
ECM inoculum was also reflected in a molecular study that sequenced
fungal DNA from forest floor samples in the monodominant and mixed
forests (McGuire et al. 2010). There were significantly fewer DNA sequences
aligning to ECM fungi in the mixed forest compared to the monodominant
forest, which reflects the reduced colonization levels of D. corymbosa
seedlings in the 2007 study. Whether D. corymbosa seedlings are subjected
to pathogen attack in the mixed forest because of lower ECM inoculum
has not been determined, but is a plausible hypothesis that may directly
relate to the lower ECM colonization levels. In other ECM trees, reduced
colonization can lead to increased vulnerability to root pathogen attack
(Marx 1972, Whipps 2001, 2004), and may be a mechanism preventing the
rapid spread of the ECM monodominant forest into the mixed forest.
Survivorship
Fig. 4. Survivorship of seeds and seedlings after one year in monodominant compared to
mixed forest. Seeds of Dicymbe corymbosa and four other species found in both forest types
were reciprocally transplanted. Data are reproduced from McGuire 2007a.
An important component to plant-soil feedback theory is that greater

density of microbial mutualists leads to greater positive feedback. The
facilitation of seedling growth and survival near the conspecifics fits within
this theoretical framework, as D. corymbosa seedlings are receiving greater
benefit than heterospecific seedlings from the accumulated symbionts
near adult D. corymbosa trees. There are numerous studies demonstrating
this linkage in other ECM systems. For example, the highly invasive
Alliariapetiolata in North America has been found to derive its success by
decreasing ECM inoculum and root colonization of native ECM trees, which
negatively affects seedling establishment of native ECM trees (Wolfe et al.
2008). Another recent study of Populusfremontii found that the presence
of an invasive neighboring plant decreased ECM inoculum and percent
colonization in the Populus trees, which resulted in decreased tree growth
(Meinhardt and Gehring 2012). In other ECM monodominant systems,
however, these mechanisms of ECM-mediated positive feedbacks have not
been explicitly tested. In more diverse tropical ECM systems, such as the
diverse dipterocarp forests of SE Asia, some trees seem to exhibit positive
feedbacks, while others do not (Brearley 2012). However, since dipterocarp
forests contain an extraordinary diversity of ECM trees, the mechanisms
operating are likely to differ from an ecosystem in which only one ECM tree
dominates. Also, most of the dipterocarp trees occur in low abundances, so
feedback models would predict that positive feedbacks are not as dramatic
as in monodominant systems.
2.3 ECM feedbacks to nutrient cycling
It has long been observed that litter decomposition in ECM forests is

generally slower than in non-ECM systems, resulting in the accumulation of
thick humic layers (Smith and Read 2008). There have been several reasons
put forth to explain these observations. One major mechanism proposed is
that that leaf litter of ECM trees is somehow more recalcitrant and resistant
to decay, as has been observed in the Pinaceae (Read 1991). However, in the
D. corymbosa system, this mechanism has not been supported. In a reciprocal
leaf litter decomposition experiment, D. corymbosa litter decomposed at
the same rate as mixed species litter in both forest types (McGuire et al.
2010). Overall, decomposition was slower in the ECM forest for all litter,
suggesting that the process of decomposition was slowed by something
intrinsic to the monodominant forest, rather than the decomposability of
the monodominant litter. This hypothesis was further supported by two
years of bi-weekly litterfall quantification, in which it was found that total
biomass of litter input to the monodominant forest was less than litter input
into the mixed forest. Thus, the litter accumulation in the D. corymbosa
forest could not be attributed to greater litter production by D. corymbosa
or to litter recalcitrance. Fungal DNA sequencing revealed that there were

significantly fewer saprotrophic fungi in the monodominant forest than in
the mixed forest and that the fungal communities were compositionally
distinct across forest types. The conclusion was, therefore, that the slowed
decomposition in the ECM forest was somehow biotically mediated.
In the ECM monodominant forest dominated by Gilbertiodendrondewevreii
in the Congo litter decomposition was also observed to beslower than in the
mixed forest (Torti et al. 2001). As in the D. corymbosa system, decomposition
rates were not driven by differences in leaf litter recalcitrance, as mixed
litter and G. dewvreii litter did not significantly differ in decomposition rates
when they were placed in the same forest type. Further evidence refuting
the hypothesis that ECM tree litter is more recalcitrant comes from a recent
meta-analysis, which revealed that ECM plants do not have consistent leaf
traits in terms of nutrient content and litter recalcitrance (Koele et al. 2012).
Instead, it appears that the majority of studies of ECM trees have been
focused on the Pinaceae and Fagales (Dickie and Moyersoen 2008), so there
has been a bias in our understanding of ECM plant traits.
Slowed decomposition in ECM monodominant forests, and perhaps
other ECM forests, must be driven by factors other than leaf chemistry. If
litter decay rates are indeed biotically driven in these forests, then there may
be some interactive feedback between ECM fungi and other decomposer
microorganisms. This line of reasoning, and the fact that ECM fungi and
decomposer fungi may be competing for some of the same resources and
share the same spatial environment, has led to the formulation of the
Gadgil hypothesis (Gadgil and Gadgil 1971, Gadgil and Gadgil 1975). This
hypothesis posits that ECM fungi suppress saprotrophic microorganisms,
which results in slowed decomposition, as ECM fungi are not able to
decompose organic litter as quickly as free-living saprotrophs (Colpaert
and van Tichelen 1996). Since ECM fungi obtain the majority of their C from
their associate plant (Hobbie et al. 2002), they have lost genes associated
with rapid C acquisition in the early stages of decomposition (Wolfe et al.
2012). However, ECM fungi do maintain the capacity to directly degrade
other forms of organic polymers such as proteins, cellulose, and fatty acids
(Abuzinadah and Read 1986, Talbot et al. 2008, Rineau et al. 2012). Therefore,
the rationale for the Gadgil hypothesis is that ECM fungi may engage in
some form of antagonistic relationship with saprotrophs so that they are
allowed enough time to gain access to organically-bound nutrients such
as N and P.
There is direct evidence supporting the Gadgil hypothesis from
laboratory studies and indirect evidence from field studies. In laboratory
microcosms, ECM fungi have been found to suppress both saprotrophic
fungal growth and bacterial growth (Olsson et al. 1996, Lindahl et al. 2001).
In one study using autoradiography, ECM fungi were found to raid the
saprotrophic fungal mycelium of P, which was subsequently transferred

to the ECM plant host (Lindahl et al. 1999). Allelopathy through common
mycorrhizal networks has also been observed (Barto et al. 2011), which may
be one mechanism by which ECM fungi directly interact with saprotrophic
fungi and bacteria.
In Guyana, molecular analyses suggest that fewer decomposer fungi
and potential bacteria reside in the monodominant forest compared to the
mixed forest (McGuire et al. 2010), which is consistent with the predictions
of the Gadgil hypothesis. Total microbial biomass was also lower in the
monodominant forest and microbial community composition was distinct,
suggesting that decomposer communities were indeed different in the ECM
forest. Since litter decay was also slower, these various lines of evidence at
least offer some indirect support for the Gadgil hypothesis. While there are
numerous alternative hypotheses that may explain the patterns observed
from field studies, it remains a plausible hypothesis worthy of further testing
in ECM monodominant systems. A further extension of this hypothesis that
is worth exploring is that ECM fungi engage in antagonistic relationships
with saprotrophs foraging for more labile forms of C, N, and P, but that
ECM fungi facilitate persistence of saprotrophs that directly degrade more
recalcitrant compounds such as polyphenols and lignin (Wu 2011). This
modification of the Gadgil hypothesis is more in line with recent findings of
the loss of lignin decay genes in ECM fungal lineages (Wolfe et al. 2012), but
is inevitably more difficult to test, as it requires species-specific knowledge
of fungal function in the environment.
3. Containment of Monodominant Forests: Can Net

Feedbacks Become Negative?
A common question put forth about monodominant forests is, “why do they
not take over all lowland tropical rain forests?” When considering the ECM-
mediated advantages of monodominant trees, some of the same mechanisms
that facilitate local dominance may be disadvantageous for dispersal and
colonization to other sites. For example, in monodominant systems where
there is adult tree facilitation of conspecific seedling growth and survival
(i.e., reverse Janzen-Connell mechanism), when a seed is dispersed away
from a parent tree it is has a disadvantage for growth and survival, as it is
denied access to the common mycorrhizal network and experiences ECM
fungal inoculum limitation. It is yet unknown how long ECM inoculum
can persist in the soil in the absence of an ECM tree host or what the
dispersal range is for ECM fungi. In general, the dispersal capabilities of
ECM fungi have not been well examined, but one recent study found that
almost all of the spores of six ECM species fell within 1 m of the sporocarp
cap (Galante et al. 2011). Another recent study evaluating landscape-level

ECM dispersal patterns across fragments of pine “islands” showed that
spore loads decreased dramatically with increasing distance from the edge
of the pine fragment (Peay et al. 2012). With the exception of one ECM
fungal species, most ECM fungi appeared to exhibit dispersal limitation.
Dispersal limitation of ECM fungi may be an important mechanism
preventing novel clumps of ECM forest from arising. The patchy nature
of ECM monodominant forests is analogous to forest islands as described
in the Peay et al. 2009 study (Degagne et al. 2009), and some of the same
dynamics related to the theory of island biogeography may be operating
with ECM fungi in these regions.
Long-term studies of ECM monodominant forest dynamics are
necessary to know whether or not these forests are expanding, contracting,
or in dynamic states of both. Plant-soil feedback theory is based on
individual plant interactions, but in ECM monodominant forests these
feedbacks may scale up to the community level. It is possible that positive
plant-soil feedbacks become stronger with increasing density of conspecifics,
but then begin to decline past a threshold density of monodominant trees.
Pathogens or herbivores may begin to accumulate and periodic outbreaks
may prevent unchecked expansion of these forests (Gross et al. 2000). A
major limitation in studying in situ monodominant forests is that there is
little opportunity to follow individual seedlings over time to observe the
accumulation pathogens versus symbionts, which is the basis for the plant-
soil feedback models. Theoretical modeling coupled with empirical studies
will likely be necessary to formulate predictions about community threshold
effects and diversity-dominance oscillations in these forests.
Future global changes due to anthropogenic activity may function to
contain the expansion of monodominant forests, as there may be more
frequent pathogen outbreaks or recurrent disturbance events that alter
competitive dynamics and plant-soil feedbacks. Regions containing ECM
monodominant forests do not experience frequent or intense disturbances
(Connell and Lowman 1989, Hart et al. 1989, Torti et al. 2001), so the role
of disturbance in containing the spread of ECM monodominant forests is
thought to be insignificant. Nonetheless, changes in precipitation, storm
surges, and other climactic factors have the potential to affect plant-fungal
mutualisms (Kiers et al. 2010), and ECM fungi are known to be sensitive to
disturbance (Ingleby et al. 1998, Waltert et al. 2002, Peay et al. 2009). Also, if
these monodominant, ECM trees are dependent on having sufficient time
for competitive exclusion to occur, more frequent disturbance events may
ultimately cause a contraction of these forests.
4. Conclusions and Future Directions

It is clear that significant empirical work is needed to test many of the
aforementioned hypotheses across ECM monodominant systems. Since
many of the monodominant forests occur in remote locations, long-term,
intensive studies are lacking in comparison to many of the more easily
accessed diverse tropical forests. In the Dicymbe corymbosa forest in Guyana,
it appears that positive plant-soil feedbacks function to maintain ECM
monodominance through seedling facilitation and alterations of nutrient
cycling pathways.
A major focus for future research in ECM monodominant forests
should be deconstructing species-level functions of the associate ECM
fungi. Feedbacks between particular species of ECM fungi and ECM trees
may play some role in the competitive dominance of these trees compared
to other ECM trees in the community. With the exception of some work
in the Dipterocarpaceae (Brearley 2012), almost no studies have been
done evaluating species-specific feedbacks, despite the strong potential
for species-level differences. For example, ECM fungal diversity in the
D. corymbosa forest is high, with more than 200 ECM fungal species estimated
to coexist in this system (Henkel et al. 2012), and these individual fungal
species may have different functional capacities and variable mutualistic
benefits to the host. It is also possible that the ECM monodominant trees
are able to support a greater diversity of ECM fungi compared to non-
dominant ECM trees, which may partially explain why these trees attain
such extreme dominance. With a greater diversity of symbionts, trees have
access to more diverse pools of soil resources. Resource partitioning, which
usually stabilizes plant species coexistence, may be functionally similar in
these ECM forests, but ECM fungi may be differentially partitioning soil
resources, rather than different species of plants. Different ECM species also
vary in their ability to protect trees from pathogen attack (Ramachela and
Theron 2010). Therefore, a major research priority for these systems should
be to determine how individual ECM fungi function with respect to genetic
capabilities, host pathogen protection, and resource acquisition. Since most
of the ECM genera in tropical forests are the same as in temperate forests
(Bâ et al. 2012), hypotheses can be formulated based on known functions
of temperate fungal taxa.
Acknowledgements
We thank Krista L. McGuire and Caitlyn Gillikin for their valuable
comments on this study.
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CHAPTER
11
Ectomycorrhiza in Forest
Rehabilitation in Indonesia
Hesti L. Tata
1. Introduction
The total forest area1 of Indonesia is 131.3 million ha, which is about 69.9%
of the total terrestrial territory of Indonesia. However, the forest cover
inside the state forest area is only 133,327.2 ha, which is only about 0.1%
of designated forest area (Ministry of Forestry 2012). This implies that
deforestation rates in Indonesia are very high. Based on satellite imagery,
the Ministry of Forestry reported that deforestation inside the state forest
area in Indonesia during 2009/2010 was 610,375.9 ha. Deforestation in
Indonesia is mainly caused by fire, conversion of forest and peat-land to
other land uses, mining, and forest degradation due to illegal logging and
poor forest management practices (Ministry of Forestry 2012). Deforestation
in the islands of Kalimantan and Sumatra was remarkably high compared
with other islands in Indonesia (Holmes 2002). In the period between 1985
and 2007, forest loss in Sumatra alone was reported to be more than 70%
of total forest area (Laumonier et al. 2010).
Centre for Conservation and Rehabilitation, Jalan Gunung Batu 5, Bogor 16151, Indonesia.
Email: hl.tata@gmail.com
1
Forest area is designated by the institutional framework of forestry and includes areas
normally forming part of the forest area which are temporarily cleared as a result of human
intervention, but which are expected to revert to forest.
Ectomycorrhiza in Forest Rehabilitation in Indonesia 201
Lowland rainforests of Sumatra and Kalimantan are dominated by

Dipterocarpaceae tree species, which produce high quality timber. Some
species also produce non-timber products, such as illepe-nuts and dammar
resin (Shiva and Jantan 1998). Most dipterocarps species naturally occur in
the lowland forests of Sumatra and Kalimantan. Some species grow in peat-
swamp areas (such as Shorea balangeran and Shorea uliginosa), while others
can grow in higher altitude (750 m to 1200 m) montane forests (such as Shorea
platyclados and Shorea ovata) (Ashton 1982, Whitmore 1984). Deforestation
commonly occurs in the lowland mixed dipterocarps forests, where
commercial timber trees grow well and the land is easily accessible.
Over the last three decades, many efforts have been made by the
Government of Indonesia to overcome the loss of forest cover (Nawir
et al. 2007). The Ministry of Forestry implemented forest rehabilitation
programs in association with local communities, such as the Rehabilitation
of Logged-Over Area (1996-2003), Community Based Forest Management
(1994–1999), Farm Forestry (1970), Programme of Specific Allocated Funds
—Reforestation Funds (2001) and the Collaborative Forest Management
Project (2001). However, the programs were not successfully implemented
due to the failure of the utilized approach, which was mainly based on
government’s instruction approach (Nawir et al. 2007).
In order to develop more integrated rehabilitation programs, the
Planning Agency of The Ministry of Forestry set up a national movement
to rehabilitate the state forest area (Gerakan Rehabilitasi Nasional, Gerhan) in
2003, totaling 3 million ha of forest in 28 provinces within 5 years.2 However,
this program failed to meet the target, owing to mismanagement of the
project’s implementation and policy failure (Nawir et al. 2007).
The success of forest rehabilitation is not only influenced by policy
and socio-economic factors, but also technical aspects of program
implementation, such as site characteristics, site-species matching, planting
stocks production, and maintenance. Production of planting stocks is an
important part of the reforestation and rehabilitation of degraded land.
Large numbers of good quality planting stocks have to be produced in
nurseries as the first step of a successful rehabilitation program.
2. Promoting Dipterocarpaceae for Rehabilitation

Enrichment planting in the area of production forests (e.g., forest areas
to produce forest products for the benefit societies and industries) using
native species has been regulated since 1993, under the silviculture system
2
http://www.dephut.go.id/INFORMASI/RRL/Benih/GN_RHL.html
of Indonesian Selective Cutting and Planting (Tebang Pilih Tanam Indonesia,

TPTI). Dipterocarpaceae is recommended for planting in the rehabilitation
of degraded forest and logged areas (Soekotjo 2005), Imperata grassland
(Adjers and Otsamo 1996) and burned forests (Anonymous 2004, Priadjati
2002, Tata et al. 2010). Despite some constraints, such as soil compactions,
loss of top-soils, high temperature, and competition with weeds and fast
growing tree species, Dipterocarpaceae trees are challenging to use in the
rehabilitation projects (Kettle 2010, Matsune et al. 2006, Okimori et al. 2006),
because they have EcM associations (Brearley 2012, Hadi 1987, Lee 1998,
Smits 1994). Mutualistic symbioses between dipterocarps trees and EcM
fungi is necessary for initial tree growth (Smits 1994, Turjaman et al. 2005,
Turjaman et al. 2006).
3. Effect of Site Factors and Disturbance on Ectomycorrhiza

The soil type of tropical rain forests in Kalimantan island is dominated
by Ultisols soils, with characteristics of high clay content, high acidity,
low nutrients, and high aluminum and iron levels (Ohta and Syarif 1996),
whereas soil types in Central Sumatra are dominated by Oxisols and Ultisols
soils (Ketterings et al. 2000, Van Noordwijk et al. 1998). Low nutrient content
but high heavy metal content in the soils may hamper the survival and
growth of dipterocarp seedlings.
Mycorrhizal inoculum potential in different land use types of Jambi
province, including forests rubber agroforests rubber monocultures small
holder oil palm cassava field annual crops Imperata grasslands, has been
studied. Tata (2008) reported that land use types affected EcM colonization
of two dipterocarp species of Shorea (Shorea selanica and Shorea lamellata) in
their nursery stage at 10 months after planting. All seedlings of S. selanica
and S. lamellata planted in soils from different land use types were colonized
by EcM fungi. The highest EcM colonization occurred on Shorea planted
in soils from rubber agroforests (RA), while the lowest EcM colonization
occurred on Shorea planted in soils from annual crops (CR) (Fig. 1). The
soils from annual crops were categorized as fertile soils, which had higher
macro nutrient contents (Pavailable, N, Na, Ca and Mg) compared with soils
from other land uses (Tata 2008). High soil nutrient content, especially P,
could inhibit colonization of EcM on the root systems (Marx et al. 1977).
Forest disturbance caused by fire affected fruiting body development of
EcM fungi in East Kalimantan (Tata et al. 2003a). Two years after a wildfire
in a mixed dipterocarp forest of Sungai Wain, East Kalimantan, no fruiting
bodies were found on the burned forest floor. However, bioassay studies on
the seedlings of Shorea pinanga and Shorea stenoptera using soil from burned
dipterocarp forests have shown that the roots of both Shorea species were
colonized with EcM fungi. EcM morphotypes encountered in the roots
from different soils were varied (Table 1). This observation implies that
mycorrhizal inoculum potential persists in the soils after wildfire. EcM
morphotypes from unburned forests were different compared to those from
burned forests (Tata et al. 2003a).
Fig. 1. Ectomycorrhizal colonization on S. selanica and S. lamellata seedlings planted on soils

from different land use types in nurseries. C=sterilized soil (control), RM= rubber monoculture,
OP=smallholder oil-palm, CA=cassava field, CR=annual crops, IM=Imperata grasslands,
FO=forest, RA=rubber agroforest. Data from Tata (2008).
Table 1. Ectomycorrhizal morphotypes on Shorea pinanga and S. stenoptera seedlings grown in

different soil sources from burned and unburned mixed dipterocarp forests in Sungai Wain,
East Kalimantan and a reforested dipterocarp site in Haurbentes, West Java.
Tree Soil sources EcM Morphotypes Hypha Clamp

Species connection
Diameter Color Diameter
(µm) (µm)
S. pinanga Burned forest Monopodial, white 4.0–4.9 hyaline 6.5–7.1
Unburned Monopodial, white 4.0–4.4 reddish 4.6–6.9
forest brown
Haurbentes Monopodial, white 4.6–5.3 reddish 6.3–8.2
brown
S. stenoptera Burned forest Monopodial, white 3.4–4.6 hyaline 5.0–8.0
Unburned Monopodial, white 4.0–4.4 hyaline 5.3–6.7
forest
Haurbentes Monopodial, white 5.3–6.3 reddish 8.2–9.0
brown
Data from Tata et al. (2003a)
Another study on the effect of heat treatment up to 150ºC on EcM

inoculum potential of forest soils from a lowland dipterocarp forest of
Jambi (Sumatra) showed that heat treatment had little effect on the growth
of tree cuttings from five species of dipterocarps (Shorea selanica, Shorea
leprosula, Shorea lamellata, Vatica sumatrana, and Hopea odorata). Although
EcM colonization in the control soils was higher than that of the soil treated
with higher temperatures (150ºC), the effect was smaller than expected
(Fig. 2). The rapid colonization of heated soils in the experiment may
imply abundant availability of EcM fungi for dipterocarp growth in field
conditions. Molecular identification based on sequences from the bioassay
studies of the Internal Transcribed Spacer (ITS) region of ectomycorrhizal
fungi on heat-treated S. selanica differed from those on S. selanica planted
in the field experiment (Tata 2008).
Logging, or timber extraction, reduces canopy cover and has been
shown to affect soil EcM communities. Studies of EcM communities on the
roots of S. parvifolia seedlings showed that the diversity of mycorrhizal types
found under open canopies caused by logging was higher than that found
under closed canopies. Mycorrhizal types from open canopies were thought
to be pioneer fungi (Ingleby et al. 1998). Similar evidence was found in S.
lamellata planted on different ages of rubber agroforests. EcM colonization
on S. lamellata grown in 1 year old rubber agroforests was higher than those
grown under a forest canopy. However, S. selanica planted under the forest
canopy had the best EcM colonization (Table 2).
Fig. 2. Ectomycorrhizal colonization on cuttings of five dipterocarps species planted on soils

that underwent a series of heat treatments. Data from Tata (2008).
Table 2. Ectomycorrhizal colonization of S. selanica and S. lamellata inoculated (+) and non-
inoculated (–) with Scleroderma columnare in nurseries planted on different land use types.
EcM colonization (%)

Land uses S. lamellata (–) S. lamellata (+) S. selanica (–) S. selanica (+)
Forest 10.0 a 17.6 a 41.5 b 59.4 c
R1_forest 20.2 a 37.5 ab 27.5 a 54.1 c
R1_RAF 21.5 a 23.1 a 47.5 a 29.8 ab
Note: R1_forest: rubber garden age 1 yr derived from forest, R1_RAF: rubber garden age 1
yr derived from RAF. Values followed by same letter are not significantly different (P< 0.05).
Data from: Tata (2008).
Complex results on the effect of Scleroderma columnare inoculation on

growth of S. selanica and S. lamellata planted in rubber agroforests have
been reported. Inoculation of Scleroderma columnare increased survival of
S. selanica and S. lamellata seedlings one year after transplanted to the field.
After two years, inoculation of EcM did not affect survival of both Shorea
species. Seedlings growth, e.g., relative growth rate of height and stem
diameter, were only increased by EcM inoculation in the second semester
of two years observation (Tata et al. 2010).
4. The Importance of EcM Inoculation on Planting Stock

Production
Dipterocarp trees can be generative and vegetative propagated. Planting
stocks of dipterocarps are produced generatively using sown seeds in
nurseries and wild seedlings collections (commonly known as wildlings)
(Smits 1994, Kettle 2010), and vegetatively using shoot cutting (Smits
1992, Subiakto et al. 2005). Many studies performed during nursery stages
showed that ectomycorrhizal fungal inoculation on dipterocarp seedlings
increase dipterocarp seedling growth (Santoso 1988, Smits 1994, Yasman
1995, Priadjati 2002, Turjaman et al. 2005, Turjaman et al. 2006, Riniarti
2010, Tata et al. 2010) and nutrient uptake (Santoso 1988, Omon 2002, Tata
et al. 2010).
In natural ecosystems, dipterocarp trees exhibit nurse effects for their
seedlings by facilitating their growth and survival through hyphal networks
in the soil (Read 1991, Smits 1992). Mimicking this habit, EcM inoculation
can be done by planting dipterocarps seedlings in nursery using topsoils
that are collected below conspecific parent trees (Smits 1992, Yasman
1995). Planting stocks from vegetative propagation, on the contrary, do not
have direct contact with the hyphal networks of parent trees. Propagation
techniques usually use sterilized rooting media to minimize diseases and
increase survival. Hence, artificial EcM inoculation prior to transplanting is

recommended (Smits 1992, Smits 1994,Yasman 1995, Omon 2002). However,
Fig. 2 shows cuttings of H. odorata, S. leprosula, S. platyclados, S. selanica and
V. sumatrana were colonized with EcM fungi at all degrees of heat
treatment on transplanted media. Heat treatment up to 150ºC might not
kill mycorrhizal inocula in the soil (Tata 2008).
Efforts to rehabilitate logged forest areas in Kalimantan encountered
difficulties due to the lack of EcM partners in the soil after forest disturbance.
Thus, rehabilitation using dipterocarps trees in East Kalimantan requires
mycorrhizal inoculation in the nursery stage (Smits 1992, Smits 1994).
5. Ectomycorrhizal Response in Field Experiment

Further studies in field conditions, however, have not been performed in
detail. Some reports showed different responses of EcM fungal inoculation
on the growth of dipterocarp seedlings. In a peat swamp forest in Central
Kalimantan, inoculation with Boletus sp. and Scleroderma sp. increased the
growth of Shorea balangeran (Turjaman et al. 2011). However, it is not clear
whether or not EcM fungi that have been inoculated in the nursery stage,
e.g., Scleroderma sp. and Boletus sp., persist in the root system of Shorea
balangeran after transplanting to the field. Another study showed that 2 years
after transplanting to a field of rubber-based agroforestry, S. selanica and
S. lamellata that were inoculated in the nursery stage with S. columnare have
different EcM communities in their root systems (Tata et al. 2010).
In logged forests in Jambi, Central Sumatra, inoculation of EcM fungi
on dipterocarps seedlings (Shorea macroptera and Shorea parvifolia) planted in
logged forests improved seedlings performance. Seedling growth showed
a positive relation with the percentage of EcM colonization (Kikuchi 2006).
This author reported that the types of EcM fungi found in planted and
natural seedling were quite different.
Priadjati (2002) reported different responses of EcM inoculation in field
experiments. Shorea leprosula cuttings planted in a secondary forest in East
Kalimantan showed that EcM inoculation did not increase performance
and tree growth. Non-inoculated cuttings, which were used as the control,
had the best performance of height and stem diameter among other EcM
inoculations (Priadjati 2002). This author found that, after transplanting
to the field, control trees had a higher percentage of EcM colonization
than the trees that had been inoculated with Amanita sp., Russula sp., and
Scleroderma sp.
Another trial performed in a rubber agroecosystem showed that EcM
inoculation had limited effects on the growth of S. selanica and S. lamellata
(Tata et al. 2010). In the initial development of rubber agroforests, natural
forest was cleared using the slash and burn technique as a low-cost
establishment method (Gouyon et al. 1993, Penot 2004). No potential host

trees for EcM fungi, such as trees belonging to the family of Dipterocarpaceae
and Fagaceae, were still alive. Rubber seedlings were planted alongside
upland paddy and other food crops. Fallow succession allowed natural
regeneration until rubber trees reached a certain stem diameter that
could be tapped (~8–10 years after planting; Gouyon et al. 1993, Joshi et
al. 2002). Tata et al. (2010) reported that two years after planting in the
rubber agroforests, the seedlings of two Shorea species, both inoculated and
un-inoculated with Scleroderma columnare, were colonized by EcM fungi.
Different EcM fungi colonized the seedlings that had been inoculated with
Scleroderma columnare in nurseries. The authors implied that mycorrhizal
inoculum potential persists in the soils of rubber agro-ecosystems, even
rubber gardens with long histories of forest clearing. Further study was
suggested to investigate whether other factors, such as squirrels and wild
pigs, play roles as dispersal agents of EcM inoculums from the remaining
forests (Tata et al. 2010).
In a comprehensive review, Brearley (2012) suggested that EcM fungi
are mostly likely to benefit the growth of dipterocarp seedlings when they
are planted in degraded areas. Therefore, inoculation of EcM seedlings at
the nursery stage is recommended in the rehabilitation of degraded areas
where mycorrhizal inoculum potential is limited.
6. Rehabilitation Techniques
Technically, rehabilitation of degraded dipterocarp forests can be achieved
through (i) enrichment planting with desirable dipterocarp seedlings or clonal
plant materials in order to increase timber productivity, and (ii) planting
fast-growing light demanding species as a nurse canopy and followed by
under-planting with dipterocarps (Kettle 2010). The former is established
under small opening of forest canopy covers and in the presence of gaps.
The latter is established under severely degraded forest conditions.
Many studies have shown techniques to rehabilitate degraded
dipterocarp forests caused by logging and over-exploitation, fires, and the
invasion of Imperata grassland (Adjers et al. 1996, Otsamo et al. 1996, Otsamo
2000, Mori 2001). Forest rehabilitation with enrichment planting can be
done in gaps resulted from logging activities. To rehabilitate burned forests
and Imperata grasslands, strip planting techniques can be adopted. Mixed
planting between fast growing trees (such as Acacia mangium) and native
tree species is the best option to rehabilitate Imperata grassland in South
Kalimantan (Otsamo 2000). However, no EcM inoculation was mentioned
in these publications.
Rehabilitation of degraded land in Haurbentes, West Java, with
various Dipterocarpaceae trees, showed a successful human constructed
dipterocarp forest outside their natural habitats. The establishment of a

reforested dipterocarp site in Haurbentes, West Java, was initiated in order
to establish a dipterocarp forest research station in 1938 (Masano et al. 1987,
Subiakto et al. 2001, Mindawati et al. 2004a, b). Mindawati et al. (2004a,b)
reported the growth and yield of several dipterocarp species from different
origins that have been planted in Haurbentes since 1940. Shorea leprosula,
Shorea mecistopteryx, Shorea stenoptera, S. selanica, Shorea balangeran, Hopea
mengerawan and H. odorata demonstrated the best growth among others
(Mindawati et al. 2004a,b). However, there is no report on the study of
inoculation EcM fungi on dipterocarp seedlings since the early development
of the Haurbentes research station in 1938. Long after the reforested
dipterocarp site was established, Santoso and Turjaman (2003) reported
that several species of EcM fungi were encountered on the forest floor of
the Haurbentes forest research station, such as Scleroderma sp., Lactarius sp.,
Boletus sp., and Cantharellus cibarius (Santoso and Turjaman 2003).
Agroforestry is another option for forest rehabilitation through
enrichment planting and mix-planting dipterocarp trees in agro-ecosystems.
Agroforestry system in Indonesia includes taungya farming system (e.g.,
growing annual agricultural crops along with the forestry species during
the early years of establishment of the forestry plantation), and tree-based
agroforestry system, such as Shorea javanica and rubber (Hevea brasiliensis)
trees. Shorea javanica, a dammar resin producer, has been planted in
agroforestry systems in Lampung, Sumatra for many years (Michon and
Bompard 1987, Torquebiau 1984). Traditional farmers who planted Shorea
javanica (locally known as dammar ‘mata kucing’) were not aware of the
technology of EcM inoculation. S. javanica seedlings were transplanted
and domesticated in dammar gardens with soil from the nursery. According
to farmers, transplantation is always successful, even without artificial
inoculation of EcM fungi in nursery. Soils from the nursery increase chances
of survival after transplantation (Michon and Bompard 1987). Nuhamara
(1987) reported that several EcM fungi were found in the S. javanica
agroforest, such as Amanita hemibapha, Cantharellus cibarius, Lactarius spp.,
Russula spp., and Scleroderma sp. This author concluded that using soils with
abundant inocula, e.g., top soils with mycorrhizal networks, as sources of
inocula resulted in better growth of dipterocarp trees compared with those
inoculated with a single EcM fungus.
Another agroforestry system, rubber agroforests, has relatively high
tree diversity (Gouyon et al. 1993, Beukema et al. 2007, Tata et al. 2008).
Low management intensity allows other beneficial trees to grow, forming an
agroforestry complex. EcM host trees such as Shorea leprosula and Lithocarpus
spp. that grow in the rubber garden maintain EcM sporocarp diversity.
Russula spp., Geastrum sp. and Scleroderma sp. were encountered on the
rubber agroforest floor in Jambi (H.L.T. unpublished data).
The taungya system has been applied to rehabilitate burned and

severely degraded forests in Bukit Suharto, East Kalimantan. Shorea
smithiana seedlings were planted along with rubber trees and five cash
crops. Three fertilizers have been used to improve soil macro-nutrients
(N, P and K), but no treatment of EcM fungi inoculation was reported. One
year after transplantation to the field, survival of S. smithiana seedlings was
85.8%, which was higher than rubber trees (80.5%) (Sutisna 2001). Although
S. smithiana is categorized as a shade demanding species, it survived and
grew in an exposed plot (Sutisna 2001). However, this author did not report
the presence of mycorrhizal colonization on the root system. The soils of the
site in East Kalimantan may still have considerable mycorrhizal inoculum
potential, as reported by Tata et al. (2003a) in a burned dipterocarp forest
in Sungai Wain, East Kalimantan.
7. Conclusions
The success of rehabilitation is determined by silvicultural and ecological
factors. Ectomycorhizas play an important role in the production of planting
stocks for forest rehabilitation. In the nursery stage, ectomycorrhizal fungal
colonization increases early growth of dipterocarp species, which benefits
planting stock production. However, after field planting, very few studies
have reported success of EcM fungal inoculations on tree growth in field
conditions. Inoculation of EcM fungi in the nursery stage requires skills and
capital in the initial nursery development. Using topsoil collected under
conspecific parent trees for planting stock productions is more applicable
for EcM fungal inoculation.
8. Future Research Needs

There are several publications on the applied research of EcM in Indonesia
relating to forest and land rehabilitation, published mostly in national
journals and national proceedings. Most of the research was conducted with
a small number of replications and inappropriate design. There has been
limited basic research performed on the physiology, taxonomy, phylogeny,
and ecology-community of ectomycorrhizas, has been studied that can
provide evidence and support for the applied research for improvement
of forest and land rehabilitation practices in Indonesia.
Another weakness of EcM studies in Indonesia is the lack of identification
of EcM fungi and ectomycorrhiza. The identity of tropical EcM fungi is
currently based on morphological characters of temperate EcM fungi and
EcM identification book guidelines (Tata et al. 2003b), which is relatively
different from sporocarps of tropical EcM fungi. Due to limited laboratory

facilities, advanced technology is not currently commonly practiced.
EcM fungi promote forest rehabilitation by increasing seedling survival
and tree performance. Other than that, EcMs and EcM fungi provide
ecosystem services to the environment by provisioning, regulating and
supporting roles. Besides silvicultural and ecological criteria, the success of
forest rehabilitation also needs to consider socio-economic criteria, such as
population size, market access, availability of food and fuel, and economic
benefit and subsidy to local people. Factors such as increased population size,
food shortages, and poverty are often the roots of forest degradation (Lamb
and Tomlinson 1994). Moreover, current major drivers of deforestation in
Sumatra is the development of oil palm plantations (Laumonier et al. 2010)
in forested areas, which has a greater economic benefit than other crops.
Therefore, forest and land rehabilitation in Indonesia should be integrated
with rural development.
Rehabilitation is not only about planting trees, but also contains multiple
and complex aspects and activities. Nowadays, rehabilitation and forest
restoration have large targets including biodiversity conservation, biomass
production, and climate change mitigation and adaptation (Ciccarese et al.
2012). Hence, to rehabilitate degraded tropical forests and lands, more efforts
to integrate multiple research disciplines are required. Public participation
in the rehabilitation of degraded lands in Indonesia has been increasing,
such as the development of small holder timber and farm forests, using
both native species and domesticated species (see, for example, Tata and
Van Noordwijk 2011, Sofiyuddin et al. 2012). Forest rehabilitation may be
more attractive when farmers see the alternative income, clear incentive,
and policy support (Roshetko et al. 2008).
Acknowledgements
The author acknowledges Meine van Noordwijk for comments on the early
draft of manuscript, anonymous reviewers for comments to improve the
manuscript and Krista L. McGuire and Caitlyn Gillikin for improving the
language.
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CHAPTER
12
The Controlled
Ectomycorrhization Practices
in Tropical Areas: Fungal
Inoculum Biotechnology,
Field Results and Research
Perspectives
Robin Duponnois,1,* Hervé Sanguin,1 Amadou Mustapha
Bâ,1,2 Antoine Galiana,1 Marc Ducousso,1 Ezékiel
Baudoin,1 Michel Lebrun1 and Yves Prin1
1. Introduction
Overexploitation of forest resources resulting from excessive industrial
exploitation, clearing for industrial purposes, and collection of firewood has
led to a dramatic deforestation during recent decades in the Mediterranean
and tropical areas (Piéri 1991). One of the detrimental effects of deforestation
is soil degradation and desertification processes resulting in alterations of
1
UMR 113 CIRAD/INRA/IRD/SUP-AGRO/UM2, Laboratoire des Symbioses Tropicales
et Méditerranéennes (LSTM). TA A-82/J, Campus International de Baillarguet. Montpellier
Cedex 5, France.
2
Bel Air, BP. 1386, CP. 18524 Dakar, Sénégal.
*Corresponding author: Robin.Duponnois@ird.fr
major physicochemical and biological soil properties (Requena et al. 2001).

The disturbances in plant cover largely contribute to increase soil erosion and
decrease soil fertility and microbial activities (Garcia et al. 1997). Numerous
studies have reported that in such conditions, indigenous inoculum levels
of mycorrhizal fungi were significantly altered (Duponnois et al. 2001,
Azcon-Aguilar et al. 2003). The mycorrhizal symbiotic establishment takes
up and transports nutrients to plant roots, improves soil aggregation in
eroded soils, and reduces water stress (Smith and Read 2008).
Mycorrhizal fungi are ubiquitous components of most ecosystems
throughout the world and are considered key ecological factors in governing
the cycles of major plant nutrients and in sustaining the vegetation cover
(Van der Heijden et al. 1998, Requena et al. 2001, Schreiner et al. 2003).
Two major forms of mycorrhizas are usually detected: the arbuscular
mycorrhizas (AMs) and the ectomycorrhizas (ECMs). The AM symbiosis
is the most widespread mycorrhizal association type with terrestrial plants
(i.e. pteridophytes, gymnosperms and angiosperms) (Read et al. 2000). The
arbuscular mycorrhizal fungi (AMF) colonize about 80–90% land plants in
natural, agricultural, and forest ecosystems (Brundrett 2002). ECMs affect
trees and shrubs, gymnosperms (Pinaceae), and angiosperms, and are
frequently the result of the association of Homobasidiomycetes with about
20 families of mainly woody plants (Smith and Read 2008). Compared to
AMs, ECMs are formed with a greater diversity of fungi, comprising 4,000
to 6,000 species of mainly Basidiomycetes and Ascomycetes (Allen et al.
1995, Valentine et al. 2004). Ectomycorrhizal fungi are not homogeneously
distributed within the ecosystem, which has important implications for
reforestation in areas with no native ectomycorrhizal vegetation (Marx
1991). The absence of ectomycorrhizal fungi results from natural (Terwilliger
and Pastor 1999) or anthropogenic disturbance (Jones et al. 2003).
The lack of ectomycorrhizal fungal formations on root systems is a
major cause of poor ectomycorrhizal plant establishment and growth in a
variety of forest landscapes. Numerous studies have reported that specific
ectomycorrhizal fungi can promote the survival and early growth of various
tree species in the field (Castellano and Molina 1989, Kropp and Langlois
1990, Marx 1991, Castellano 1996, Roldan et al. 1996, Garbaye and Churin
1997, Duponnois et al. 2005a, 2007). Since mycorrhizal associations are
estimated to occur in 95% of native undisturbed vegetation, whereas they
are found in less than 1% of vegetation from disturbed sites, improving
mycorrhizal soil potential may increase the benefit of the mycorrhizal effect
on plant growth. This objective can be achieved by inoculating seedlings
before they are transferred into disturbed sites. In order to improve the
performance of afforestation, it is necessary that nurseries produce tree
Controlled Ectomycorrhization Practices 217
seedlings associated with ectomycorrhizal fungi that are ecologically

compatible with the tree species and characteristics of the planting sites.
According to these conditions, different methods of controlled inoculation
have been identified to optimize the effect on plant growth.
Controlled mycorrhization is based on the use of mycorrhizal strains
best suited to host plant species which will rapidly colonize their root
systems and are well adapted to the environmental conditions of the
planting site (Duponnois et al. 2007). However, this practice requires high
volumes of ectomycorrhizal fungal inoculum. Hence this biotechnological
process is generally associated with large investment of resources that limits
its use in forest nurseries, especially in developing countries.
As the production of fungal inoculum is costly, it has been suggested
that Mycorrhization Helper Bacteria in the rhizosphere could stimulate
the establishment of the fungal inoculant and consequently, minimize the
volume of required fungal inoculum (Duponnois et al. 1993). It has also
been demonstrated that the termite mounds of Macrotermes subhyalinus (a
litter-foraging termite frequently found in tropical areas) were colonized by
a specific microflora that promotes the ectomycorrhizal fungal development
(Duponnois 2006). Thus M. subhyalinus mound amendment could be used
as a biological tool to improve the efficiency of controlled mycorrhization
in forest nurseries and to minimize the technical and financial investment
of this biotechnology.
The main objectives of this chapter are to describe the processes required
to produce large quantities of efficient fungal inocula and to report tree
growth data resulting from the use of controlled ectomycorrhization in
nursery and field conditions in tropical and Mediterranean areas. In addition,
innovative processes of mycorrhizal inoculation using Mycorrhization
Helper Bacteria (MHB) and termite mounds of M. subhyalinus as catalysts
for mycorrhization will be described.
2. Biotechnological Processes used in Controlled

Ectomycorrhization
2.1 Criteria to adopt an inoculum formulation
Different criteria have to be taken in account to adopt an inoculum

formulation. The selected ectomycorrhizal fungi must provide a positive
effect on the growth and the survival of tree species used for plantation. The
fungal propagules must be kept viable during storage to preserve the fungal
efficiency on plant growth after inoculation. Finally ectomycorrhizal fungal
inocula have to be cost-effective in order to be used in nursery practices.
2.2 Spore-based fungal inoculum
Spores from fruiting bodies can be considered as a natural source of

inocula. This inoculation practice has been widely used in forest nurseries
(Castellano 1994). However, this inoculation technique is only effective with
fungal species that produce abundant sporocarps with large spore numbers
(i.e., Pisolithus and Scleroderma).
Numerous formulations of spore inoculums have been described.
Sporocarps are collected, carefully identified, and kept in paper bags. They
are then brushed free of adhering soil. This inoculum type is frequently
based on dry spores obtained by air drying fruiting bodies at temperatures
below 35°C. The dried sporocarps are crushed in plastic bags and sieved
through 200 and 500 Om sieves, respectively. The dried spores can then be
used in different formulations. Spore powder can be mixed with sterilized
fine sand (1:100, w/w) and used to inoculate containers by either mixing
the soil substrate with this fungal mixture or by adding a small quantity of
the fungal inoculum at the base of seedlings. Ectomycorrhizal inoculation
can be achieved by adding spore powder into an inert carrier (i.e., clay) to
form pellets that are placed at the base of seedlings (de la Cruz et al. 1990).
According to Turjaman et al. 2005, in order to determine the influence of
ectomycorrhizal fungi on the growth of dipterocarp species in peat soils,
the sporocarps were crushed manually in plastic bags to minimize loss
of spores and cross-contamination between fungi (de la Cruz et al. 1990).
Crushed sporocarps and clay were mixed and formed into pellets with a
ratio of 1:100 (w/w). Inoculation was made 10 days after seed germination.
One hole was made in each pot and a tablet (0.4 g) of fungal inoculum was
applied to a potted seedling 1 cm below the soil surface, at the proximity of
the root. Another process of ectomycorrhizal spore inoculation is developed
by coating seeds with a mixture of spores and a binding agent such as clay
(Marx et al. 1984).
The beneficial effects of ectomycorrhizal spore inoculation on plant
growth in nursery conditions have been frequently recorded (Table 1).
Several disadvantages have also been reported. It is difficult to collect large
quantities of fruit bodies with some ectomycorrhizal fungal species. In
addition, low efficiency of the fungal inoculums due to slow germination
or low spore viability is frequently recorded. Besides these critiques, this
inoculation process is easy to apply and remains an efficient method for
storage and transport of spores for some mycorrhizal fungal genera such
as Pisolithus or Scleroderma.
Table 1. Effect of ectomycorrhizal spore inoculation on plant growth in nursery conditions.
Ref. (1) Fungal strain Plant species Inoculum Effects on plant growth parameters (%) and ectomycorrhizal
formulation colonization
Height Shoot Root Ecto. colonization (%)
biomass biomass
A Pisolithus arhizus Shorea pinanga Ectomycorrhizal spore + 46.1 (2) + 66.7 nd(3) 87
pellets
A Scleroderma sp. S. pinanga Ectomycorrhizal spore + 41.3 + 60.5 nd 86
pellets
B P. tinctorius Acacia mangium Ectomycorrhizal spore nd + 29.1 + 40.7 52
tablets
C Rhizopogon roseolus Pinus halepensis Spore suspension –30.1 nd nd 48
C Suillus collinitus Pinus halepensis Spore suspension + 27.8 nd nd 75
D Scleroderma albidum Eucalyptus globulus Spore suspension + 32.1 + 19.7 + 42.5 nd
D S. areolatum E. globulus Spore suspension + 17.5 + 9.1 +33.5 nd
D S. cepa E. globulus Spore suspension + 30.8 + 8.8 + 2.0 nd
D S. albidum E. urophylla Spore suspension – 3.6 + 3.1 –0.7 nd
D S. areolatum E. urophylla Spore suspension + 7.2 + 4.9 + 4.0 nd
D S. cepa E. urophylla Spore suspension + 6.3 + 13.0 + 1.4 nd
E P. tinctorius P. halepensis Spore suspension + 37.1 + 44.9 + 41.7 55.4
E R. roseolus P. halepensis Spore suspension + 38.3 + 44.6 + 28.6 39.5
E Suillus collinitus P. halepensis Spore suspension + 35.1 + 28.2 + 30.6 28.9
A: Turjaman et al. 2005. B: Aggangan et al. 2010. C: Rincon et al. 2007. D: Chen et al. 2006. E: Torres and Honrubia 1994
(1)
Ref.: Reference. (2) (mean value of ectomycorrhizal plants—mean value of the non ectomycorrhizal plants) x 100)/(mean value of the ectomycorrhizal
plants). (3)nd: not determined.
2.3 Mycelium-based fungal inoculum
These inoculum formulations need the isolation of fungal symbionts in

axenic conditions to obtain a pure sample of a single fungal strain. These
axenic cultures are often obtained from fruit bodies of ectomycorrhizal
fungi collected in the field but they can also be performed by collecting
mycorrhizal roots, sclerotia, rhizomorphs and spores (Molina and Palmer
1982).
Isolating fungi from from fruiting bodies is the most successful method
for obtaining pure fungal cultures. Sporocarps are of adhering soil and
carefully broken in a laminar flow hood. A small amount of tissue is then
removed with fine forceps and placed on a nutrient medium agar. As growth
requirements vary between ectomycorrhizal fungi, different nutrient media
can be used (Table 2). The fungal cultures are incubated at 25°C in the dark
and subcultured until all contaminating microorganisms are eliminated.
Fungal cultures are usually maintained in Petri dishes on a required nutrient
agar medium at 20 and 25°C, respectively. Fresh subcultures are made every
6 to 12 weeks depending on fungal growth rates.
The formulation of mycelium-based inoculum is only feasible and
economical with some fungal species that can grow in cultures. Two main
cultural supports are usually used to produce fungal inocula: (i) mixture
of peat/vermiculite and (ii) hydrogel (i.e., calcium alginate gel).
Glass jars (1.6 liter) containing 1.3 liter expanded vermiculite-sphagnum
peat mixture (4:5 and 1:5, v:v, pH = 5.5) are heat disinfected (120°C, 20
min) (Marx and Bryan 1975). Another ratio of vermiculite-peat can be
used (2:3 and 1:3) but at this ratio, the peat can release substances that are
toxic for fungal growth. Hence, the first ratio mixtures are usually used.
The mixture is then moistened to field capacity with 600 ml of a modified
liquid nutrient medium (see Table 2). The jars are closed with lids with a
1-cm diameter hole. This hole is fitted with a 4-cm long tube filled with
cotton wool. The jars are then heat disinfected a second time (120°C for
20 min). After cooling, approximately eight mycelial plugs are placed on
top of the substrate. The mycelia grow down into the substrate, which is
completely colonized after 6 to 10 weeks at 25°C. For faster growth, jars can
be filled with a smaller quantity of substrate and shaken after mycelia have
colonized a few centimeters: in this manner, mycelium is evenly distributed
throughout the substrate and incubation time is shortened. This inoculum
can be stored at 4°C for upto 6 months.
Dommergues et al. (1979) and Le Tacon et al. (1983, 1985) first reported the
process of including fungal mycelium in polymeric gels (especially calcium
alginate). This type of inoculum is more efficient than the vermiculite-peat
formulation (Mortier et al. 1988) since the gel protects the mycelium from
physical stresses (e.g., water stress) and from competing microorganisms.
Table 2. Composition of nutrient media commonly used for the isolation and culture of
ectomycorrhizal fungi. Data from Brundrett et al. 1996.
Components Nutrient media

MMN (1) Pachlewski (2) FDA (3)
–1
Mineral nutrients (mg.l )
(NH4)2HPO4 250
NH4Cl 500
C4H12N2O6(4) 500
KH2PO4 500 1000 500
MgSO47H2O 150 500 500
CaCl22H2O 50 50
NaCl 25
Fe EDTA 20 20
H3BO3 2.8
MnCl22H2O 3.0
ZnSO47H2O 2.3
CuCl22H2O 0.63
Na2Mo42H2O 0.27
Carbohydrate source (g.l–1)
Maltose 5
Glucose 10 20 20
Malt extract 3
Vitamins (Og.l–1)
Thiamine HCl 0.1 0.1
Agar (g.l–1) 20 20 20
pH
Adjusted pH to 5.8 5.4 5.0
(1)
MMN: Modified Menin Norkrans medium (Marx 1969). (2) Pachlewski medium (Pachlewski
and Pachlewski 1974). (3) Ferry and Das (1968). (4) Ammonium tartrate
Using this process, it is also possible to accurately determine the weight of

mycelium or the number of living propagules contained in the inoculum.
Different methods have been evaluated to measure the quantity of mycelium
in the vermiculite-peat inoculum, such as the ergosterol assay (Martin et al.
1990) and chitin assay (Vignon et al. 1986), but none of them gave reliable
results (Mortier et al. 1988) since the peat interferes with colorimetric
measurements. With fungal strains that are able to grow from fragmented
hyphae, a mycelial suspension is produced in Erlenmeyer flasks or in a
fermentor filled with the liquid nutrient medium. The mycelium is then
washed in tap water to remove remaining nutrients, homogenized in a
Waring blender for about 10s, and suspended in sterile distilled water.
This type of fungal inoculum is quantified by measuring the fungal dry
weight per ml or by counting living propagules (determination of colony
forming units by spreading 1 ml of suspension on a 6-cm Petri dish with
nutrient agar). The hyphal fragment suspension is then mixed (1:1, v:v) with
distilled water containing 20 g.l–1 sodium alginate and 50 g.l–1 autoclaved
dry powdered sphagnum peat. The final solution is pumped through a
pipe with 2-mm holes. The drops fall into a 100 g.l–1 CaCl2 solution and
form beads of reticulated calcium alginate gel (Mauperin et al. 1987). The
beads are kept in CaCl2 for 24h at room temperature to ensure complete
reticulation. Then they are washed with tap water to eliminate NaCl and
CaCl2 and stored in air-tight containers at 4°C to prevent drying. This type
of inoculum can be kept up to 9 months in these conditions. The beads
are prepared with 1–2 g mycelium (dry weight) per liter of final solution
(Mortier et al. 1988).
3. Controlled Ectomycorrhization in Glasshouse, Nursery and

Field Conditions
The aims of the various technical procedures are to screen fungal isolates
for their compatibility with targeted host plants and to determine plant
growth promotion so the most efficient fungal isolate can be selected for
use in nurseries. This topic will be mainly illustrated by some data resulting
from controlled ectomycorrhization trials with Australian Acacia species
and tropical ECM fungal strains.
Seedlings can grow in soil (disinfected or not after autoclaving) or
in vermiculite-peat mixture (1:1, v:v). Several kinds of containers can be
used for growing seedlings in the greenhouse depending on the aim of the
experiment. The soil was then mixed with the ectomycorrhizal inoculum
(10:1, v:v). The fungal inoculum control (free of any treatment) received
an autoclaved mixture of vermiculite and peat moss moistened in nutrient
medium at the same rate. Then the pots were arranged in a randomized
complete block design. Numerous studies have reported that measuring the
promoting effects of fungal inoculation on the growth and nutrient status
of tree seedlings recorded in controlled conditions is the best way to select
the most promising and potential mycorrhizal inoculants for large-scale
inoculation programs in degraded lands in the tropics and Mediterranean
areas (Table 3). Unfortunately, although several methods of inoculation
can be used in practice, only some species of the genera Laccaria, Hebeloma,
Paxillus, Pisolithus, Scleroderma, Suillus and Rhizopogon have been routinely
used (Castellano 1996).
Table 3. Growth of some Australian Acacia species inoculated with different ectomycorrhizal
fungal strains (peat-vermiculite formulation) after 4 months culturing in glasshouse
conditions.
Fungal species Acacia species Effects on plant growth parameters References

(%) and ectomycorrhizal colonization
Shoot Root Ectomycorrhizal
biomass biomass colonization (%)
Pisolithus albus IR100 A. auriculiformis + 42.1 (1) + 38.6 45.2 A
P. albus IR100 A. mangium + 35.9 + 44.1 20.1 A
P. albus IR100 A. platycarpa + 43.1 + 9.8 31.6 A
Pisolithus sp. SL2 A. holosericea + 56.7 + 10.6 48.3 A
P. albus COI007 A. holosericea + 55.6 + 25.3 43.8 A
P. albus COI024 A. holosericea + 50.4 + 17.1 10.8 A
Pisolithus sp. COI032 A. holosericea + 54.9 + 12.6 15.0 A
P. albus IR100 A. holosericea + 57.1 + 48.9 25.2 A
P. tinctorius GEMAS A. holosericea + 57.8 + 14.1 43.1 A
Scleroderma A. holosericea + 52.9 + 21.0 53.4 A
dictyosporum IR109
S. verrucosum IR500 A. holosericea + 64.4 + 14.1 13.8 A
P. tinctorius GEMAS A. crassicarpa + 77.1 + 52.3 49.4 B
P. albus COI024 A. mangium + 54.5 + 52.1 41.7 C
Scleroderma sp. IR408 A. holosericea + 82.1 + 89.6 13.8 D
S. dictyosporum IR412 A. holosericea + 72.9 + 82.6 12.5 D
(1)
(mean value of ectomycorrhizal plants—mean value of the non ectomycorrhizal plants) x
100)/(mean value of the ectomycorrhizal plants).
A: Duponnois and Plenchette 2003. B: Lesueur and Duponnois 2005. C: Duponnois et al. 2002.
D: Duponnois et al. 2006
Numerous studies have reported that controlled mycorrhizal symbiosis

can significantly improve the growth of tree species in degraded soils
in situ (Smith and Read 2008). However, most of these experiments have been
performed in controlled glasshouse conditions. Data on the sustainability
of the positive effect of mycorrhiza on plant growth in field conditions
are lacking, especially in tropical areas. Controlled ectomycorrhization
experiments have been set up in Senegal using an Australian Acacia
holosericea and the ectomycorrhizal fungus Pisolithus albus strain IR100
(Duponnois et al. 2007). A positive effect of mycorrhizal inoculation on
A. holosericea development has been recorded in all of the experiments
(Fig. 1). These results suggest that without suitable symbionts, this species
has poor ability to scavenge for P under P-limiting conditions (Dommergues
et al. 1999) and clearly shows that the controlled mycorrhization of
A. holosericea could be a beneficial tool in improving the survival and
productivity of Acacia species. However, in order to evaluate the possible
Fig. 1. Field performance of A. holosericea trees (expressed in tons of wood biomass per ha with
1200 planted trees per ha) inoculated or not with Pisolithus albus strain IR100 in field trials
conducted in Senegal after 18 months of plantation. Data from Duponnois et al. 2007.
economic advantage of ectomycorrhizal inoculation, it is essential to carry

out field experiments in a range of situations representative of all major
types of plantations.
4. Mycorrhization Helper Bacteria (MHB)

The existence of this bacterial group was reported for the first time by
Garbaye and Bowen (1989). The MHB are commonly defined as telluric
bacteria enhancing the establishment of mycorrhizal symbiosis. To
isolate MHB, ECMs were surface-sterilized to only isolate bacteria closely
associated with the ectomycorrhizas. According to the results of Garbaye
and Bowen (1989), most of the bacteria isolated from Rhizopogonluteolus/
Pinusradiata ectomycorrhizas belong to the fluorescent pseudomonads group
and about 80% of the bacterial strains tested in this experiment stimulated
mycorrhiza formation, whereas 20% had neutral or negative influence.
Duponnois and Garbaye (1991) showed that among some bacteria isolated
from sporocarps of Laccariabicolor and ectomycorrhizas of Douglas fir with
L. bicolor, 14 bacterial isolates have significantly stimulated the
ectomycorrhizal colonization of the Douglas fir root systems with L. bicolor

strain S238 after 4 months of culture time in glasshouse conditions. These
authors did not find any links between the bacterial effect on mycorrhiza
formation and the origin of the bacteria or their taxonomic position. A bacterial
strain of Bacillus subtilis, isolated from Douglas/L. bicolor ectomycorrhizas,
has increased the ectomycorrhizal rate (percent of short roots mycorrhized
with L. bicolor) to 97.3% vs. 67.3% ectomycorrhizal rate recorded in the
control treatment (inoculated fungus without bacteria), while a bacterial
strains isolated from a L. bicolor sporocarp (Pseudomonas fluorescens strain
BBc6) exerts the same positive effect on mycorrhiza formation. This MHB
effect has been recorded in different environmental conditions (glasshouse
and nursery conditions) showing the high competitiveness of the bacterial
inoculant against the native microflora, but also in axenic conditions that
showed that the stimulation is an intrinsic property of each bacterial strain
and did not result from interactions within the microbial community in the
rhizosphere (Duponnois 1992).
MHB have been isolated from different plant-fungal combinations such
as arbuscular mycorrhizal fungi (herbaceous plants) or ectomycorrhizal
fungi (trees). To date, numerous studies have reported that many
bacterial strains could stimulate either the establishment of arbuscular
or ectomycorrhizal symbiosis (Table 4) (Frey-Klett et al. 2007). All these
results show that the MHB concept is generic and that it is not subjected to
the type of the mycorrhizal symbiosis nor to the taxonomic position of the
MHB strains (Frey-Klett et al. 2007).
The MHB effect on ectomycorrhizal symbiosis has been mainly
investigated with northern hemisphere tree species like Pseudotsugamenziesii,
Quercusrobur (Duponnois and Garbaye 1991), Picea abies, Pinus sylvestris
(Schrey et al. 2005), Pinus radiata (Garbaye and Bowen 1989) and some
ectomycorrhizal fungi, principally L. bicolor (Frey-Klett et al. 2007), Suillus
luteus (Bending et al. 2002). However, it has also been demonstrated that
some bacteria (Fluorescent pseudomonads) could help ectomycorrhizal
formation in tropical conditions with Australian Acacia species (Founoune
et al. 2002a,b).
MHBs belonging to the fluorescent pseudomonad group have been
frequently studied for their effect on ectomycorrhiza establishment in
glasshouse conditions. For example, the inoculation of a MHB, Pseudomonas
monteilii strain HR 13 has been studied on the mycorrhization of (i) an
Australian Acacia, A. holosericea, by several ectomycorrhizal fungi and one
endomycorrhizal fungus Glomusintraradices, and of (ii) several Australian
Acacia species by Pisolithusalbus strain IR 100 under glasshouse conditions
(Duponnois and Plenchette 2003). A promoting effect of HR13 on the
ectomycorrhizal establishment has been recorded with all the fungal isolates
(strains of Pisolithus and Scleroderma) (Fig. 2).
Table 4. MHB impacts on mycorrhiza establishment reported from the literature (From Frey-Klett et al. 2007).
226
Mycorrhizal fungi MHB species Host plant References

Ectomycorrhizal fungi
Amanita muscaria, Suillus bovinus Sptreptomyces Picea abies, Pinus sylvestris Schrey et al. 2005
Hebelomecrustuliniforme Unidentified bacteria Fagus sylvatica De Oliveira 1988
Laccaria bicolor Pseudomonas fluorescens, Pseudomonas Pseudotsuga menziesii Duponnois and Garbaye 1991
sp., Bacillus sp.
Laccaria fraterna, Laccaria laccata Bacillus sp., Pseudomonas sp. Eucalyptus diversicolor Dunstan et al. 1998
Lactariusrufus Paenibacillus sp., Burkholderia sp. Pinus sylvestris Poole et al. 2001
Pisolithus albus Pseudomonas monteilii Acacia holosericea Founoune et al. 2002a
Pseudomonas resinovorans
Pisolithus sp. Fluorescent Pseudomonads Acacia holosericea Founoune et al. 2002b
Rhizopogon luteolus Unidentified bacteria Pinus radiata Garbaye and Bowen 1989
Rhizopogon vinicolor, Laccaria laccata Arthrobacter sp. Pinus sylvestris Rózycki et al. 1994
Scleroderma spp., Pisolithus spp. Pseudomonas monteilii Australian Acacia species Duponnois and Plenchette
(2003)
Suillus luteus Bacillus sp. Pinus sylvestris Bending et al. 2002
Arbuscular mycorrhizal fungi
Endogone sp. Pseudomonas sp. Trifolium spp., Cucumis sativum, Mosse 1962
Allium cepa
Gigaspora margarita Azospirillum brasilense Pennidetum americanum Rao et al. 1985
Glomus clarum Azotobacter diazotrophicus, Klebsiella Ipomoea batatas Paula et al. 1992
sp.
Glomus deserticola Klebsiella pneumoniae, Alcaligenes Unicola paniculata Will and Sylvia 1990
denetrificans
Glomus fasciculatum Azotobacter chroococcum Lycopersicum esculentum Bagyaraj and Menge 1978
Glomus fasciculatum, Glomusmosseae Rhizobium meliloti Medicago sativa Azcón-Aguilar et al. 2003
Glomus fasciculatum, Glomusmosseae, Bacillus coagulans Morus alba, Carica papaya Mamatha et al. 2002
Glomus caledonium
Glomus fistulosum Pseudomonas putida Zea mays, Solunum tuberosum Vosatka and Gryndler (1999)
Glomus intraradices Bacillus subtilis, Enterobacter sp. Allium cepa Toro et al. 1997
Glomus intraradices Pseudomonas monteilii Acacia holosericea Duponnois and Plenchette 2003
Glomus intraradices Rhizobium Anthyllis cytisoides Requena et al. 1997
Glomus intraradices Agrobacterium rhizogenes, Hordeum vulgare, Triticum aestivum Fester et al. 1999
Pseudomonas fluorescens, Rhizobium
leguminosarum
Glomus intraradices Streptomyces coelicolor Sorghum Abdel-Fattah and Mohamedin
2000
Glomus mosseae Paenibacillus sp. Sorghum bicolor Budi et al. 1999
Glomus mosseae Pseudomonas sp. Lycopersicum esculentum Barea et al. 1998
Glomus mosseae Bradhyrhizobium japonicum Glycine max Xie et al. 1995
Glomus mosseae Pseudomonas fluorescens Lycopersicum esculentum Gamalero et al. 2004
Glomus mosseae Brevibacillus sp. Trifolium pratense Vivas et al. 2003
Glomus mosseae, Glomus intraradices Paenibacillus brasilensis Trifolium Artursson 2005
Complex of AMF (1) Pseudomonas putida Trifolium Meyer and Linderman (1986)
Complex of indigenous AMF Pseudomonas sp. Triticum aestivum Babana and Antoun 2005
Complex of AMF Bacillus mycoides Herbaceous plant species von Alten et al. 1993
(1)
AMF: Arbuscular Mycorrhizal Fungi.
Ectomycorrhizal isolates
Fig. 2. Effect of Pseudomonas monteilii isolate HR13 on ectomycorrhiza formation of

Acaciaholosericea with ectomycorrhizal fungi after 4 months of culturing under glasshouse
conditions.
*: Significant effect of P. monteilii isolate on ectomycorrhizal formation for each fungal strain.
: Fungal strain inoculated alone. : Fungal strain inoculated with P. monteilii HR13.
The MHB P. monteilii strain HR13 has also significantly increased the
ectomycorrhizal colonization for all the Australian Acacia species tested
in this experiment (A. auriculiformis, A. eriopoda, A. holosericea, A. mangium
and A. platycarpa) (Fig. 3).
MHB have also been tested for their influence on ectomycorrhizal
formations between Douglas and L. bicolor strain S238 in nursery conditions.
One bacterial isolate (Pseudomonas fluorescens isolate BBc6) increased
mycorrhiza formation from around 60% (per cent of short roots mycorrhized
with L. bicolor S238N without bacterial inoculation) to 87%. This experiment
was performed with a peat-vermiculite fungal inoculum. Similar positive
effects were recorded when this bacterial strain was mixed together with the
fungus in alginate beads (Duponnois 1992). After 4 months of plantation,
BBc6 stimulated ectomycorrhizal infection from 42% to 75%. Frey-Klett et
al. (1999) showed that five months after inoculation and sowing, bacterial
inoculation significantly improved the percentage of ectomycorrhizal short
roots on plants inoculated with two doses of fungal inoculum of L. bicolor
(50 and 100 mg m–2 dry weight mycelium entrapped in alginate beads
mixed into the soil at a constant dose of 1 litre m–2) in a nursery (Fig. 4). The
Acacia species
Fig. 3. Effect of Pseudomonas monteilii isolate HR13 on ectomycorrhiza formation with Pisolithus
albus isolate IR100 of five Australian Acacia species after 4 months of culturing in glasshouse
conditions. For the legend, see Fig. 2.
Fig. 4. Dose effect of P. fluorescens isolate BBcR8 on the percentage of Douglas-fir short roots
mycorrhizal with L. bicolor S238, 23 weeks after fungal and bacterial inoculations. : Fungal
inoculation dose: 50 mg dry weight mycelium m–2. : Fungal inoculation dose: 100 mg dry
weight mycelium m–2. For each fungal inoculation dose, columns indexed by the same letters
are not significantly different according to a Scheffe test. Data from Frey-Klett et al. (1999).
lowest bacterial dose increased mycorrhizal colonization from 45 to 70%

in plants inoculated with the lower amount of fungal inoculum, and from
64 to 77% in plants inoculated with the higher amount of fungal inoculum.
The lowest bacterial dose increased mycorrhizal colonization more than
the highest bacterial dose (Fig. 4).
5. Termite Mounds of Macrothermes subhyalinus as a

Catalyst of Ectomycorrhizal Formation: Islands of Microbial
Fertility in Tropical Forest Ecosystem
Termite (Isoptera) mounds are frequently observed in tropical ecosystems,
particularly in savanna environments. Termite activities result in the
translocation of large amounts of soil from different depths (Holt and
Lepage 2000). These biological disturbances strongly impact their local
environment and have a major influence on soil’s physical and chemical
characteristics (Holt and Lepage 2000) demonstrating the termite role as
ecosystem engineers.
Previous microbiological studies of termite mounds have described the
microbial communities in grass-, litter- and soil-feeding termite mounds
(Duponnois et al. 2005b). Fluorescent pseudomonads have only been
detected in mound powder of one species of termite, M. subhyalinus (Table
5).
The phylogenetic analysis done on these fluorescent pseudomonads
showed that these bacteria mostly belonged to Pseudomonas monteillii
species (Duponnois et al. 2006). The dual inoculation of termite mound
and ectomycorrhizal fungus (Scleroderma sp. isolate IR408 or S. dictyosporum
isolate IR412) significantly enhanced root growth of A. holosericea seedlings
after 4 months of culturing in greenhouse conditions (Table 6). In addition,
termite mound amendment significantly increased ectomycorrhizal
formation of A. holosericea with both of the fungal isolates tested in this
study (Table 6). It has been also reported that an isolate of P. monteilii (isolate
HR13) could stimulate ectomycorrhizal formation between A. holosericea and
different fungal isolates (Scleroderma dictyosporum, S. verrucosum, P. albus
and P. tinctorius) (Founoune et al. 2002a, Duponnois and Plenchette 2003)
as well as AM establishment between Acacia species and Glomus intraradices
(Duponnois and Plenchette 2003).
It has also been shown that another strain of P. monteillii, strain KR9,
isolated from M. subhyalinus termite mounds, stimulated ectomycorrhizal
formation between S. dictyosporum IR412 and A. holosericea and the growth
of the host plant (Duponnois et al. 2006) (Table 7).
As the termite mound powder exerted a significant positive effect on
mycorrhizal establishment and plant growth, an experiment was conducted
Table 5. Biological and chemical characteristics of Cubitermes, Macrotermes and Trinervitermes

mound powders. Data from Duponnois et al. 2005b.
Cubitermes sp. Macrotermes Trinervitermes sp.

subhyalinus
NH4+ (Og N g–1 of dry mound powder) 40.9 b * 9.4 a 37.1 b
NO3– (Og N g–1 of dry mound powder) 206.9 a 3408.9 b 42.3 a
Available P (Og g–1 of dry mound powder) 7.7 b 3.5 a 10.2 c
Microbial biomass (Og C g–1 of dry mound 17.0 a 22.5 ab 36.5 b
powder)
Fluorescent pseudomonads (x 102 CFU g–1 <1a 79.3 b <1a
of dry mound powder)
Actinomycetes (x 102 CFU g–1 of dry 37.3 b 39.5 b 22.5 a
mound powder)
Ergosterol (Og g–1 of dry mound powder) 1.381 b 0.316 a 1.717 b
*: Data in the same line followed by the same letter are not significantly different according
to Student’s “t” test (p < 0.05).
Table 6. Impacts of fungal inoculation and Macrotermes subhyalinus amendment on Acacia

holosericea growth and ectomycorrhizal establishment after 4 months of culturing in greenhouse
conditions.
Treatments Shoot biomass Root biomass Ectomycorrhizal

(mg dry weight) (mg dry weight) colonization (%)
Control 261 a (1) 33 a 0
Scleroderma sp. IR408 1458 c 318 bc 12.8 a
S. disctyosporum IR412 964 b 190 ab 14.2 a
M. subhyalinus (MS) 1288 bc 238 b 0
IR 408 + MS 1051 b 432 cd 23.6 b
IR412 + MS 1140 bc 606 d 25.8 b
(1)
Data in the same column followed by the same letters are not significantly different according
to the Student’s “t” test (p < 0.05).
Table 7. Effect of the Pseudomonas monteilii strain (isolate KR9) and/or Scleroderma disctyosporum
IR 412 on mycorrhiza formation, rhizobial development, and growth of Acacia holosericea after
4 months of culturing under glasshouse conditions.
Treatments
Control P. monteilii S. dictyosporum KR9 +
KR9 IR412 IR412
Shoot biomass (mg dry weight) 532 a (1) 553 a 1236 b 1786 c
Root biomass (mg dry weight) 184 a 198 a 536 b 868 c
Number of nodules per plant 4.2 a 4.6 a 8.3 b 12.4 c
Total nodule weight per plant (mg) 6.8 a 7.1 a 15.9 b 25.3 c
Ectomycorrhizal colonization (%) 0 0 28.3 a 48.5 c
(1)
Data in the same line followed by the same letter are not significantly different according
to the Student’s “t” test (p < 0.05).
to test a new mycorrhizal inoculation process using a “catalyser” of the

mycorrhizal establishment (termite mounds of M. subhyalinus) to minimize
the required volume of fungal inoculum added to the cultural substrate
(Duponnois 2006). The effects of the termite mound powder have been
explored on the mycorrhiza formation of an Australian Acacia, Acacia
holosericea, by an ectomycorrhizal fungus (Pisolithus albus IR 100) and an
arbuscular mycorrhizal (AM) fungus (Glomus intraradices). For this, seeds of
A. holosericea were surface-sterilized with 95% sulfuric acid for 60 minutes
and transferred aseptically to Petri dishes filled with 1% (w/v) agar/water
medium. These plates were incubated at 25°C in the dark. The germinating
seeds were used when rootlets reached 1–2 cm long. A disinfected (120°C,
40 min) sandy soil was mixed with 0, 1, 5 and 10% (v/v) of M. subhyalinus
mound powder. Ectomycorrhizal and AM inoculation were performed by
mixing the soils with either the fungal inoculum (10/1, v/v) or a control:
an autoclaved mixture of moistened vermiculite/peat moss at the same
rate (ectomycorrhizal control) or with non-mycorrhizal millet roots and
their rhizosphere soil (AM control). Plastic containers (30 x 30 x 5 cm)
filled with soil mixtures were each planted with 100 pre-germinated
seeds of A. holosericea. Seedlings were kept in a glasshouse under natural
light (daylight approximately 12 h, mean daytime temperature 30°C) and
watered daily without fertilizer. After 2 months of culturing, the seedlings
were uprooted from the containers and transferred into 1 liter pots (one
seedling per pot) filled with the same sterilized soil but without termite
mound powder amendment and fungal inoculation. Two months after the
seedling transplantation, A. holosericea plants were uprooted and the root
systems gently washed. Their growth was measured as well as the extent
of ectomycorrhizal or arbuscular mycorrhizal colonization. Beside this
experiment, the conventional process used for controlled mycorrhization in
forest nurseries was performed by planting A. holosericea seedlings in 11 pots
filled with the same disinfected soil previously used. For ectomycorrhizal
inoculation, the soil was mixed with P. albus IR100 fungal inoculum (10/1,
v/v) whereas the control treatment (without fungus) received an autoclaved
mixture of moistened (MMN medium) vermiculite/peat moss at the same
rate. For arbuscular mycorrhizal inoculation, a hole was made in each
pot and filled with 1 g fresh millet root (mycorrhizal or not for the control
treatment without fungus). The A. holosericea seedlings were kept in a
glasshouse under natural light. After 4 month’s culture time, A. holosericea
plants were uprooted and their shoot and root biomass, mycorrhizal indices
were measured as described earlier. The results showed that termite mound
powder amendment significantly stimulated the mycorrhizal formation
from both types of fungal isolates (Tables 8 and 9).
This promoting effect could result from (i) the increase of plant growth
induced by nutrients added with termite mound powder (particularly root
Table 8. Influence of arbuscular mycorrhizal inoculation and termite mound powder

amendment rates on A. holosericea growth and on mycorrhiza formation after 2 months of
culturing in 1-liter pots.
Factor (A) Shoot biomass (mg Root biomass (mg Mycorrhizal
dry weight) dry weight) colonization (%)
Fungal inoculum rate (%)
0 190.1 (22.6) (1) a (2) 70.5 (8.5) a 0
1 783.5 (41.5) b 238.1 (18.4) b 41.8 (2.5) a
5 1236.1 (48.9) c 357.1 (20.3) c 70.1 (2.6) c
10 1510.1 (73.9) d 334.5 (35.9) c 55.5 (4.3) b
Termite mound amendment rate (%)
0 709.6 (101.1) a 175.0 (25.6) a 31.0 (5.3) a
1 1000.5 (142.9) b 274.5 (43.3) b 47.3 (7.3) b
5 1019.5 (125.2) b 294.5 (30.7) b 41.5 (6.4) b
10 988.5 (113.4) b 256.1 (29.9) ab 47.5 (6.6) b
Fungal inoculum rate (FIR) S S S
Termite mound amendment rate (TAR) S S S
FIR x TAR NS S S
S: significant (p < 0.05), NS: not significant (p < 0.05). (1) Standard error of the mean. (2) Data in
the same column and for each factor followed by the same letter are not significantly different
according to the Newman-Keuls test (p < 0.05). (A) Values are means of 20 replicates for fungal
inoculum and termite mound amendment rates. Fungal inoculum rate factor is for all termite
mound amendment rate treatments combined; the termite amendment factor is for all fungal
inoculum rate treatments combined.
growth) and (ii) the addition via the termite mound of a bacterial group
(i.e., fluorescent pseudomonads) that could act as Mycorrhization Helper
Bacteria (MHB) (Duponnois and Plenchette 2003). The data recorded from
this study were similar to those obtained from the cultural practice using
the conventional process of controlled mycorrhization (Table 10).
One of the main problems encountered with the conventionally
controlled mycorrhization of forest planting stocks is the large quantity of
fungal inoculum required for the production of high quality mycorrhizal
plants in nursery conditions. In tropical and Mediterranean areas, the
mycorrhizal inoculum dose added per plant to the cultural substrate is
Table 9. Effect of ectomycorrhizal inoculation and termite mound powder amendment rates
on A. holosericea growth and on mycorrhiza formation after 2 months of culturing in pots of
1 liter.
Factor (A) Shoot biomass Root biomass Mycorrhizal

Fungal inoculum rate (%)
0 191.0 (27.9) (1) a (2) 74.5 (12.9) a 0
1 556.5 (61.4) b 166.1 (15.2) b 57.6 (2.1) a
5 745.5 (43.6) c 221.1 (17.4) c 61.1 (1.5) a
10 1197.5 (96.6) d 364.5 (27.1) d 61.6 (1.9) a
Termite mound amendment rate (%)
0 501.0 (77.1) a 167.0 (27.4) a 41.1 (5.5) a
1 612.2 (78.1) ab 195.0 (30.2) ab 41.6 (5.6) a
5 858.6 (96.9) c 250.5 (27.8) b 51.2 (6.8) c
10 719.1 (135.5) bc 213.5 (33.6) b 46.3 (6.4) b
Fungal inoculum rate (FIR) S S S
Termite mound amendment rate S S S
(TAR)
FIR x TAR S S S
S: significant (p < 0.05), NS: not significant (p < 0.05). Standard error of the mean. (2) Data in
(1)
the same column and for each factor followed by the same letter are not significantly different
according to the Newman-Keuls test (p < 0.05). (A) Values are means of 20 replicates for fungal
inoculum and termite mound amendment rates. Fungal inoculum rate factor is for all termite
mound amendment rate treatments combined; the termite amendment factor is for all fungal
inoculum rate treatments combined.
usually of 0.1 liter per liter of soil (ectomycorrhizal inoculation) and 1 g

fresh weight of mycorrhizal root per litre of soil (arbuscular mycorrhizal
inoculation) (Duponnois et al. 2007). With this innovative process, these
volumes are drastically lowered since, with only 450 ml of ectomycorrhizal
inoculum or arbuscular mycorrhizal inoculum (mixture of spores,
mycorrhizal roots, and rhizosphere soil), about 100 mycorrhized A.
holosericea plants can be produced with the same growth and mycorrhizal
colonization as mycorrhized plants produced with the conventional
controlled mycorrhization procedure. Therefore the inoculation cost could
be greatly decreased since termite mounds are commonly found in tropical
ecosystems while requiring very low fungal amounts.
Table 10. Influence of ectomycorrhizal or AM inoculations on growth of A. holosericea and

on mycorrhiza formation after 4 months of culturing in glasshouse conditions with the
conventional process of controlled mycorrhization.
Treatments Shoot biomass Root biomass Mycorrhizal

AM inoculation
Uninoculated control 648.1 a (1) 312.2 a 0
Glomus intraradices 1834.1 b 546.3 b 59.3
Ectomycorrhizal inoculation
Uninoculated control 550 a 290 a 0
Pisolithus albus IR100 1120.1 b 560.2 b 35.6
(1)
For each type of fungal inoculation, data in the same column followed by the same letter
are not significantly different according to the Newman Keuls test (p< 0.05).
6. Conclusion
Numerous studies have reported the benefits that result from using fungal
inoculants to enhance the development of tree seedlings in glasshouse and
nursery conditions. In the same way, numerous technical procedures have
been described to produce cost-effective mycorrhizal inocula. The results
presented in this chapter suggest that inoculation with ectomycorrhizal
fungi can improve the early growth of the most planted tree species in
tropical and Mediterranean forests and that this technique will accelerate the
rehabilitation of degraded forests. Unfortunately, mycorrhizal inoculation
remains underexploited in nursery practices, although the management of
tree mycorrhizal status could have very important implications for tropical
reforestation programs in developing countries. Controlled mycorrhization
could be highly improved if the fungal inoculation is associated with
Mycorrhization Helper Bacteria or termite mound powder of M. subhyalinus.
These innovative procedures decrease the necessary amount of fungal
inoculum while keeping the same mycorrhizal establishment that is usually
measured with the conventional process of controlled mycorrhization.
Hence the use of these cultural practices could be facilitated and encouraged
in forest nurseries. Future research should prioritize the generalizability of
these controlled experiments to long-term applications in tree plantations
and reforestation projects.
Acknowledgements
We thank the anonymous referees for their valuable comments on this
study, and Krista L. McGuire and Caitlyn Gillikin for improving the
language.
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CHAPTER
13
Biodiversity and Sustainable
Use of Wild Edible Fungi in the
Sudanian Centre of Endemism:
A Plea for Valorisation
Nourou Soulemane Yorou,1,2* N’Golo Abdoulaye Koné,3
Marie-Laure Guissou,4 Atsu Kudzo Guelly,5 Dao Lamèga
Maba,2,5 Marius R.M. Ekué6 and André De Kesel7
1. Introduction
Wild Edible Fungi (WEF) play an important role in the livelihood of local
inhabitants (Boa 2004). In tropical Africa, picking wild edible mushrooms is a
lucrative activity and involves hundreds of rural women (Boa 2004, Degreef
et al. 1997, Buyck 1994a). Over 300 edible mushrooms are recorded in sub-
Saharan Africa (Rammeloo and Walleyn 1993, Walleyn and Rammeloo 1994).
Still, mycophagy greatly varies from one country to another, even within
ethnic groups of the same area (Yorou and De Kesel 2002, Guissou et al.
2008). Annual consumptions of over 30 kg per habitant have been recorded
in Central and East Africa (Degreef et al. 1997). In rural tropical Africa, WEF
are important in terms of species richness and consumed quantities, but
also as a potential source of proteins, vitamins, and minerals (Degreef et
al. 1997, Adewusi et al. 1993, Ogundana and Fagade 1981, 1982, De Kesel
and Malaisse 2010).
Authors’ affiliations given at the end of the chapter.

In tropical Africa, ethnomycological knowledge appears to be most

elaborate and widespread in the Zambesian miombo forests where Degreef
et al. (1997) and De Kesel and Malaisse (2010) enumerated 42 edible
species in Katanga (Democratic Republic of Congo). Other important
ethnomycological contributions from that region are available for Tanzania
(Härkönen 1992, Härkönen et al. 1994, 2003), Zambia (Pegler and Piearce
1980, Piearce 1987), Malawi (Morris 1984, 1994), and Burundi (Buyck 1994a,
Buyck and Nzigidahera 1995). The contributions from Eyi-Ndong (2009),
Eyi-Ndong and Degreef (2009, 2010), Eyi-Ndong et al. (2011), combined
with those from Mossebo and Amougou (2002), Mossebo et al. (2002), and
Malaisse et al. (2008), provide an extensive documentation of WEF in the
Guineo-Congolese area. As far as West Africa is concerned, investigations
on WEF along with subsequent ethnomycological knowledge have been
initiated only recently in a few countries such as Benin (De Kesel et al.
2002, Yorou 2000, 2010, Yorou and De Kesel 2002, Yorou et al. 2002a,b),
Burkina Faso (Guissou et al. 2005, 2008), Senegal (Bâ et al. 2011), Niger
(Hama et al. 2010), and Ivory Coast (Koné et al. 2010a,b). Though fungi are
widespread and commonly used in rural areas of West Africa, numerous
investigations demonstrated that women and in some cases children are
the traditional harvesters, sellers of WEF, and keepers of ethnomycological
information (Buyck 1994a, De Kesel et al. 2002, Guissou et al. 2008). In WA,
local mycological knowledge is diverse and full of instructions, including
patterns of collecting, drying, processing, and cooking techniques (Yorou
and De Kesel 2002, Guissou et al. 2005, 2008). Yorou and De Kesel (2002)
and De Kesel et al. (2002) demonstrated that folk classification of WEF
by Nagot people in Central Benin challenges modern taxonomy, whilst
local nomenclature carries important information related to the ecology
(either saprotrophic or symbiotic), growth habit (solitary or gregarious),
morphology/size, colour, flesh context, and taste of the mushroom.
Unfortunately, local usage and subsequent ethnomycological know-how
are actively eroding and/or disappearing from one generation to another
(Guissou et al. 2008), mostly due to urbanization and modern schooling.
In tropical Africa, WEF occur predominantly in the Zambesian and
Sudanian Centres of Endemism (Ducousso et al. 2002, De Kesel et al. 2002,
Härkönen et al. 2003, De Kesel and Malaisse 2010). Still, the dense evergreen
forests of the Guineo-Congolese domain host an important diversity of WEF
(Eyi-Ndong et al. 2011). Though a few species may occur throughout the
year, the occurrence of edible ectomycorrhizal (EcM) species is seasonal.
Whilst numerous species appear just after the first rains, many others
need persistent rains over a longer period. In seasonal forests of tropical
Africa, natural production of WEF is related to the presence, health, and
photosynthetic performance of partner tree species. The latter are mainly
Isoberlinia spp., Berlinia spp., Uapaca spp., and Anthonotha spp., among
Wild Edible Fungi of West Africa 243
others (Bâ et al. 2012, Ducousso et al. 2002). Unfortunately, many such
EcM trees are disappearing at an alarming rate (Neuenschwander et al.
2011), either through large scale deforestation or selective logging of timber
species. The process of deforestation is due mostly to shifting cultivation,
timber exploitation, land conversion to pastures, and increasing urban
demands of wood fuel (charcoal). Shifting cultivation usually results in
the slashing, burning and conversion of large forest areas into croplands,
whilst timber exploitation consists primarily of selective logging the
economically important aforementioned EcM trees. In any case, the removal
and destruction of partner tree species not only erodes the diversity of the
area, but also severely hampers the natural production of locally exploited
WEF. In Benin, about 28 WEF are threatened with extinction (Yorou and De
Kesel 2011), chiefly because of forest fragmentation, habitat disturbance, and
timber exploitation for charcoal. In a similar way, numerous fungi-rich WA
ecosystems are undergoing an unrestrained process of fragmentation (Hahn-
Hadjali et al. 2010, Wegmann et al. 2010), along with the disappearance
of economically important timber species and their associated WEF. This
process deeply affects the lives of the people living in surrounding villages
and cities. Due to such uncontrolled deforestation, the forest ecosystems
of WA are not only losing their potential as providers of Non Timber
Forest Products, but also their ability to improve the livelihood of rural
communities. As WEF are diverse and of local economic importance, the
installation of an economic sector based on WEF can serve as a model to
promote awareness among rural people, forest officers/administration,
and decision makers. The main goal of establishing an economic sector
of WEF is to face poverty and to conserve the hosts and the ecosystems
of fungal species all alike. In the present contribution, we are attempting
(1) to explore the diversity of WEF and fungal hotspots in West Africa
and (2) to highlight the natural productions, phenology, nutritional, and
socio-economic exploitation of WEF in West Africa. Data are assembled
from intensive mycological investigations in some West African countries
(Benin, Togo, Burkina Faso…) over the last 10 years, but also from extensive
mycological exploration in various ecosystems from other countries like
Ghana, Mali, Ivory Coast, and Guinea (started in 2009). Using a case study
from Benin, we will analyse different steps towards the establishment of
an economic sector based on WEF in West Africa.
2. Fungal Hotspots and WEF-rich Ecosystems in West Africa

WEF are generally composed of two major biological groups, notably the
symbiotic species and the saprotrophic ones. In West Africa, there is a
north-south gradient in diversity, ecological groups (symbiotic, parasitic,
or saprotrophic), and natural productions of WEF. The dense southern
forests of the coastal provinces of countries like Benin, Togo, Ghana, and
Ivory Coast exhibit a rather low diversity of edible, mostly saprotrophic
fungi. Most of the native edible taxa are members of Pleurotus, Marasmiellus,
Lentinus, Cookeina, and Armillaria. Plantations of exotic tree species, man-
made ecosystems, fallows, and to some extent disturbed habitats generally
harbour few edible fungi of the genera Chlorophyllum, Macrolepiota,
Volvariealla, and Agaricus. Towards the North, i.e., entering the ecological
zone where woodlands and gallery forests of the Guineo-Sudanian type start
to dominate (White 1986), a high diversity of edible EcM fungi is observed
(Bâ et al. 2011, De Kesel et al. 2002, Ducousso et al. 2002, Yorou et al. 2002a,b).
For landlocked countries such as Burkina Faso, Niger, and Mali, WEF are
most diverse in the southern Sudanian woodlands (Guissou 2005, Sanon
et al. 1997). The Sudanian Centre of Endemism (SCE) is home to hundreds
of WEF including notably representatives of Russula, Lactarius, Lactifluus,
Amanita, and Boletus. Numerous termites’ fungi (Termitomyces spp.) are
present in West Africa. These taxa are considered an important resource
and they play a seasonal but substantial role in the local diet, medicine,
and cash incomes for native villagers. The SCE is a narrow vegetation band
stretching from East (Soudan) to West (Senegal) between 9° and 11° North
latitudes (White 1986). Vegetation is composed of a mosaic of woodlands,
savannahs and gallery forests, mostly dominated by tree species belonging
to the Caesalpiniaceae, Phyllantaceae and Dipterocarps. In this area, some
timber species such as Afzelia spp., Isoberlinia spp., Uapaca spp., Berlinia
grandiflora and Anthonotha spp. form EcM symbiosis with numerous WEF.
Ectomycorrhizal fungi are predominant not only in terms of species richness,
but also in terms of fresh biomass, a large proportion of which is consumed
by local inhabitants (Table 1). Though woodlands form vast areas in the SCE,
they are actually fragmented and regressing annually (FAO 2010). More
conserved and semi-natural woodland ecosystems are located in numerous
forest reserves. Due to their conservation status as natural or semi-natural
ecosystems, Caesalpinioid and Phyllantioid-dominated woodlands and
gallery forests of the SCE emerge as fungi-rich ecosystems in West Africa.
The presence in their flora of important EcM tree species such as Berlinia spp.
and Uapaca spp. makes them remarkable WEF-rich ecosystems, generally
acting as refuge ecosystems for numerous unique and rare species (Yorou
and De Kesel 2011). Their generally fragmented size (Natta et al. 2002,
Sparovek et al. 2002) coupled with the unique and rare mycoflora they host
make the gallery forests important fungal hotspots with top conservation
priority. Because of the presence in its flora of numerous EcM trees such as
Anthonotha spp., Gilbertiodendron spp. Paramacrolobium coeruleum (to quote
only a few), Guinea harbours the highest diversity of potential WEF that
have been scantily documented ethnomycologically.
Table 1. Contrasting relative dominance of fungal flora between two dense forests (FD =
dense forest and FR = riparian forest) lacking EcM trees and 4 woodland ecosystems (SA =
shrub savannah, SB = wooded savannah, FC = Woodland of Uapaca spp. and FI = woodland
of Isoberlinia spp.) dominated by EcM trees in Sudanian area.
FD FR SA SB FC FI
Total Biomass 8.6 ± 5.2 26.7 ± 4.7 241.7 ± 13.88 223.7 ± 293.9 ± 285.7 ± 16
(kg/ha/year) 15.4 16,1
% Ectomycorrhizal 6 6.8 88.3 98.9 95.0 90.0
fungi from the total
biomass
% Saprotrophic 92.0 93.2 11.7 1.1 4.0 10.0
species from the total
biomass
% Wild edible 2.3 4.1 85.4 87.7 73.1 78.9
Fungi from the total
biomass
Source: Yorou NS (2000), unpublished data.
3. Diversity of WEF in West Africa with Special Emphasis on

the Most Abundant Genera
The specific diversity of WEF in a given country depends on a range of
factors, including its surface area and the diversity in ecosystems and
floristic composition. West Africa counts conservative estimates of about
55 to 70 edible fungal species (Bâ et al. 2011). Whilst monographs are
progressing in a few countries such as Benin (De Kesel et al. 2002) and
Burkina Faso (Guissou 2005), numerous other countries have never been
investigated ethnomycologically. De Kesel et al. (2002) recorded about 50
edible species in Benin. In Burkina Faso, Guissou et al. (2008) reported on
about 40 useful wild fungi of which 21 are consumed by various ethnic
groups. Over 70 EcM fungi were recorded in Ivory Coast by Bâ et al. 2011,
a large proportion of which are edible. Similarly, about 110 EcM fungi,
composed mostly of edible ones, are present in various ecosystems of
Guinea (Rivière et al. 2007). According to Bâ et al. (2011), a non exhaustive
list of edible species from Senegal includes 45 species. The total list of 72
species whose edibility is confirmed in Benin, Burkina Faso, Ivory Coast
and Togo is presented in Table 2.
In general, genera Lactarius s. l. (including Lactifluus), Amanita,
Termitomyces and Russula are the most common WEF in WA (Fig. 1).
All four genera Lactifluus, Lactarius, Russula and Amanita form EcM
symbioses with the above-mentioned tree species. Termitomyces are not
ectomycorrhizal, but they form mutualistic symbiosis with many termite
species belonging to the Macrotermitinae subfamily, the so-called fungus
growing termites.
Table 2. List of WEF confirmed as edible in some West African countries
Fungal Species Biological

group Countries
Benin Burkina Ivory Togo
Faso Coast
Afroboletus luteolus (Heinemann) Symbiotic + +
Pegler & Young
Afrocantharellus platyphyllus Symbiotic + +
(Heinem.) Tibuhwa
Agaricus sp1 Saprotrophic +
Agaricus sp2 Saprotrophic +
Agaricus goossensiae Heinem. Saprotrophic + +
Agaricus subsaharianus L.A. Parra, Saprotrophic + + + +
Hama & De Kesel
Agaricus volvatulus Heinem. & Goos Saprotrophic +
Amanita aff. craseoderma Bas Symbiotic +
Amanita crassiconus Bas Symbiotic +
Amanita massasiensis Härk. & Saarim. Symbiotic + + +
Amanita strobilaceovolvata Beeli Symbiotic +
Amanita subviscosa Beeli Symbiotic + + +
Amanita cf. rubescens (Pers.: Fr) S. F. Symbiotic + +
Gray
Amanita cf. xanthogala Bas Symbiotic +
Auricularia cornea Ehrenb. Saprotrophic + +
Boletus loosii Heinem. Symbiotic +
Cantharellus congolensis Beeli Symbiotic + +
Cantharellus addaiensis Heinem. Symbiotic + + +
Cantharellus aff. rufopunctatus var Symbiotic + +
rufopunctatus (Beeli) Hein
Cantharellus pseudofriesii Heinem. Symbiotic +
Macrolepiota cf. rachodes Saprotrophic + +
Cookeina sulcipes (Berk. ) Kuntze Saprotrophic + +
Marasmiellus inoderma (Berk.) Singer Saprotrophic + +
Gymnopus sp. Saprotrophic +
Hygrophoropsis aurantiaca (Wulfen) Saprotrophe +
Maire
Hygrophoropsis sp. Saprotrophic +
Lactarius baliophaeus Pegler Symbiotic +
Lactifluus denigricans (Verbeken & Symbiotic +
Karhula) Verbeken
Lactifluus densifolius (Verbeken & Symbiotic +
Karhula) Verbeken
Table 2. contd....
Table 2. contd.

group Countries
Faso Coast
Lactifluus edulis (Verbeken & Buyck) Symbiotic + +
Buyck
Lactifluus flammans (Verbeken) Symbiotic +
Verbeken
Lactifluus gymnocarpus (Heim ex Symbiotic + + +
Singer) Verbeken
Lactiflluus gymnocarpoides (Verbeken) Symbiotic + + + +
Verbeken
Lactifluus luteopus (Verbeken) Symbiotic + + +
Verbeken
Lactarius pseudogymnocarpus Symbiotic + +
(Verbeken) Verbeken
Lactifluus meduase Verbeken Symbiotic +
Lactarius saponaceus Verbeken Symbiotic +
Lactarius tenellus Verbeken & Walleyn Symbiotic + +
Lactifluus sudanicus nom. prov. symbiotic +
(MD105)
Lentinus squarrosulus Mont. Saprotrophic + + +
Lentinus aff. atrobrunneus Pegler Saprotrophic +
Lentinus aff. brunneofloccosus Pegler. Saprotrophic +
Pleurotus tuber-regium (Fr.) Fr. Saprotrophic + + + +
Leucoagaricus cf. bresadolae (Schulz.) Saprotrophic +
Bon & Boiffard
Leucocoprinus cretatus Locq. ex Saprotrophic + +
Lanzoni
Macrocybe lobayensis (R. Heim) Pegler Saprotrophic + + +
& Lodge
Marasmius heinemaniannus V. Antonin Saprotrophic +
Phlebopus sudanicus (Har. & Pat) Symbiotic + + + +
Heinem.
Pleurotus sp1 Saprotrophic +
Pleurotus sp2 Saprotrophic +
Pleurotus cystidiosus Muller Saprotrophic +
Psathyrella tuberculata (Pat) A. H. Sm Saprotrophic + + + +
Russula sp1 Symbiotic +
Russula cellulata Buyck Symbiotic +
Russula congoana Pat. Symbiotic + + +
Table 2. contd....
Table 2. contd.

group Countries
Faso Coast
Russula oleifera Buyck Symbiotic + +
Russula aff. rubroalba (Singer) Symbiotic + +
Romagnesi
Russula sesenagula Beeli Symbiotic +
Schizophyllum commune (L.) Fr Saprotrophic +
Termitomyces clypeatus R. Heim Symbiotic + +
Termitomyces eurrhizus (Berk.) R. Symbiotic + + +
Heim
Termitomyces fuliginosus R. Heim Symbiotic + + + +
Termitomyces letestui (Pat.) R. Heim Symbiotic + + +
Termitomyces medius R. Heim et Symbiotic + + +
Grassé
Termitomyces microcarpus (Berk. & Symbiotic + +
Broom) Heim
Termitomyces robustus (Beeli) Heim Symbiotic + +
Termitomyces schimperi (Pat.) R. Heim Symbiotic + + +
Termitomyces striatus (Beeli) Heim Symbiotic + + +
Tubosaeta brunneosetosa (Singer) Symbiotic +
E. Horak
Volvariella volvacea (Bull.) Singer Saprotrophic + + +
Volvariella earlei (Murr.) Shaffer Saprotrophic + + +
Volvariella acystidiata Pathak Saprotrophic +
Source (De Kesel et al. 2002, Guissou et al. 2008, Yorou et al. 2002b). Symbiotic species include
ectomycorrhizal and termite fungi.
Fig. 1. Frequency of some WEF genera in West Africa (data from Table 2).
3.1 Common ectomycorrhizal edible genera
3.1.1 The genera Lactifluus and Lactarius
In tropical Africa and in West Africa in particular, the milkcaps (Lactifluus

and Lactarius, see Verbeken et al. 2011, 2012) play an important role in the
growth and regeneration of numerous timber species and ecosystems. Both
genera contain approximately 90 described species in tropical Africa for an
estimated total richness of 150, of which 60 species might be common to
the Sudano-Zambezian domains (Verbeken 1996, 1998a,b, 2000, Verbeken
and Walleyn 2000, 2010). In both ecozones, many (if not all) Lactifluus and
Lactarius species are used by local inhabitants as food (Härkönen et al.
2003, Verbeken and Walleyn 2010). In WA, Lactifluus and Lactarius species
are the most common, making up 16% of the total number of WEF species.
A total of 22 Lactifluus and Lactarius species, composed mostly of edible
ones, is recorded in Benin alone (van Rooij et al. 2003). In this country,
a higher diversity may be expected since the sampling effort during
repetitive investigations was biased toward central and western Benin. In
Benin (Yorou and De Kesel 2011, van Rooij et al. 2003) and in Burkina Faso
(pers. observation), many rare and edible species are confined and may
be ecologically specific to gallery forests dominated by Berlinia grandiflora,
including notably the one in Bassila (Central Benin), the gallery forests
of Kou, and the Forest Reserve of Mouhoun in South-western Burkina
Faso. Many Lactarius and Lactifluus, notably Lactifluus edulis, Lactifluus
gymnocarpoides, Lactifluus medusae and Lactifluus flammans, number among
the most appreciated WEF in Benin, Togo and in West Africa in general.
Some representatives of edible Lactifluus and Lactarius from West Africa
are presented in Fig. 2.
3.1.2 The genus Amanita
Though the genus Amanita is worldwide reputed to encompass many

poisonous species, representatives of this genus are commonly used as
food in West Africa and in tropical Africa (Buyck 1994a, Härkönen et al.
2003, Boa 2004). In the Democratic Republic of Congo, for example, Parent
and Thoen (1977) mentioned that the genus Amanita ranks second in terms
of annually consumed biomass whilst Morris (1984) reported on Amanita
loosii as the most sold edible species in local markets. According to current
records, a total of 7 Amanita species are consumed in West Africa, i.e. 10% of
all WEF. Three species (A. subviscosa, A. masasiensis, A. cf. xanthogala) are well
known and common in the culinary history of the Nagot people in central
Benin. In the neighbouring country of Burkina Faso, Guissou et al. (2008)
reported three edible species. Härkönen et al. (1994) mentioned a few cases
Fig. 2. Some edible Lactarius species from West Africa. a. Lactarius flammans (Kou Forest Reserve,
Burkina Faso), b. L. gymnocarpus (Baro Forest Reserve, Upper Guinea), c. L. foetens (Mouhoun
Forest Reserve, Burkina Faso), d. L. medusae (Fazao National Park, Togo), e. L. cf. pelliculatus
(Forest Guinea), f. L. tenellus (from Benin).
where temperate Amanita species (introduced in tropical Africa together

with partner Pinus trees) have been misidentified by local people and caused
fatal deaths. In many West African countries, exotic Pinus plantations are
absent and it is almost impossible to record temperate species. In Benin,
Togo, and even in central Africa (De Kesel et al. 2002, Eyi-Ndong et al. 2011),
one species related to the temperate and boreal species A. rubescens occurs.
Molecular data are needed to clarify the genetic affinity between tropical
African specimens and temperate (and boreal) specimens of A. rubescens.
Our ethnomycological investigations reveal that this sibling species of
A. rubescens is eaten by local people without trouble. Pinus plantations are
established in the mountains of the Fouta Djallon area in Guinea. So far
there is no known report about poisonings due to misidentifications. It is
actually very difficult to accurately discuss the diversity and edibility of

Amanita species in West Africa. Not only is mycophagy rarely investigated
in many West African countries, but the genus itself suffers from numerous
taxonomical ambiguities (Bas 1969, Härkönen et al. 1994, 1995). Amanita
is probably more diverse than actually reported. A total of 55 species are
described and accepted for conservative estimates of 70 Amanita species
in tropical Africa.
3.1.3 The genus Russula
Russula is probably one of the most ecologically important fungal groups

in tropical Africa, where it emerges as the most diverse EcM genus with
conspicuous fruit bodies. All species are known to form EcM symbiosis
with native forest trees in Sudano-Zambezian woodlands and dense
evergreen forests as well (Buyck 1994b,c, 1997, Diédhiou et al. 2010).
Reportedly, Russula includes about 160 identified/described species from
a conservative estimate of 290 species (Buyck 1994b,c, 1997, Härkönen et
al. 1994). In general, the majority (if not all) of Russula species are edible
though many species are not consumed by local people due to a repulsive
smell or taste. A total of about 40 species are consumed in tropical Africa
according to Buyck (1994). Six (6) edible species have been recorded so
far in West Africa (see Table 2 above), including notably R. congoana and
R. oleifera (Fig. 3), making it 9% of the total WEF so far inventory in this
Fig. 3. Some edible Russula species from West Africa. a. Russula congoana (Moussaya Forest
Reserve, Upper Guinea), b. Russula sp1. (Ziama National Park, Guinea), c. Russula oleifera
(Bui national Park, Ghana).
region. In Benin, De Kesel et al. (2002) mentioned 4 edible species. Three

edible species are known from Togo whilst Guissou et al. (2008) reported
only two from Burkina Faso.
3.1.4 The genus Cantharellus
A total of 46 Cantharellus (including the new genus Afrocantharellus according

to Tibuhwa et al. 2012) species are known from tropical Africa (Tibuhwa et
al. 2008, 2012, Buyck et al. 2013, De Kesel et al. 2011, Eyssartier and Buyck
1998). Conservative estimates of 60 species are suggested by Verbeken and
Buyck (2001) for the whole of tropical Africa. Cantharellus encompasses
several well-known and highly esteemed edible species (Eyssartier 2003).
In the Zambesian ecozone, numerous species are widely collected and huge
quantities are frequently sold at local markets and along the roadsides
(Buyck 1994a, Malaisse 1997, Buyck et al. 2000, De Kesel and Malaisse 2010).
According to Parent and Thoen (1977), Cantharellus ranks first in terms of
diversity and annually harvested biomass. A total of 11 species are well
known edible species in Western Burundi (Buyck and Nzigidahera 1995)
whilst Malaisse (1997) reported over 16 species. Eyi-Ndong et al. (2011)
reports a total of 8 species in central African rain forests. Unlike in the
Zambesian ecozone, the genus Cantharellus is much less diverse in Sudanian
woodlands. This is probably due to the absence in West Africa of 14
dominant tree species in Brachystegia and two more in the genus Julbernardia.
Both these genera are important and typical partners of Cantharellus in the
Zambesian area. Because of the low species diversity, their scarcity in natural
areas, and the confinement to specific habitats (Yorou and De Kesel 2011),
the use of Cantharellus species as food is rather unpopular in West Africa.
Only one Cantharellus species (Cantharellus addaiensis) is widespread in
Isoberlinia-dominated Sudanian woodlands (De Kesel et al. 2002, Yorou et
al. 2002a,b). In contrast, Berlinia- and Uapaca-dominated forest galleries host
a larger diversity of species (Yorou and De Kesel 2011); including notably
Afrocantharellus platyphyllus, Cantharellus congolensis, C. conspicuus and
C. solidus (De Kesel et al. 2002, 2011, Eyssartier et al. 2002) in Benin and Togo.
There is no doubt that gallery forests of the neighboring countries—Ghana,
Ivory Coast, Burkina Faso, Senegal and more importantly Guinea, host more
and possibly different Cantharellus species. A few representatives of edible
Cantharellus species from tropical Africa are given in Fig. 4.
Fig. 4. Some edible Cantharellus species from West Africa. a. Cantharellus addaiensis (Wari-Maro
Forest Reserve, Benin), b. Cantharellus congolensis (Aledjo Forest Reserve, Togo), Afrocantharellus
platyphyllus (Kota gallery Forest, Benin), d. Cantharellus cf. conspicuus (Bassila gallery forest,
Benin).
3.2 Special focus: Termitomyces as a delicacy in West Africa and

elsewhere
The associated fungi of termites (Termitomyces) exhibit the highest food

value among WEF in tropical Africa (Parent and Thoen 1977, Degreef et al.
1997, Malaisse 1997). Termitomyces have a paleo-tropical (tropical Africa and
Asia) distribution. Molecular studies including both partners gave strong
evidence of its unique African rain forests origin (Aanen and Eggleton
2005, Nobre et al. 2011). They display a large range of morphological
features, including great variability in their size (small, medium and
large), pseudorhizes (absent or not), and perforatorium (shape, size, see
Fig. 5). Whilst species such as T. microcarpus exhibit small-sized-fruit bodies
(1–1.5 cm cap diameter, stipe 1–3 cm long x 1–2 mm diameter), numerous
others such as T. schimperi and T. letestui present larger fruit bodies (cap
up to 20 cm diameter, stipe 15 cm long x 2–4 cm diameter). A total of about
20 Termitomyces species have been recorded throughout tropical Africa
(Heim 1977, Mossebo et al. 2002). The largest Termitomyces in tropical
Africa, Termitomyces titanicus, is not yet recorded in West Africa. Buyck
(1994) mentioned 4 species in western Burundi, Pegler and Piearce (1980)
Fig. 5. A few edible Termitomyces from West Africa. a. Termitomyces sp. (Fazao National Park),
b. Termitomyces microcarpus, c. Termitomyces schimperi (Adjengré region, Togo), d. Termitomyces
letestui (Pèrèré region, Benin).
enumerated 6 species in Zambia, and Morris (1986) reported 8 species for

Malawi. Mossebo et al. (2002) inventoried 14 species in Cameroon. West
African ecosystems host numerous Termitomyces species, many of which are
still waiting for descriptions, as attested by numerous specimens from Togo
and Guinea with deviating anatomo-morphological features (pers. observ.)
than those already described (De Kesel et al. 2002, Mossebo et al. 2002).
With a total richness of 9 species so far recorded, the genus Termitomyces
makes about 20% of WEF in Benin (De Kesel et al. 2002, Yorou et al.
2002b). It represents about 13% of all WEF recorded for West Africa. Seven
Termitomyces species are recorded in the southern part of Ivory Coast (Koné
et al. 2010a,b). Repetitive mycological investigations generated more than
8 Termitomyces species in Togo (A. Guelly, unpublished data). According to
Koné (2013) and taking into account all available data on fungus growing
termites and their symbiotic fungi, a conservative diversity of at least 20
species can be estimated in West Africa. In addition, the published sequence
data (COI and ITS rDNA sequences) for both fungus-growing termites and
their symbiontes allocate the centre of termite agriculture to western Africa
(Nobré et al. 2011, Koné et al. 2011).
Termitomyces range among the top 3 most appreciated WEF in tropical
Africa (Rammeloo and Walleyn 1993). They remain a delicacy in West
Africa where many species are highly appreciated because of their taste
(De Kesel et al. 2002, Heim 1977). In West Africa, Termitomyces sporophores
are commonly used as food, but highly appreciated species are commonly
traded at local and regional markets (Fig. 6), contributing to secure cash
incomes for local people. In Ivory Coast for example, fruit bodies of
Termitomyces letestui are intensively collected and sold, with approximate
annual harvests amounting 7000 kg per year. Seasonal income (i.e., February
to April each year) of participants in the marketing chain varies from
US$58.38 to US$341.62, thus playing a crucial role for the livelihood of rural
communities (Koné et al. 2010b).
Fig. 6. Trade of Termitomyces letestui in Ivory Coast. a. from harvesters to dwellers, b and c.
Trade by women along roadside.
4. Natural Productions and Phenology of WEF in West Africa

Sudanian woodlands and gallery forests display not only a high diversity
of WEF, but also harbour large quantities of fungal biomass. Quantitative
investigations on the natural production of WEF in West Africa are rare,
and very little (if not at all) is known about effective annual harvests and
consumption by rural communities. However, taking into consideration
the high diversity of WEF and that West Africa woodlands generally cover
hundreds of thousands of hectares each, there is no doubt that annual natural
productions are far higher than seasonal harvests. Parent and Thoen (1977)
mentioned annual harvests of 1000 kg in Upper-Shaba region (Democratic
Republic of Congo). In a recent study, Yorou et al. (2002a) measured an
annual natural fresh biomass of up to 220 kg/ha for a total of 30 WEF in
woodlands of Central Benin. As the study was executed during three months
(covering 15th June to 15th September), and thus it covers only a part of
the fruiting period, effective natural productions of over 300 kg per hectare
may be expected. However, not all vegetation types produce such large
fungal biomass. Significant difference (F(5,107) = 179,13, p < 0,01) was recorded
within investigated ecosystems (Yorou et al. 2002a), with annual productions
ranging from 8.6 ± 5.2 kg/ha (for dense forests poor in EcM trees) to 293.9 ±
16.1 kg/ha (for Caesalpinioid-dominated woodlands). The study suggests
that Caesalpinioid- and Phyllantioid-dominated woodlands harbour larger
biomass of EcM WEF (up to 99%) in comparison to Caesalpinioid-poor
dense forests (Fig. 7). Natural productions are mostly dominated by species
members of Lactarius (+ Lactifluus), Russula and Amanita. Genera Lactarius
Fig. 7. Proportion of EcM versus saprotrophic WEF biomass within six different vegetation
types. Caesalpinioid-dominated woodlands (SA, SB, FC, FI) are dominated (up to 99%) by
EcM edible fungi in comparison to Caesalpinioid-poor dense forests (FD and FR), where
saprotrophic fungi are predominant. Source Yorou: 2000 (unpublished).
and Lactifluus are not only the most common in term of species richness,
but the most dominant in woodlands and gallery forests as well (Yorou et
al. 2002a). In Sudanian ecozones, a single hectare of woodlands dominated
by Isoberlinia spp. and/or Uapaca spp. may produce over 6000 sporophores,
and up to 121 kg fresh biomass of Lactifluus gymnocarpoides annually, making
it the most dominant WEF in Sudanian woodlands.
The Sudanian ecozones are characterised by a rainy season of up to 5–6
months (May to October) which contrasts strongly with a long severe dry
season. While the dry season is generally the harvest period of many crops,
the rainy season is particularly a difficult time for numerous farmers because
of the lack of harvested crops. The beginning of the rainy/wet season (May
to early July) represents a difficult period for local inhabitants. It is known
as a shortage period because stocks are empty or sold out and new crops
still unavailable. It is during this period that WEF play an important role
as suppliers of food (De Kesel and Malaisse 2010, Yorou and De Kesel 2002)
and essential proteins (Adewusi et al. 1993, Guissou et al. 2005). Yorou et
al. (2002a) demonstrated that more than 70% of total annual productions
of WEF are obtained at the beginning of the raining season (May to early
July), which coincides with the aforementioned shortage period, thus
contributing substantially to improve the food security of vast numbers
of WA people. Natural productions of WEF significantly vary throughout
the fruiting period (F(10,107) =157,57; p<0,01) and fit with an J-inverse curve
(Fig. 8). The first production peak is obtained with the first intense rains
(usually in May and June). Ecologically, the first rains generally break
Fig. 8. Temporal variability (F(10,107) =157,57; p<0,01) over 6 months (11 sampled weeks I to XI,
2-week interval from May till October) of total natural productions of ectomycorrhizal WEF
in Sudanian woodlands (SA, SB, FC and FI). Natural production fits a J-inverse curve with
two peaks of productions. Source: Yorou 2000 (unpublished).
water stress of the myco-rhizosphere enabling the re-growth of below-

ground fungal propagules (ectomycorrhizal root tips, mycelium, sclerotia,
spores.....). Water availability within the myco-rhizosphere, coupled with
the re-growth of EcM mycelium and the intensification of photosynthesis
of partner trees provoke intensive fructifications of numerous species at the
same time. As the soil becomes more humid in July, August, and September,
natural productions of WEF decline. Another smaller production peak
is obtained again towards the end of the rainy season, generally ending
September to early October when soils gradually become drier. Temporal
variability of natural production may also be related to specific preferences
and phenology of both fungal and host species. Though species such as
Cantharellus addaiensis and Amanita cf. craseoderma occur throughout the
rainy season with no significant variability (F(10, 121) = 1.74, p = 0.07 and F(10,
121)
= 1.5, p = 0.15 respectively), many other species members of Lactifluus,
Lactarius, Russula and Amanita occur predominantly at the beginning of
the rainy season and become less frequent towards the end of it. More
information on the phenology of EcM species is needed to accurately
explain temporal variability of natural productions and the phenology of
individual species.
5. Steps Towards Establishing an Economic Sector based

on WEF in West Africa: Challenges from a Case Study in
Central Benin
Since more than 70 % of natural productions are available in a relatively short
span of time and that local uses are so far lower than natural availability, it is
essential to implement policies that maximize the use of WEF throughout the
year. This is particularly justified by the fact that (1) the use of WEF as food
is a common practice (Fig. 9), (2) it is also common to observe WEF being
sold at local markets and/or at roadsides, contributing hereby to secure cash
incomes for villagers, and importantly (3) to promote awareness among
villagers, foresters and decision makers about the potential of using WEF
as an income generating activity. An economic sector based on WEF may
act as an incentive to make villagers gain interest in forest conservation and
sustainable use. In West Africa, WEF are harvested, exploited and sold in
a rather traditional way. Assistance is needed in order to help villagers get
a maximum profit from these forests. Many aspects need to be addressed,
i.e. ranging from the ones related to the basic and sustainable harvesting
towards transformations and the end-users of mushrooms located either
in the forest villages or in and around cities.
Fig. 9. Use of wild mushrooms as food by local people in Ivory Coast. a. Processing dried
mushrooms before cooking, b and c. Cleaning Termitomyces letestui for cooking.
To examine the effectiveness of an economic sector based on WEF in

Benin (West Africa) and its relevance to improve the livelihood of villagers,
a case study was implemented in Bassila region in central Benin. Because
of both the variability of vegetation types and the presence of several
ethnic groups, the Bassila region was regarded as suitable for such a study.
Through this case study, we would like to illustrate (1) the different steps
we followed in this study and (2) address some of the constraints we faced.
A simplified diagram will be suggested for scientists willing to establish a
value chain of WEF in rural communities.
5.1 Ethnomycological surveys to select priority WEF in Bassila

region
Ethnomycological surveys were conducted according to Yorou and De Kesel

(2002). A total of 6 ethnic groups were considered in the study. The aim is
to identify species that are appreciated by local people and are sold at local
markets and that can be promoted in a value chain according to various
considerations/criteria. During the first step of this study, forest walks were
undertaken with villagers to monograph and identify all WEF they used as
food. Repeated interviews were conducted in order to check the reliability

of the information and to eliminate incoherent stories. The purpose was to
get a reliable dataset enabling extraction of specific ethnic profile lists of
WEF. We recorded a total of 21 edible species (for all ethnic groups). The
diversity of WEF varied from one group to another, i.e., 7 species for Peuhl
people to 16 species for Nagot people. In the second step, each ethnic group
was requested to make a simple and matrix ranking (Rastogi 1999, Martin
1995) of the species they commonly use. Simple ranking is performed by
asking each group to rank the top 10 species according to their nutritional
importance (gustative appreciation). A matrix ranking (on a 1 to 8 points
scale) of all top 10 species identified from simple ranking is performed
against 4 criteria (local price per kg, preference for selling or own use, spatial
and temporal availability in nature, and drying requirement of the species).
Matrix ranking allowed the identification of the top 8 priority species,
notably Termitomyces letestui, T. schimperi, T. clypeatus, Lactifluus flammans,
Lactifluus gymnocarpoides, Lactifluus medusae, Cantharellus aff. rufopunctatus
and Psathyrella tuberculata. These 8 species have been unanimously pointed
out as interesting target species to act as a backbone for a regional economic
sector based on wild edible fungi.
5.2 Identification of WEF-rich habitats and assessment of natural

productions and phenology
The purpose of this activity is to identify the habitats of the identified priority
species, phenology, and natural availability. This is of great importance since
knowing target species’ preferred habitat, distribution, and phenology will
greatly reduce harvest effort of villagers. Except for Psathyrella tuberculata,
all above mentioned species include termite fungi (T. letestui, T. schimperi
and T. clypeatus) and EcM ones (Lactifluus flammans, L. gymnocarpoides
Lactifluus medusae and Cantharellus aff. rufopunctatus). Ecologically, Bassila
region harbours Caesalpinioid- and Phyllantioid-dominated woodlands
for which quantitative data on natural production and species distribution
are available (Yorou et al. 2002a). Annual productions of Termitomyces
are difficult to assess since their fructification is sporadic and relies upon
many environmental factors. Our numerous attempts to assess natural
productions of Termitomyces within permanent plots were unsuccessful.
However, Koné et al. (2010a,b) reported an annual estimation of over 7000
kg for T. letestui in southern Ivory Coast. Cantharellus aff. rufopunctatus is a
species that is confined to gallery forests dominated by Berlinia grandiflora.
Annual productions of this species amounts 178 kg/ha, on the basis of direct
weekly biomass measure of 8.9 kg during 5 fruiting months. In summary,
woodlands and gallery forests dominated by Isoberlinia spp. and Uapaca
spp. were identified during the study as important target habitats to deploy
sustainable harvesting in the context of a WEF-based local economical
activity ( in Central Benin).
5.3 Market surveys, identifying potential end users and assessing

economic balance: The challenges
Market surveys are aiming at identifying species that are jointly appreciated
by the harvesters and the end users as well. Market surveys should
allow making an economic examination of the sector, taking into account
harvesting effort, but also costs analysis related to mushroom drying,
labelling, and transportation from the forest villages to the location of
potential users. Such factors, coupled with the variability of environmental
factors, remain the most challenging aspects in the establishment and
continuity of a regional economic sector based on WEF. To be efficient and
visible, this economic activity not only needs to identify good target species,
but also needs to be available throughout the year. It is clear that both fresh
and dried specimens should be available and that they should meet the
users’ expectation. This goes from personal criteria and requirements about
transformed fungi (dried, in oil) to the way the edible fungi are packed,
labelled and presented. In addition, costs for collecting in the forest, drying
and transportation from rural to urban markets should be as low as possible
to allow a rapid flow at reasonable price.
In this particular case (Bassila region), though we were able to identify
priority species along with their production and distribution, a successful
mushroom-based sector could not be initiated because of (1) the lack of
potential buyers within and outside the villages, (2) lack of assistantship to
promote awareness and to coach villagers from harvesting to the selling of
WEF and finally (3) consumers’ preferences of fresh mushrooms over dried
ones. Because mushrooms are available in their immediate environment,
no villager from the study area can imagine buying mushrooms except the
very rare and highly prized species such as Termitomyces. Many villagers
harvest the little quantities of WEF needed for their own personal uses, with
very limited lucrative ambition. As WEF are collected only for personal use,
and that they are mostly appreciated in fresh conditions, there is no need
for collecting and drying more than daily required. We hardly observed
villagers drying surplus specimens for a long term use (neither for personal
consumption nor for sales). However, drying mushrooms seems not to
be a major worry in tropical rural areas. Wild edible mushrooms can be
either dried under the sun or smoked together with bush meat (Fig. 10). In
the neighbouring country Burkina Faso, the highly appreciated Phlebopus
sudanicus is harvested and commonly dried under the sun by Bobo people
for long term exploitation (Guissou et al. 2005).
Fig. 10. Drying wild edible mushrooms. a and b. Drying of Psathyrella tuberculata and Volvariella
volvacea under sun, c. Smoking of Termitomyces letestui together with bush meat.
Another problem we faced during our investigations was the paucity

of highly esteemed species in natural areas. Termitomyces species usually
present a high economical output, as can be confirmed in the study areas
where several women were observed selling T. fuliginosus specimens.
Whenever available in large quantities, Termitomyces species could
successfully support a large scale sector of WEF in West Africa, as the
appreciation and demand is high and most end users (either in villages
or in cities) are prepared to buy them at a relatively high price (Koné et
al. 2010b). Exactly the same phenomenon is observed with Termitomyces
letestui in the termite rich miombo areas of Lubumbashi (DR Congo, De
Kesel pers. obs.). Unfortunately, because of their dependence on termites
species their fructification being moderated by various environmental
factors (Koné 2013), Termitomyces species are not constantly available in
nature to justify a long lasting value chain. Unless grown under controlled
conditions, it will remain a moderately predictable but still highly seasonal
edible product from the forest.
One important challenge in the study area, and probably in many West
African cities, is to revive culinary habits of citizens so as to introduce WEF
in their daily dishes. Though WEF are valuable by villagers throughout the
study areas, they hardly gained attention of citizens of the neighbouring
cities. Generally, there is a decline in the use of WEF from one generation
to another and when moving from forest villages to the cities (Yorou and
De Kesel 2002, Degreef et al. 1997). This is probably a general fact in the
whole of West Africa (Guissou et al. 2008), and it may negatively influence
the promotion of value chain of WEF. Reversing, changing, or widening
culinary habits of citizens is probably the most tedious task in the actual West
Africa context, as firstly, many imported foodstuffs are actually regarded as
the best, and secondly, wild fungi are usually considered to be poisonous.
If Termitomyces can attract the interest of many villagers and citizens, much
effort is needed to increase the attention of the people in cities towards EcM
edible mushrooms. This can be successfully performed through large scale
awareness-raising programme about the culinary and nutritional benefits
of WEF, and importance of WEF to improve food security among villagers
and citizens.
5.4 Recommended protocol for the establishment of an economic

sector based on WEF from tropical Africa.
For an enterprise to be successful, basic studies and some considerations

should be made. We propose a simplified diagram (Fig. 11) including
important steps and studies to be considered for the establishment of a value
chain of WEF in tropical Africa. Most required studies include:
1) Studies of the diversity, distributions and natural production of WEF:
this will allow the generation of annual availability of WEF as well as
identification of WEF-rich habitats,
2) Ethnomycological investigations: to record patterns of local uses but
also to identify priority species,
3) Studies on different ways to locally dry and store WEF. It is evident that
these techniques should be adapted to the local needs and possibilities.
They should be economic, efficient, low-tech, affordable, safe and
reliable.
4) Market studies to demonstrate the need for and the efficiency of the
sector. What is the demand and from where comes the demand. The
market potential is qualitative, quantitative and surely not evenly
factors
Fig. 11. Simplified diagram showing different steps and most needed investigations to be
considered in erecting an economic sector based on WEF.
distributed. Is the sector solid enough to overcome drawbacks in

production due to seasonal fluctuations? A thorough analysis is needed
to know who is winning and who is losing. There is no doubt that a
small scale pilot project will be needed in order to extrapolate and
simulate a large scale market.
6. Concluding Remarks
Wild edible fungi are mostly EcM fungi and they are a seasonal but abundant
and important source of food. In parts of West Africa, they represent a
modest but stable income for rural people. The socio-economic potential
of these wild edible fungi is high because our studies have indicated that
the naturally produced quantities largely surpass the demand and local
consumption. Wild edible fungi are under-utilized in West Africa. This
potential is however currently being hampered or even annihilated by
wide scale and devastating human activities. Let there be no doubt that the
massive production of charcoal has become the biggest threat to all EcM
forests in the whole of tropical Africa. Although forest products, including
edible fungi, are often highly valued by local inhabitants, much effort is still
needed to promote awareness among people that harvesting and marketing
of edible fungi can only become an income generating activity if EcM forests
are maintained and preserved. Multi-disciplinary investigations including
taxonomists, ecologists, food scientists, food technologists, and economists
should be implemented in order to secure a better exploitation of EcM
forests and their generally associated edible fungi. There is very little doubt
that sustainable use and preservation of West African forests, rather than
cutting them down for charcoal,will yield a long-term improvement in the
livelihood of millions of rural people.
Key words: Wild Edible Fungi, diversity, natural productions, phenology,
local uses, economic sector, West Africa
7. Aknowledgement
We are grateful to local communities who provided valuable ethnomycological
information. Yorou NS is grateful to the German Research Foundation for
financial supports (DFG project Ag7/19-1 and Yo 174/2-1). Koné NA is
undebted to the International Foundation for Science (Grant D/4982-1). The
authors acknowledge Krista L. McGuire and Caitlyn Gillikin for improving
the language.
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champignons comestibles de la forêt classée de Wari-Maro (Bénin, Afrique de l’Ouest).
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625.
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Abomey-Calavi (Benin) October 30th to November 18th 2002, pp. 231–240.
1
Department of Natural Resources Management, Faculty of Agronomy, University of Parakou,
BP 123, Parakou, Benin.
2
Department Biology I, Ludwig-Maximilians-Universität München, Menzinger Str. 67, 80638,
Munich, Germany.
3
URF des Sciences de la Nature et de l’Environnement, Station d’Écologie de Lamto, Université
Nangui Abrogoua (Côte d’Ivoire), BP 28 N’Douci.
4
École Normale Supérieure, Université de Koudougou, BP 376, Koudougou, Burkina Faso.
5
Département de Botanique et Écologie Végétale, Faculté des Sciences, Université de Lomé,
081 BP 1515 Lomé, Togo.
6
Laboratoire d´Écologie Appliquée, Faculté des Sciences Agronomiques, Université d’Abomey-
Calavi, 01 BP 526, Cotonou, Bénin.
7
Department of Cryptogamy (Bryophyta & Thallophyta), National Botanic Garden of Belgium,
Domein van Bouchout, B-1860, Belgium.
*Corresponding author: n.s.yorou@bio.lmu.de
Color Plate Section
Chapter 1
Fig. 1. Distribution of EcM trees in tropical Africa: (1) rainforests in the Guinea-Congo region; (2)
open forests in the Sudanian and Zambezian regions; (3) savanna woodlands in the Sudanian
and Zambezian regions (Bâ et al. 2012).
Chapter 2
Fig. 1. Micrographs of mycorrhizas of Neea species 1 (a-e), Neea aff. floribunda (g-h), Pisonia sp.
(g-h), a. Russula puiggarii − Neea species 1, b. Russula-Lactarius − Neea species 1, c. Tomentella-
Thelephora species 1 − Neea species 1, d. Tomentella-Thelephora species 2 − Neea species 1, e.
Ascomycete − Neea species 1, f. Tomentella-Thelephora species 3 − Neea aff. floribunda, g.,h. .
Tomentella-Thelephora species 3 − Pisonia sp., g. overview: long roots partially with hyphal
mantle (arrows), partially with root hairs and no hyphal mantle (*), h. one root shown at higher
magnification: distal portion with root hairs (*), proximal portion with hyphal mantle (arrows).
Scale bars: a.,b.,f.,h. 1 mm; c.-e. 0,5 mm; g. 5 mm. Data from Haug et al. 2005.
Chapter 6
Fig. 3. (A) Podoserpula miranda and (B) Cantharellus garnieri. Photos by courtesy from Ducousso
Marc, CIRAD.
Fig. 4. Pisolithus albus from New Caledonia. A: Pisolithus albus MD07-117 from the Koniambo
massif; B: Pisolithus albus MD07-228 from the Ouen-Toro, Noumea; C: cross section of Pisolithus
albus MD07-166 from Pindjen water-fall and D: globose spores (8.77 to 9.62 µm) of Pisolithus
albus MD06-379 from Poum, erected spines (1.2 µm) are clearly visible. From Jourand et al.
(2010a).
Fig. 9. Eucalyptus globulus seedlings after 12-weeks growth. A and A’ mycorrhizal; B and B’:
non-mycorrhizal (controls). A and B: no nickel added; A’ and B‘ seedlings treated with Ni.
From Jourand et al. (2010b).
Chapter 7
Fig. 1. Climatic regions of Burkina Faso and localization of prospected areas. Data from Sanon
et al. (2009a).
Chapter 8
Fig. 1 Sporocarps of S. sinnamariense. Bar is 2.5 mm.
Fig. 3. Yellow Gnetum EM root tip formed with S. sinnamariense, showing extensive extramatrical
mycelia. Bar is 0.3 mm.
Chapter 9
Fig. 1. Distribution of three Coccoloba species (Coccoloba uvifera, C. swartzii and C. pubescens) and
their putative ectomycorrhizal fungi along a salinity gradient in Martinique and Guadeloupe
(Lesser Antilles).
Chapter 10
Fig. 1. Map illustrating examples of monodominant and co-dominant forests with the tree
species or genus listed and the tree family in parentheses. This diagram is not an exhaustive
list of monodominant species, but it illustrates how monodominant forests are found in all
tropical regions.
Chapter 13
Fig. 2. Some edible Lactarius species from West Africa. a. Lactarius flammans (Kou Forest Reserve,
Burkina Faso), b. L. gymnocarpus (Baro Forest Reserve, Upper Guinea), c. L. foetens (Mouhoun
Forest Reserve, Burkina Faso), d. L. medusae (Fazao National Park, Togo), e. L. cf. pelliculatus
(Forest Guinea), f. L. tenellus (from Benin).
Fig. 4. Some edible Cantharellus species from West Africa. a. Cantharellus floridulus (Wari-Maro
Forest Reserve, Benin), b. Cantharellus congolensis (Aledjo Forest Reserve, Togo), Afrocantharellus
platyphyllus (Kota gallery Forest, Benin), d. Cantharellus cf. conspicuus (Bassila gallery forest,
Benin).
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Suite 300, Boca Raton, FL 33487
711 Third Avenue
New York, NY 10017 9 781 466 59 468 5
an informa business 2 Park Square, Milton Park 9 781 466 59 468 5
Abingdon, Oxon OX14 4RN, UK 9 781 466 59 468 5 A SCIENCE PUBLISHERS BOOK
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Ectomycorrhizal (ECM) symbiosis is a mutualistic plant-

Ectomycorrhizal Symbioses in Tropical

Ectomycorrhizal Symbioses in Tropical

and Neotropical Forests

No claim to original U.S. Government works

International Standard Book Number-13: 978-1-4665-9469-2 (eBook - PDF)

Ectomycorrhizal symbioses in tropical and neotropical forests: A major

Gnetum (a liana probably related to Pinaceae; Bechem, Chapter 8) on the

As a ‘primarily temperate’ mycorrhizologist, I have always been

Ectomycorrhizal (EcM) symbioses are mutualistic plant-fungus associations

protein-rich food for local communities who depend on non-timber forest

From this finding, it should be evident that further research is

6. Abundance, Distribution, and Function of Pisolithus albus 100

Authors’ affiliations given at the end of the chapter.

ectomycorrhizal symbioses. EcMs are the most widespread mycorrhizal

EcM associations are found mainly on Caesalpinioideae, Dipterocarpaceae,

of South-Eastern Cameroon we described the EcM and sporocarp diversity,

Entangrophragma congoensi, Erismadelphus exsul var. platyphyllus, Fernandoa

3. General Traits of the EcM Fungal Communities from the

fungi fell into three phyla: Ascomycota, Basidiomycota and Zygomycota

Type of forest: Mixed Monodominant

as observed in the suillus-rhizopogon lineage assumed to be specific to

found only in EcMs, whereas the/cantharellus,/coltricia, and/inocybe were

4. Comparisons of the EcM Community between the Mixed

Monodominant forest Mixed forest

Letouzey, R. 1985. Notice de la carte phytogéographique du Cameroun au 1/500.000, Institut

Towards global patterns in the diversity and community structure of ectomycorrhizal

1993). Species of Neea were found to be ectomycorrhizal in the Amazonian

2. Ectomycorrhizas of Nyctaginaceae in the Tropical Mountain

2.1 Ectomycorrhizas of Neea species 1

Molecular analyses of the ectomycorrhizal partners of Neea species 1

2.2 Ectomycorrhizas of Neea aff. floribunda Poepp. & Endl.

and inner layers are compactly plectenchymatous (Fig. 2b). A prominent

2.3 Ectomycorrhizas of Pisonia sp.

2.4 Ectomycorrhizas of Nyctaginaceae

The morphology of ectomycorrhizas found on the short roots of Neea species

Table 1. Ectomycorrhizal types of three Nyctaginacean species in South Ecuador.

Host plant Fungal partner Morphology Sequences

hair formation that is not suppressed, occasional intracellular penetration

(formerly Uapacaceae), Sarcolaenaceae, Sapotaceae, and Gnetaceae. This

2. Study Sites and Selection of Vegetation Types

Table 1. Localization, elevation, rainfall and soil physicochemical characteristics of forest

Research sites Ebimimbang Ebom Nyangong Bityili

Rainfall (mm) 1556 1987 1677 1800

an ectomycorrhizal plant, was considered important as this liana might

3. Sampling and Identification of Sporocarps

4. Morphological and Anatomical Description of Root Tips

5. Effects of Agriculture and Logging Practices on ECM

6. Experimental Design and Statistical Analysis

7. Habitat Types of Ectomycorrhizal Associations in Cameroon

8. Cameroonian Ectomycorrhizae and Sporocarp Diversity

Table 2. List of ectomycorrhizal tree species by types of ectomycorrhizal forest habitats in

Habitat types Family Species Pilot name

Basal stem Mixed Gilbertiodendron Uapaca Microberlinia

Ectomycorrhizal Color Shape Texture Texture of mantle Thickness Presence of Presence of

Ectomycorrhizal Color Shape Texture Texture of mantle Thickness Presence of Presence of

Amanitaceae Boletaceae Russulaceae

Inocybe Cantharellaceae Lactarius

Ten provisionary keys to Cameroonian species were developed for

9. Effect of Disturbance Regimes on Ectomycorrhizal

Table 6. Two-way analysis of variance of site and disturbance stages on ectomycorrhizal

Table 7. Ectomycorrhizal colonization (percent root length colonized) of seedlings of

Forest types Ebimimbang Ebom Nyangong Bityili

Forest clump 48bc 22a 32cd 5c 81a 34a 89a 53a