UNIVERSITE D’ANTANANARIVO FACULTE DES SCIENCES DEPARTEMENT DE BIOCHIMIE FONDAMENTALE ET APPLIQUEE HABILITATION A DIRIGER DES RECHERCHES EN SCIENCES DE LA VIE PRODUCTIONS SCIENTIFIQUES Présentée par RAMANANKIERANA Heriniaina Maître de Recherches Soutenue devant la commission d’examen composée de Président : Professeur JEANNODA Victor Rapporteur interne : Professeur RAHERIMANDIMBY Marson Rapporteur externe : Professeur RAZANAKA Samuel Examinateurs : Professeur RAZAFINJARA Lala Professeur ANDRIANARISOA Blandine Date de soutenance : 02 Novembre 2012
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UNIVERSITE D’ANTANANARIVO
FACULTE DES SCIENCES DEPARTEMENT DE BIOCHIMIE
FONDAMENTALE ET APPLIQUEE
HABILITATION A DIRIGER DES RECHERCHES
EN SCIENCES DE LA VIE
PRODUCTIONS SCIENTIFIQUES
Présentée par RAMANANKIERANA Heriniaina
Maître de Recherches
Soutenue devant la commission d’examen composée de
Thème : Importance de la communauté de champignons ectomycorhiziens associés
aux espèces arbustives pionnières des zones forestières dégradées sur la régénération
d’essences endémiques de Madagascar
21
PROJETS DE RECHERCHE
Après mes études universitaires, j’ai participé au montage, à la soumission et à la réalisation
de six (6) projets de recherche pour lesquels, j’ai été porteur du projet pour quatre (4) projets.
2000 - 2002 : Projet de valorisation des plantes médicinales et aromatiques de Madagascar
Projet financé par le Gouvernement Malagasy
Porteur du projet : Dr RAMAROSON Luciano, LME/CNRE
Ce projet financé par le Gouvernement Malagasy constitue la suite des activités menées
auparavant dans le cadre du programme PLARM. L’objectif principal du projet a été d’isoler
des molécules biologiquement actives à partir des plantes aromatiques ou médicinales
préalablement identifiées suite aux enquêtes ethnobotaniques effectuées auprès des tradi-
praticiens dans plusieurs régions de Madagascar. La région Est (Moramanga - Bekorakaka et
Sud (Ifotaka) de Madagascar ont été particulièrement concernée par les activités du
programme. Ce projet constitue également un des premiers programmes réalisés au sein du
LME, nouvellement construit à l’époque, à l’issu desquels, il a été constaté que peu d’attention
ont été portées sur la gestion rationnelle et la conservation des plantes aromatiques et
médicinales de Madagascar. Ainsi, ma responsabilité dans le programme a été orientée sur la
préservation des plantes à haute valeur ajoutée et menacées de disparition via l’exploitation de
la potentialité des techniques de micropropagation.
2001 - 2005 : Maîtrise de la symbiose ectomycorhizienne pour améliorer le développement
d’essences ligneuses endémiques de Madagascar
Projet CORUS 1 financé par le Ministère Français des Affaires Etrangères.
Porteurs du projet : Dr RAMAROSON Luciano, LME/CNRE
Dr DUPONNOIS Robin, LSTM/IRD
C’est au cours de la réalisation de ce projet que nous avons commencé à travailler sur les
mycorhizes associées aux arbres autochtones et/ou endémiques de Madagascar. Les résultats de
ce projet nous ont permis d’avoir des idées préliminaires sur l’importance de la symbiose
ectomycorhizienne dans la conservation et la régénération d’essences endémiques. Ainsi, le
statut mycorhizien d’une dizaine d’essences ligneuses endémiques de Madagascar a été décrit.
De plus, la technique d’ectomycorhization contrôlée mise au point pour la première fois avec
une essence ligneuse endémique de Madagascar (Uapaca bojeri) a donné des résultats
22
intéressants aussi bien sur le développement de la plante en pépinière que sur la reprise de sa
croissance après transplantation en milieu naturel. Ce projet a été mené en collaboration avec le
Département d’Ecologie et Biologie Végétale de la Faculté des Sciences de l’Université
d’Antananarivo et le Laboratoire des Symbioses Tropicales et Méditerranéennes de l’IRD
Montpellier.
2006 – 2009 : Maîtrise de la symbiose mycorhizienne pour la régénération et conservation
de quelques essences ligneuses des forêts sclérophylles de la haute et moyenne altitude de
Madagascar
Projet financé par International Foundation for Science.
Porteur du projet : Dr RAMANANKIERANA Heriniaina, LME/CNRE
Résumé : Ce projet proposait la gestion de la symbiose mycorhizienne et son interaction avec
les microorganismes de la rhizosphère dans l'objectif d'améliorer le développement des arbres
autochtones et/ou endémiques en vue d'une revégétalisation des zones nues et restaurer ainsi la
fertilité du sol. La gestion de cette symbiose est d'un interêt fondamental pour la réussite des
programmes de reboisement, d'association arbres et cultures annuelles dans le cadre d'un
système d’agroforesterie et dans la réactivation des sols nus abandonés. Ce programme
concernait deux sites situés sur le haut plateau de Madagascar à savoir la forêt sclérophylle
d’Arivonimamo et d’Ambohimanjaka (col à Tapia). Dans cet esprit, le projet a été divisé en
quatre volets : (i) Description du statut mycorhizien in situ des essences autochtones formant
la strate arborée des sites d'étude, (ii) Determination du cortège mycorhizien associé aux
espèces ligneuses pendant les premiers mois de développement de l'arbre (iii) Isolement,
purification et étude du spectre d'hôte des isolats fongiques les plus représentatifs de la
communauté fongique récoltée dans chaque site (iv) Description des modifications induites par
la gestion de cette symbiose mycorhizienne au niveau du biofonctionnement du sol et de la
croissance de la plante. Cette approche a fait appel à plusieurs disciplines allant de l'écologie
des microorganismes du sol et des champignons mycorhiziens, passant par des caractérisations
des souches microbiennes et leur rôle dans la régénération des plantes et la fertilité du sol,
jusqu'à la production et suivi des plantules inoculées en péninière et en condition contrôlée. Les
résultats du projet ont permis dans un premier temps d’apprécier la grande diversité de
champignons ectomycorhiziens associés aux essences ligneuses de ces deux formations
sclérophylles. Ces résultats ont été pourtant obtenus en considérant seulement la population
épigée de ces champignons (carpophores). C’est pourquoi et pour pouvoir exploiter ces
23
résultats, tous les carpophores appartenant au groupe de champignons précoces (early stage)
ont fait l’objet d’isolement de souche. Ces souches constituent les premiers éléments de la
collection de souches ectomycorhiziennes au sein du LME. Utilisant ces souches fongiques
pour la mycorhization de Uapaca bojeri sur le sol stérilisé et non stérilisé, nous avons pu
décrire l’influence de la mycorhization sur la structure et le fonctionnement des
microorganismes dans différents compartiments du sol rhizosphérique.
2007 – 2009: Ectomycorrhizal host shrubs as an important nurse plant to tree successional
processes and ecology restoration in haighland of Madagascar
Projet financé par British Ecological Society.
Porteur du projet : Dr RAMANANKIERANA Heriniaina, LME/CNRE
Résumé : La régénération des plantules pourrait être inhibée ou stimulée par des plantes
préexistantes dans le milieu. En milieu tropical, les connaissances relatives aux potentialités
des plantes pionnières à faciliter l’établissement des plantules des essences ligneuses restent
encore fragmentaires. L’objectif principal de ce projet a été de décrire la contribution des
arbustes ectotrophes pionnières des zones dégradées à la succession végétale et à la
régénération d’essences ligneuses. Le projet a concerné deux sites d’étude situé au sein de la
formation sclérophylle du haut plateau de Madagascar à savoir à Ambohimanjaka et
Ambatofinandrahana. Dans les deux sites d’étude, la communauté de champignons
ectomycorhiziens associés aux arbustes ectotrophes a été décrite et comparée avec celle
associée aux essences ligneuses dont principalement Uapaca bojeri. La capacité de chaque
espèce arbustive ectotrophe et dominante dans chaque site à stimuler la régénération d’Uapaca
bojeri a été évaluée sous condition de serre et de pépinière. Les résultats ont montré que la
présence préalable de Leptolaena bojeriana et Sarcolaena oblongifolia, respectivement
dominante à Ambohimanjaka et Ambatofinandrahana, a facilité l’établissement des plantules
d’Uapaca bojeri et a stimulé leur développement sous condition de serre et de pépinière. Les
approches adoptées lors de ce projet ont permis de démontrer que certains arbustes pionniers
des zones dégradées tiennent des rôles importants dans le phénomène de succession secondaire
ou de l’établissement des plantules d’essences ligneuses. Ce phénomène de facilitation plante-
plante, peu considéré dans les opérations de reboisement ou de restauration écologique, est
d’un intérêt fondamental pour sauvegarder les essences ligneuses endémiques de Madagascar.
Abstract: The establishment of seedlings may be both inhibited and facilitated by established
plants. In tropical ecosystem, little is known about the potentiality of early-established plant to
24
facilitate seedling establishment of tree. The main objective of this research project is to
advance understanding of the contribution of early-established ectomycorrhizal shrubs to tree
successional and forest regeneration processes. This research project will be conducted in two
study sites located in disturbed sclerophyllous forest areas in the highland of Madagascar. In
each of two study sites, ecology of ectomycorrhizal communities associated with these shrubs
species will be investigated with an emphasis on their implications on the establishment of
native tree seedling. Then, relationship between dominant ectomycorrhizal shrubs species in
disturbed area and ecology restoration processes will be assessed. This ecological approach
was never considered in regeneration strategies and in protection program of important or rare
endemic tree species in Madagascar
2009– 2013 : Analyses des paramètres biotiques et abiotiques déterminant l’évolution
spatio-temporelle du potentiel infectieux ectomycorhizogène des sols à Madagascar.
Projet financé par l’Institut de Recherche pour le Développement (IRD) à travers le
programme « Jeunes Equipes Associées à l’IRD »
Porteur du projet : Dr RAMANANKIERANA Heriniaina, LME/CNRE
Résumé : L’écosystème terrestre malagasy est connu pour être un des plus riches et divers de
la planète avec de nombreuses espèces végétales et animales endémiques de la Grande Ile.
Cette diversité végétale a été particulièrement recherchée et exploitée au cours de ces dernières
décennies (production de bois précieux, d’huiles essentielles, etc). La dégradation et la
surexploitation de ces ressources n’ont cessé de progresser au cours de ces dernières décennies
aboutissant à une dégradation spectaculaire du paysage originel. Il a été estimé que moins de
15% de la forêt naturelle malagasy subsiste encore dans son état plus ou moins originel. Le
reste a été exploité par les populations locales ou a été dégradée par le bétail ou par les
incendies (Ex : culture sur brulis).
Parmi toutes les options techniques et scientifiques susceptibles de remédier à cette situation, la
gestion et la valorisation des ressources microbiennes telluriques pour améliorer les
performances des programmes de reboisement sont encore relativement ignorées. Or, il est
connu que les communautés microbiennes telluriques sont des composantes majeures dans le
développement des cycles biogéochimiques majeurs (Cycles du carbone, phosphore et azote).
Parmi tous ces groupes microbiens, les champignons mycorhiziens occupent une position
centrale dans ces phénomènes interactifs et complexes régissant l’évolution spatio-temporelle
des écosystèmes terrestres. En conséquence, la compréhension du rôle des paramètres
écologiques dans le fonctionnement durable de ce phénomène symbiotique et leur maîtrise,
25
constituent des préalables indispensables à la conception d’itinéraires techniques susceptibles
d’assurer une réhabilitation durable de ces milieux dégradés.
2009 – 2014 : Production de champignons comestibles à Madagascar
Projet financé simultanément par l’Institut de Recherche pour le Développement
(IRD) à travers le programme « Maturation de projet innovant » du Département Expertise
et Valorisation, par l’Incubateur Bond’innov et par le Service International d’Appui au
Développement
Porteurs du projet : Dr RAMANANKIERANA Heriniaina, LME/CNRE
Dr DUPONNOIS Robin, LSTM/IRD
Description de la technologie valorisée
Les champignons comestibles saprophytes manifestent différentes activités enzymatiques
(cellulolytique, pectinolytique, chitinolytique, etc) qui leur permettent de se développer sur des
substrats organiques en catabolisant des molécules complexes (cellulose, pectine, etc) et/ou en
mobilisant des macroéléments inorganiques (micas, feldspath, etc).
Du fait du savoir faire technologique de l’équipe impliquée dans ce projet, des ressources en
champignons comestibles endémiques de la Grande Ile, du caractère innovant de la
méthodologie proposée (valorisation des souches de champignons pour leur fructification et en
tant que bio-fertilisants), les objectifs de ce projet ont été les suivants : (i) adoption d’une
technique culturale standard identifiée en fonction des résultats acquis, (ii) une diversification
de la production (élargissement de la gamme de produits), (iii) une protection de la technique
de production et de valorisation des produits et sous-produits de l’itinéraire cultural
(proposition de dépôt de brevet) et enfin une description plus précise des potentialités
économiques de ce type de production sur le marché national et international.
La technologie retenue dans ce projet vise (i) à multiplier le champignon sur des résidus de
culture (paille de riz) et des particules minérales (Podzollane) puis stimuler sa fructification par
un choc thermique et (ii) en fin de phase de fructification, à valoriser le substrat colonisé par la
souche fongique en tant que bio-fertilisant et bio-pesticide pour améliorer durablement la
productivité des cultures maraîchères à Madagascar.
26
PRODUCTIONS SCIENTIFIQUES DANS DES JOURNAUX A FACTEUR D’IMPACT
Article (1) : Duponnois R., Assikbetse K., Ramanankierana H., Kisa M., Thioulouse J. & Lepage M.
(2005). Litter-forager termite mounds enhance the ectomycorrhizal symbiosis between Acacia
holosericea A. Cunn. Ex G. Don and Scleroderma dictyosporum isolates. FEMS Microbiol
Ecol. 56: 292 – 303.
Article (2) : Ramanankierana H., Rakotoarimanga N., Thioulouse J., Kisa M., Randrianjohany E.,
Ramaroson L. & Duponnois R. (2006). The ctomycorrhizosphere effect influences functional
diversity of soil microflora. International Journal of Soil Sciences. 1 (1): 8 - 19
Article (3) : Ramanankierana H., Ducousso M., Rakotoarimanga N., Prin Y., Thioulouse J.,
Randrianjohany E., Ramaroson L., Kisa M., Galiana A. & Duponnois R. (2007). Arbuscular
mycorrhizas and ectomycorrhizas of Uapaca bojeri L. (Euphorbiaceae) : sporophore diversity,
patterns of root colonization and effects on seedling growth and soil microbial catabolic
diversity. Mycorrhiza 17: 195 – 208
Article (4) : Ducousso M., Ramanankierana H., Duponnois R., Rabevohitra R., Randrihasipara L.,
Vincelette M., Dreyfus B. & Prin Y. (2008). Mycorrhizal status of native trees and shrubs from
eastern Madagascar littoral forests with special emphasis on one new ectomycorrhizal endemic
family, the Asteropeiaceae. New Phytologist 178: 233 – 238
Article (5) : Baohanta R., Thioulouse J., Ramanankierana H., Prin Y., Rasolomampianina R., Baudouin
E., Rakotoarimanga N., Galiana A., Randriambanona H. Lebrun M. & Duponnois R. (2012). Restoring native forest ecosystems after exotic tree plantation in Madagascar: contribution of
the local ectotrophic species Leptolaena bojeriana and Uapaca bojeri mitigates the negative
influence of the exotic species Eucalyptus camaldulensis and Pinus patula. Biol. Invasion. In
press. DOI 10.1007/s10530-012-0238-5
BREVET
Ramanankierana H., Baohanta R., Duponnois R. Prin Y. Reforestation of a soil area with co-
culture of tree species and nurse plant. European Patent Office. Avril 2012
Litter-forager termitemoundsenhance the ectomycorrhizalsymbiosis betweenAcacia holosericea A.Cunn.ExG.DonandSclerodermadictyosporum isolatesRobin Duponnois1, Komi Assikbetse2, Heriniaina Ramanankierana3, Marija Kisa1, Jean Thioulouse4 &Michel Lepage5,6
1Institut de Recherche pour le Developpement, Laboratoire des Symbioses Tropicales et Mediterraneennes, Montpellier, France; 2Institut de Recherche
pour le Developpement, Dakar, Senegal; 3Laboratoire de Microbiologie de l’Environnement, Centre National de Recherches sur l’Environnement,
Antananarivo, Madagascar; 4Laboratoire de Biometrie et Biologie Evolutive, Universite Lyon 1, Villeubanne Cedex, France; 5Institut de Recherche pour le
Developpement, Ouagadougou, Burkina Faso; and 6Laboratoire d’Ecologie, Ecole Normale Superieure, Paris Cedex, France
The hypothesis of the present study was that the termite mounds of Macrotermes
subhyalinus (MS) (a litter–forager termite) were inhabited by a specific microflora
that could enhance with the ectomycorrhizal fungal development. We tested the
effect of this feeding group mound material on (i) the ectomycorrhization
symbiosis between Acacia holosericea (an Australian Acacia introduced in the
sahelian areas) and two ectomycorrhizal fungal isolates of Scleroderma dictyospo-
rum (IR408 and IR412) in greenhouse conditions, (ii) the functional diversity of
soil microflora and (iii) the diversity of fluorescent pseudomonads. The results
showed that the termite mound amendment significantly increased the ectomy-
corrhizal expansion. MS mound amendment and ectomycorrhizal inoculation
induced strong modifications of the soil functional microbial diversity by
promoting the multiplication of carboxylic acid catabolizing microorganisms.
The phylogenetic analysis showed that fluorescent pseudomonads mostly belong
to the Pseudomonads monteillii species. One of these, P. monteillii isolate KR9,
increased the ectomycorrhizal development between S. dictyosporum IR412 and
A. holosericea. The occurrence of MS termite mounds could be involved in the
expansion of ectomycorrhizal symbiosis and could be implicated in nutrient flow
and local diversity.
Introduction
In recent decades, there has been increasing evidence that
soil microorganisms have an important effect on soil fertility
and plant health (Gianinazzi & Schuepp, 1994). Amongst
the microbial populations living in the rhizosphere, myco-
rrhizal fungi have been found to be essential components of
sustainable soil–plant systems (Amato & Ladd, 1988; Beth-
lenfalvay & Linderman, 1992; Hooker & Black, 1995; Van
der Hejden et al., 1998; Hart et al., 2003; Dickie & Reich,
2005). Over 80% of all land plants form some type of
symbiotic association with mycorrhizal fungi. By increasing
the absorptive surface area of their host plant, this fungal
symbiosis influences plant growth and the uptake of nu-
trients, particularly phosphorus, a highly immobile element
in the soil, which thus frequently limits plant growth in
tropical areas. In addition to this positive effect on plant
growth, the hyphae that grow outwards from the mycorrhizae
into the surrounding soil interact with other soil microorgan-
isms and constitute an important pathway for the transloca-
tion of energy-rich plant compounds to the soil. The
expanding mycorrhizal mycelium exploits a larger volume of
soil that would otherwise be inaccessible to plant roots. As
mycorrhizal symbiosis modifies the microbial communities of
its surrounding soil through changes in root exudation, this
microbial compartment is usually named the ‘mycorrhizo-
sphere’ (Linderman, 1988), rather than the rhizosphere. The
mycorrhizosphere includes the more specific term ‘hypho-
sphere’, which refers only to the zone surrounding individual
hyphae. Numerous studies have described the effect of the
mycorrhizosphere on bacterial communities, such as fluores-
cent pseudomonads (Frey et al., 1997; Founoune et al., 2002a)
or rhizobia (Duponnois & Plenchette, 2003). However, some
bacteria belonging to the mycorrhizosphere compartment may
FEMS Microbiol Ecol 56 (2006) 292–303c� 2006 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. No claim to original French government works
promote the development of mycorrhizal symbiosis (Garbaye,
1994). These bacterial strains have been named mycorrhiza
helper bacteria (MHB), and the MHB effect has been recorded
in different plant–fungus combinations (Dunstan et al., 1998;
Founoune et al., 2002b; Duponnois & Plenchette, 2003).
Mycorrhizal establishment usually depends on the plant
species, soil type, soil phosphorus and mycorrhizal fungal
species (Smith & Read, 1997). The mycorrhizosphere effect
will therefore be influenced by soil disturbance (grazing or
erosion) and by the impact of natural events in ecosystem
functioning. For instance, the structures produced by the
soil fauna strongly determine the diversity of the functional
groups in their spheres of influence, at specific space and
time scales (Lavelle, 1996). Termites, as ecosystem engineers,
modulate the availability of resources for other species, such
as microorganisms and plants (Lavelle, 1997). For example,
fruit bodies of the ectomycorrhizal fungus Scleroderma spp.
are regularly observed around the termite mounds of
Macrotermes subhyalinus (a litter–forager termite) in the
south of Burkina Faso (K. Sanon, pers. commun.) and
Australia (Spain et al., 2004). In order to explain this
positive effect of the termite mound on fungal fructification,
we hypothesized that the epigeal mound material was
inhabited by a specific microflora that enhanced ectomyco-
rrhizal fungal development.
In order to verify this hypothesis, we tested the effect of
the mound material of this feeding group on the ectomyco-
rrhizal symbiosis between Acacia holosericea (an Australian
Acacia introduced in sahelian areas) and two ectomyco-
rrhizal fungal isolates of Scleroderma dictyosporum (isolates
IR408 and IR412), which are known to form ectomyco-
rrhizae with A. holosericea seedlings in pot experiments. The
influence of mound material amendment on the functional
diversity of soil microflora was also assessed. As it has been
demonstrated previously that most MHB belong to the
fluorescent pseudomonad group (Frey-Klett et al., 1997),
and that termite mounds of M. subhyalinus are inhabited by
this bacterial genus (Duponnois et al., 2005), we investigated
their diversity and their effect on IR412 ectomycorrhizal
establishment.
Materials and methods
Chemical and microbiological analysis of thesampled epigeal mounds
Five termite mounds of Macrotermes subhyalinus were
collected in a shrubby savanna, 50 km north of Ouagadou-
gou, near the village of Yaktenga (Burkina Faso). The soil
was shallow and rich in gravel above the hardpan level. Large
hydromorphic spots intertwined with the deepest soils
characterized the landscape. Macrotermes mounds were
predominantly localized on deeper soils. Termite mounds
(about 5 kg each) were crushed and passed through a 2 mm
sieve before use.
The chemical and microbiological analyses have been
described in a previous study (Table 1) (Duponnois et al.,
2005). The NH41 and NO3
� contents were measured
according to the method of Bremner, 1965, whereas avail-
able phosphorus was determined according to Olsen et al.
(1954). The content of ergosterol was determined using the
method of Grant & West (1986). The fumigation–extraction
method was used to estimate the microbial biomass (Amato
& Ladd, 1988). The enumeration of colony-forming units
was carried out on King’s B agar medium for the fluorescent
pseudomonads (King et al., 1954) and on actinomycete
isolation agar medium (Difco Laboratories, Detroit, MI)
for the actinomycetes. The isolates of fluorescent pseudo-
monads were randomly selected (18 bacterial strains),
purified, subcultured on King’s B medium and cryopre-
served at � 80 1C in glycerol 60%-TSB (tryptic soy broth,
3 g L�1) culture [1/1, volume in volume (v/v)].
Molecular characterization of fluorescentpseudomonad isolates
Fluorescent pseudomonads were grown overnight on TSB
agar plates at 28 1C. For each strain, a single colony was
picked up and suspended in 50 mL of lysis buffer [0.05 M
NaOH, 0.25% sodium dodecylsulphate (SDS)], vortexed for
60 s, heated to 95 1C for 15 min and centrifuged at 2400 g.
for 10 min. The lysate cell suspensions were diluted (1/10, v/
v) with sterile distilled water. The primers rD1 (50-AAGCT-
TAAGGAGGTGATCCAGCC-30) and fD1 (50-AGAGTTT-
GATCCTGGCTCAG-30) were used to amplify the 16S
rDNA gene (Frey-Klett et al., 1997). PCR was performed in
a GeneAmp PCR System 2400 thermal cycler (Perking-
Elmer, Foster City, CA) using PureTaq Ready-To-Go PCR
beads (Amersham Biosciences, Orsay, France), 1 mM of each
primer and 3 mL of bacterial cell suspension in 25 mL
reaction mixtures. The mixture was submitted to 5 min of
initial denaturation, followed by 35 cycles at 94 1C for 1 min,
55 1C for 45 s and 72 1C for 1.5 min. A final elongation step
Table 1. Biological and chemical characteristics of Macrotermes sub-
hyalinus mound powder
Biological and chemical characteristics M. subhyalinus
NH41 (mg N g�1 of dry mound powder) 9.4
NO3� (mg N g�1 of dry mound powder) 3408.9
Available P (mg g�1 of dry mound powder) 3.5
Microbial biomass (mg C g�1 of dry mound powder) 22.5
Fluorescent pseudomonads
(�102 CFU g�1 of dry mound powder)
79.3
Actinomycetes (�102 CFU g�1 of dry mound powder) 39.5
Ergosterol (mg g�1 of dry mound powder) 0.316
FEMS Microbiol Ecol 56 (2006) 292–303 c� 2006 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. No claim to original French government works
nitrogen, 5.2; soluble phosphorus, 0.0043 mg g�1; total
phosphorus, 0.116 mg g�1. The soil was mixed with 10%
(v/v) of mound powder and/or 10% (v/v) IR408 or IR412
fungal inoculum. The control treatment was not mixed with
either mound powder or fungal inoculum. There were six
treatments: control (C), fungal isolate inoculation (IR408
and IR412), termite mound amendment (MS) and fungal
inoculum and termite mound added together to the soil
(IR4081MS and IR412 1 MS). The plants were placed in a
glasshouse (25 1C by day, 15 1C by night, 10 h photoperiod)
and watered regularly with tap water without the addition of
fertilizer. They were arranged in a randomized complete
block design with eight replicates per treatment.
After 4 months of culture, the plants were collected and
their root systems were gently washed under running tap
water. The oven dry weight (1 week at 65 1C) of the shoot
was measured. Some nodules were detected along the root
systems despite disinfection of the soil and the seed surface.
FEMS Microbiol Ecol 56 (2006) 292–303c� 2006 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. No claim to original French government works
294 R. Duponnois et al.
The root nodules were counted. The root systems were cut
into 1 cm root pieces and mixed. The percentage of ectomy-
corrhizal colonization [(number of ectomycorrhizal short
roots/total number of short roots)� 100] was determined
under a stereomicroscope at �40 magnification on a
random sample of at least 100 short roots per root system.
The arbuscular mycorrhizal fungal colonization was assessed
after clearing and staining the root pieces according to the
method of Phillips & Hayman (1970). The root pieces were
placed on a slide for microscopic observations at � 250
magnification (Brundrett et al., 1985). About 50 1 cm root
pieces were observed per plant. Arbuscular mycorrhizal
colonization was expressed in terms of the fraction of the
root length with mycorrhizal internal structures (vesicles or
hyphae): [(length of colonized root fragments/total length
of root fragments)� 100]. The dry weight (65 1C, 1 week) of
the roots was then determined.
The soil from each pot was mixed and kept at 4 1C for the
assessment of the catabolic diversity of microbial commu-
nities.
Assessment of the catabolic diversity of microbialcommunities
The microbial functional diversity in soil treatments was
assessed by the determination of the in situ catabolic
potential patterns of microbial communities (Degens &
Harris, 1997). A range of amino acids, carbohydrates,
organic acids, amides and a polymer were screened for
differences in substrate-induced respiration (SIR) respon-
siveness between soil treatments (Table 2). The substrate
concentrations providing optimum SIR responses are in-
dicated in Table 2 (Degens & Harris, 1997). Four replicates
(soil samples) were randomly chosen from each treatment.
One gram equivalent of dry weight soil for each sample was
suspended in 2 mL of sterile distilled water in 10 mL bottles
(West & Sparling, 1986). CO2 production from basal
respiratory activity in the soil samples was also determined
by adding 2 mL of sterile distilled water to 1 g equivalent of
dry weight soil. The bottles were immediately closed and
kept at 28 1C for 4 h after the addition of the substrate
solutions to the soil samples. CO2 fluxes from the soils were
assessed using an infrared gas analyser (Polytron IR CO2,
Drager, Germany) in combination with a thermal flow
meter (Heinemeyer et al., 1989). Results were expressed as
micrograms of CO2 per gram of soil per hour.
Effect of the fluorescent pseudomonad isolateKR9 on IR412 ectomycorrhizal development
Seeds of A. holosericea were prepared as described above; A.
holosericea seedlings were individually grown in 1 L pots
filled with the same autoclaved sandy soil (140 1C, 40 min)
as used in the previous glasshouse experiment. The substrate
was mixed with 10% (v/v) IR412 fungal inoculum or with a
10% vermiculite–peat mixture (4/1, v/v) for treatments
without fungus. Immediately after planting, the young
seedlings from the experimental group were inoculated with
5 mL of fluorescent pseudomonad KR9 suspension (108
bacterial cells), whereas those from the control group were
inoculated with 5 mL of 0.1 M MgSO4 solution. The plants
were placed in a glasshouse (25 1C by day, 15 1C by night,
10-h photoperiod) and watered regularly with tap water
without the addition of fertilizer. The pots were arranged in
a randomized complete block design with eight replicates
per treatment.
After 4 months of culture, the shoot and root biomass,
the number of nodules and the percentage of ectomycor-
rhizal colonization were determined for each plant in each
treatment, as described above.
Statistical analysis
The data were treated with one-way analysis of variance.
Means were compared using Fisher’s protected least signifi-
cant difference test (Po 0.05). The percentages of myco-
rrhizal colonization were transformed by arcsin (sqrt) before
statistical analysis. Co-inertia analysis was performed for
plant growth, mycorrhizal colonization indices and SIR
Doledec & Chessel, 1994) is a multivariate analysis
Table 2. Organic compounds and their concentrations used to assess
patterns of ISCP of soil treatments
Organic substrates Organic substrates
Amino acids (15 mM) Carboxylic acids (100 mM)
L-Glutamine 2-Keto-glutaric acid
L-Arginine 3-Hydroxybutyric acid
L-Serine Ascorbic acid
L-Histidine D-quinic acid
Phenylalanine DL-malic acid
L-Asparagine Formic acid
L-Tyrosine Fumaric acid
L-Glutamic acid Gallic acid
L-Lysine Gluconic acid
L-Cystein Ketobutyric acid
Malonic acid
Carbohydrates (75 mM) Oxalic acid
D-Glucose Succinic acid
D-Mannose Tartaric acid
Sucrose Tri-sodium citrate
Uric acid
Amides (15 mM)
D-Glucosamine Polymer (100 mM)
N-methyl-D-Glucamine Cyclohexane
Succinamide
ICSP, in situ catabolic potential.
FEMS Microbiol Ecol 56 (2006) 292–303 c� 2006 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. No claim to original French government works
Catabolic diversity of microbial communities insoil treatments
Co-inertia analysis of the relationship between plant growth,
mycorrhizal formation and SIR responses is shown in Fig. 4.
The four figures (Fig. 4a–d) can be superimposed to allow
700
200100
M 1 2 3 4 5 6 7 8 9 10 11
800
500
100
M 1 2 3 4 5 6 7 8 9 10 11
(a)
(b)
Fig. 1. Gel electrophoresis of PCR-amplified 16S rDNA fragments of
fluorescent pseudomonad isolates digested with HaeIII (a) and MspI (b).
Lanes 1–11: fluorescent pseudomonads isolated from termite mounds of
Macrotermes subhyalinus. Lane M, 100 bp molecular size ladder.
FEMS Microbiol Ecol 56 (2006) 292–303c� 2006 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. No claim to original French government works
296 R. Duponnois et al.
the analysis of the relationships between these variables. The
Monte-Carlo test showed that there was a statistically
significant, although not extremely strong, relationship
(P = 0.025). Figure 4a and 4c shows the positive effect of
fungal inoculation on plant growth: the points correspond-
ing to the inoculated treatments (IR408 and IR412) are
shifted towards the right of the figures, which correspond to
higher root and shoot biomass. The positive effect of
Macrotermes subhyalinus mound powder amendment on
plant growth is also clearly visible: treatments MS, IR408 1
P. FLUORESCENS (AJ308308)
P. veronii (AY081814)
P. marginalis (AJ308309)
P. tolaasii (AJ308317)
P. mandeliia (F058286)
P. syringae (AJ308316)
P. chlororaphis (AJ308301)
P. aurantiaca (AJ308299)
P. taetrolens (D84027)
P. cichorii (AJ308302)
P. jessenii (F068259)
P. agarici (AJ308298)
P. gingeri (AF332511)
P. fulva (D84015)Pseudomonas sp. KR9
P. monteillii HR 13 (AY032725) P. mosselii (AF072688)
P. putida (AB029257)
P. plecoglossicida (AB009457)
P. monteilii (AF064458)
P. mevalonii (AJ299216)
P. flavescens (AJ308320)
P. mendocina (AJ308310)
P. stutzeri (AB126690)
P. fragi (AB021413)
P. denitrificans (AB021419)
P. alcaligenes (D84006)
P. resinovorans (AJ308314)
P. aeruginosa (AJ308297)
E. coli (AJ01859)
0.000.020.040.06
Fig. 2. Dendrogram showing neighbour-joining
analysis of 16S rDNA from some fluorescent pseu-
domonads retrieved from the Ribosome Database
Project. The sequence obtained in this study is
indicated in bold. Accession numbers are indicated
in parentheses.
Table 3. Effects of fungal inoculation and Macrotermes subhyalinus mound powder amendment on the Acacia holosericea growth, on the total
number of nodules per plant and on the arbuscular mycorrhizal colonization after 4 months of culturing in greenhouse conditions
Treatments
Shoot biomass
(mg dry weight)
Root biomass
(mg dry weight)
Arbuscular mycorrhizal
colonization index (%)
Total number of
nodules per plant
Control 261 a� 33 a 0 a 0.5 a
Scleroderma sp. IR408 1458 c 318 bc 0 a 2.3 a
S. dictyosporum IR 412 964 b 190 ab 0 a 2.5 a
M. subhyalinus (MS) 1288 bc 238 b 0.5 a 1.2 a
IR 4081MS 1051 b 432 cd 0.5 a 0.5 a
IR 4121MS 1140 bc 606 d 1.8 a 1.0 a
�Data in the same column followed by the same letter are not significantly different according to the one-way analysis of variance (Po 0.05).
FEMS Microbiol Ecol 56 (2006) 292–303 c� 2006 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. No claim to original French government works
malonic, oxalic, succinic, tartaric and uric acids, trisodium
citrate and cyclohexane (Table 4). The SIR response with
gallic acid was significantly higher when termite mound and
ectomycorrhizal inoculum were both added to the soil
(Table 4). The highest catabolic richness was recorded in
the IR412 treatment, whereas the highest catabolic evenness
was recorded in the IR4081MS treatment (Table 4).
Effect of a fluorescent pseudomonad strain(isolate KR9) on IR412 ectomycorrhizaldevelopment
After 4 months of culture, S. dictyosporum IR412 had
colonized A. holosericea seedlings and had significantly
increased shoot and root growth (Table 5). By contrast, no
significant effect of the bacterial inoculant KR9 was recorded
on plant growth. When KR9 was co-inoculated with IR412,
plant growth was significantly higher than that measured
when IR412 was inoculated alone; ectomycorrhizal coloni-
zation was also significantly increased (from 28.3% to
48.5%) (Table 5). The total biomass of the plants correlated
significantly with the mycorrhizal rates (r2 = 0.78). Nodules
were observed in all treatments. Ectomycorrhizal inocula-
tion significantly enhanced the number and total weight of
nodules per plant. This fungal positive effect was signifi-
cantly increased when S. dictyosporum was co-inoculated
with KR9 (Table 5). The number and total biomass of
nodules per plant were significantly linked with the myco-
rrhizal rates (r2 = 0.76 and r2 = 0.79, respectively).
Discussion
The main objectives of this study were to test the effect of a
Macrotermes subhyalinus mound structure amendment on
the formation of ectomycorrhizae between Acacia holo-
sericea and two isolates of Scleroderma dictyosporum and to
evaluate the role of fluorescent pseudomonads inhabiting
the mound in these interactions.
In a previous study, spores of ectomycorrhizal fungi were
detected in the mounds of wood-, litter- and grass-feeding
termites (Spain et al., 2004). The authors showed that there
was a greater diversity and more concentrated populations
of ectomycorrhizal fungal spores in the mounds than in the
surrounding soil. They also detected basidiocarps of the
common genera Pisolithus and Scleroderma species on the
mound surfaces. This localization of fruit bodies indicated
that the hyphae in the mounds originated from the nearest
putative host plants. In our study, no ectomycorrhizal short
roots were detected in the M. subhyalinus treatment without
ectomycorrhizal fungal inoculation. This result seems to
contradict the conclusions of Spain et al. (Spain et al., 2004).
However, the termite mounds of M. subhyalinus were
collected in a shrubby savanna where all the plant species
were associated with arbuscular mycorrhizal fungi (Dupon-
nois et al., 2001). As no potential ectomycorrhizal host tree
species was present in these areas, termite mounds could be
overspread by ectomycorrhizal short roots. In addition, in a
previous study (Duponnois & Lesueur, 2005), the formation
of ectomycorrhizae was not observed after 4 months of
culture when spores of ectomycorrhizal fungi were inocu-
lated in the soil.
In the present study, termite mound amendment signifi-
cantly enhanced the ectomycorrhizal expansion of both
fungal isolates. This promoting effect could be attributed
to: (1) the enhancement of plant growth (particularly root
growth) induced by termite mound amendment; (2) inocu-
lation (via the termite mound) by a bacterial group (i.e.
fluorescent pseudomonads) that could act as MHB (Du-
ponnois & Plenchette, 2003); and (3) the development of
0
5
10
15
20
25
30
Con
trol
+ M
S
IR40
8
IR41
2
IR40
8+M
S
IR41
2+M
S
Ect
omyc
orrh
izal
col
oniz
atio
n (%
)
a a
b b
c c
Fig. 3. Ectomycorrhizal formation of Scleroderma sp. IR408 and Scle-
roderma dictyosporum IR412 on Acacia holosericea root systems in soil
amended and not amended with Macrotermes subhyalinus mound
powder after 4 months of culture in glasshouse conditions. Columns
indicated by the same letter are not significantly different according to
one-way analysis of variance (P o 0.05). MS, M. subhyalinus mound
powder amendment.
FEMS Microbiol Ecol 56 (2006) 292–303c� 2006 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. No claim to original French government works
298 R. Duponnois et al.
multitrophic interactions between the ectomycorrhizal sym-
biosis and the soil microflora.
Termite mounds (Isoptera) are a ubiquitous feature of
tropical ecosystems, especially in savanna environments.
Through termite activities, large amounts of soil are trans-
located from various depths of the soil profile (Holt &
Lepage, 2000). In some areas, such termitaria represent a
soil volume of more than 300 m3 above the ground. These
structures strongly influence their environment. In their
review, Lobry de Bruyn & Conacher (1990) reported a soil
quantity of up to 4.7 tonnes ha�1 year�1. This termite activity
has a considerable influence on soil physical and chemical
properties (Lee & Wood, 1971; Lobry de Bruyn & Conacher,
1990; Black & Okwakol, 1997; Holt & Lepage, 2000), and
largely explains the termite role as ecosystem engineers. In
the present study, termite mound amendment stimulated
root growth, probably through an enhanced supply of
nitrogen, which, in turn, increased the number of fungal
infection sites.
Recent studies have suggested that termite mounds could
be sites of great bacterial and fungal diversity. Termite nests
generally contain a diversity of fungi (Sannasi & Sundara-
Rajulu, 1967; Mohindra & Mukerji, 1982). In Macrotermes
bellicosus mound soil in Nigeria, Thomas (Thomas, 1987a)
found 21 species of fungi. Other authors have found large
populations of active bacteria in termite mounds, different
from those of the parent soil: eight functional bacterial
groups were found in a Macrotermes mound in Rhodesia
(Meiklejohn, 1965). The higher microbial diversity in ter-
mite mounds was attributed to higher organic matter levels
Table 4. Effect of ectomycorrhizal inoculation and Macrotermes subhyalinus mound powder amendment on in situ catabolic potential (ISCP) of
microbial communities and catabolic richness, catabolic evenness in soil treatments
Organic substrates
Treatments
Control IR 408 IR 412 M. subhyalinus (MS) IR 4081MS IR 4121MS
L-Glutamine 5.44 ab� 4.16 ab 4.13 ab 3.89 a 8.05 b 4.20 ab
L-Arginine 8.48 ab 14.14 c 15.45 c 6.09 a 16.53 c 11.96 bc
L-Serine 1.96 ab 3.24 bc 1.96 ab 1.96 ab 3.48 c 1.52 a
L-Histidine 0.0 a 0.0 a 0.87 b 0.22 ab 0.0 a 0.87 b
Phenylalanine 0.26 ab 0.70 ab 0.47 ab 0.02 a 1.79 b 0.89 ab
L-Asparagine 4.64 a 7.41 a 7.84 a 6.09 a 4.79 a 6.53 a
L-Tyrosine 3.79 c 3.58 bc 2.93 bc 0.94 a 2.93 bc 1.84 ab
L-Glutamic acid 4.76 a 4.15 a 3.72 a 3.89 a 5.11 a 4.19 a
L-Lysine 3.26 ab 2.61 ab 2.61 ab 3.92 b 1.96 a 3.05 ab
D-Glucose 5.44 a 6.96 ab 11.5 b 7.6 ab 9.13 ab 11.31 b
D-Mannose 2.61 a 3.48 a 3.26 a 2.61 a 3.26 a 2.83 a
Sucrose 2.39 a 3.26 a 3.26 a 6.09 b 7.18 b 6.75 b
D-Glucosamine 5.66 a 6.31 a 8.49 a 18.5 b 11.3 a 5.87 a
N-methyl-D-Glucamine 3.51 ab 3.72 b 3.50 ab 3.50 ab 2.89 a 3.94 b
Succinamide 3.26 abc 4.57 c 2.17 ab 2.83 abc 4.14 bc 1.52 a
2-Keto-glutaric acid 66.61 a 70.71 ab 75.74 ab 90.77 c 73.14 ab 84.05 bc
3-Hydroxybutyric acid 1.23 ab 0.87 a 1.09 a 4.57 c 3.92 bc 3.92 bc
Ascorbic acid 1.96 a 3.05 a 2.61 a 6.09 b 5.44 b 6.21 b
D-Quinic acid 1.52 a 1.74 a 4.13 a 13.49 b 14.81 b 15.01 b
DL-Malic acid 1.52 ab 3.05 b 2.39 b 0.0 a 2.87 b 2.39 b
Formic acid 7.35 b 9.74 c 6.69 b 2.34 a 10.46 c 4.09 b
Fumaric acid 0.65 a 1.31 a 0.43 a 4.13 b 2.61 ab 1.96 ab
Gallic acid 5.66 a 6.53 ab 5.88 a 5.22 a 10.01 c 9.36 bc
Gluconic acid 3.92 a 4.13 a 7.18 ab 10.88 b 10.01 b 9.58 b
Ketobutyric acid 59.86 a 65.3 a 62.47 a 87.72 b 65.08 a 82.28 b
Malonic acid 3.23 a 4.57 ab 4.78 ab 20.68 c 11.07 ab 12.61 bc
Oxalic acid 19.22 ab 18.94 a 26.34 ab 38.53 c 25.69 ab 28.08 b
Succinic acid 1.96 a 4.35 a 2.61 a 8.05 b 4.57 a 4.12 a
Tartaric acid 2.39 a 3.70 a 3.51 a 13.49 c 11.75 c 7.57 b
Tri-sodium citrate 3.71 a 3.27 a 3.71 a 9.79 c 6.96 b 9.36 c
Uric acid 5.88 a 8.05 ab 8.71 abc 11.10 bc 14.98 d 11.97 cd
Cyclohexane 4.35 a 4.79 a 3.71 a 6.96 b 6.96 b 7.18 b
Catabolic richness 30.7 ab 31.2 b 32.0 b 29.8 a 31.0 ab 30.8 ab
Catabolic eveness 2.55 a 2.65 a 2.62 a 2.62 a 2.84 b 2.66 a
Data are expressed as mg CO2 g�1 soil h�1.�Data in the same line followed by the same letter are not significantly different according to the one-way analysis of variance (Po 0.05).
FEMS Microbiol Ecol 56 (2006) 292–303 c� 2006 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. No claim to original French government works
The population and composition of microbial groups
appear to vary according to the mound compartment
considered (Brauman, 2000). Increasing evidence demon-
strates that termites are able to control the number of
2
11
5
3
2122 7
166 10ECI
AMI
NN
SB
RB
−2 −1 2
826
17 23
28
30 25
19
141 4
27
9
12 20
1318 24
29
31
32
33
−0.8
1.1−0.8 1.1
IR 408
IR 408 + MSIR 412
IR 412 + MS
MS
C
−3
3−3 3
IR 408 IR 408 + MS
IR 412
IR 412 + MS
MS
C
−4
6−5 5
(a) (b)
(c) (d)
1
Fig. 4. Co-inertia analysis of substrate-induced respiration (SIR) responses of soils inoculated or not with Scleroderma dictyosporum isolates IR408 and
IR412 and amended or not with mound powder. In the four panels (a–d), the top-right inset gives the minimum and maximum of the horizontal and
vertical coordinates. (a) Factor map of plant growth. Mycorrhizal and rhizobial variables: SB, shoot biomass; RB, root biomass; AMI, arbuscular
mycorrhizal colonization index; ECI, ectomycorrhizal colonization index; NN, number of nodules per plant. (b) Factor map of SIR responses. 1,
Microbial and soil sample variables: C, control; MS, soil amended with Macrotermes subhyalinus mound powder; IR408, soil inoculated with S.
dictyosporum strain IR408; IR412, soil inoculated with S. dictyosporum strain IR412; IR4081MS, soil inoculated with S. dictyosporum strain IR408 and
amended with M. subhyalinus mound powder; IR4121MS, soil inoculated with S. dictyosporum strain IR412 and amended with M. subhyalinus mound
powder. The star-like diagrams represent the four replicates of each treatment, and the dot inside each star is the mean of these replicates. (d) Factor
map of SIR responses of soil samples (for details, see c).
Table 5. Effect of Scleroderma dictyosporum IR412 and/or the fluorescent pseudomonad strain, isolate KR9, on mycorrhiza formation, rhizobial
development growth of Acacia holosericea after 4 months culture under glasshouse conditions
Treatments
Shoot biomass
(mg dry weight)
Root biomass
(mg dry weight)
Number of nodules
per plant
Total nodule weight
per plant (mg)
Ectomycorrhizal
colonization (%)
Control 532 a� 184 a 4.2 a 6.8 a 0 a
Isolate KR9 553 a 198 a 4.6 a 7.1 a 0 a
S. dictyosporum IR 412 1236 b 536 b 8.3 b 15.9 b 28.3 b
S. dictyosporum IR 4121Isolate KR9 1786 c 868 c 12.4 c 25.3 c 48.5 c
�Data in the same column followed by the same letter are not significantly different according to the one-way analysis of variance (Po 0.05).
FEMS Microbiol Ecol 56 (2006) 292–303c� 2006 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. No claim to original French government works
300 R. Duponnois et al.
microorganisms, and probably their diversity, in selected
parts of their mounds (Sannasi & Sundara-Rajulu, 1967;
Holt & Lepage, 2000). Previous microbiological studies of
termite mounds have been carried out to compare the
cultures of microbial communities in grass-, litter- and
soil-feeding termite mounds (Duponnois et al., 2005).
Fluorescent pseudomonads have been detected only in M.
subhyalinus mound powder. The phylogenetic analysis per-
formed in this study showed that these fluorescent pseudo-
monads mostly belonged to Pseudomonas monteillii species.
It has been demonstrated that one isolate of P. monteillii
(isolate HR13) can stimulate the establishment of ectomy-
corrhizal symbiosis in tropical conditions (Founoune et al.,
2002b) and is considered as an MHB. This MHB effect has
been recorded with different fungal isolates, such as S.
dictyosporum, S. verrucosum, Pisolithus albus and P. tinctor-
ius, on A. holosericea and other Australian Acacia species
(Duponnois & Plenchette, 2003). As P. monteillii isolate KR9
stimulated ectomycorrhizal formation between S. dictyos-
porum IR412 and A. holosericea, these bacterial strains
present in M. subhyalinus mounds could also be involved in
the enhancement of ectomycorrhizal formation recorded in
the present study.
Macrotermes subhyalinus mound amendment and ecto-
mycorrhizal inoculation induced strong modifications of
functional microbial diversity. In particular, important soil
microflora, able to use carboxylic acids, were detected
through high SIR responsiveness with these compounds.
Biological and biochemical weathering is mediated by
microorganisms that excrete organic acids, phenolic com-
pounds, protons and siderophores (Drever & Vance, 1994).
For instance, it is well known that many different fungal
species produce these organic acids as the strongest chelators
of trivalent metals (oxalate, malate and citrate) (Dutton &
Evans, 1996; Gadd, 1999). In addition, amongst termites,
the Macrotermitinae subfamily (also called ‘fungus-growing
termites’) plays a major role in African ecosystem function-
ing, mainly in arid and semi-arid areas. The effect of these
termites on soil microbiology is not only due to their
influence on nonmutualistic microorganisms, but also to
their specific exosymbiotic relationship with the fungus
Termitomyces, which is only found in special structures
within the mound, called ‘fungus combs’ (Sands, 1969;
2000). It is suggested that these fungal communities (sapro-
phytic and ectomycorrhizal fungi) could exert a selective
influence on the soil microflora by promoting the multi-
plication of carboxylic acid catabolizing microorganisms.
Macrotermitinae-built structures constitute patches in the
landscape in which the availability of soil nutrients for plants
is improved (Jouquet, 2002). Associations between fungus-
growing nests and grasses have recently been found in West
African savanna (Jouquet et al., 2004). Mounds of grass- and
litter-feeding termites form fertile ‘islands’ in the savanna,
maintaining fertility in these, mostly highly weathered, soils
(Okello-Oloya et al., 1985, 1986; Lobry de Bruyn & Con-
acher, 1990). This positive effect is generally attributed to
the activity of termites, which translocate nutrient elements
in food into their mounds. However, another translocation
could be proposed, from the termite mound to the host
plant, mediated by ectomycorrhizal roots.
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FEMS Microbiol Ecol 56 (2006) 292–303 c� 2006 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. No claim to original French government works
Arbuscular mycorrhizas and ectomycorrhizas of Uapacabojeri L. (Euphorbiaceae): sporophore diversity, patternsof root colonization, and effects on seedling growthand soil microbial catabolic diversity
Naina Ramanankierana & Marc Ducousso &
Nirina Rakotoarimanga & Yves Prin & Jean Thioulouse &
Emile Randrianjohany & Luciano Ramaroson &
Marija Kisa & Antoine Galiana & Robin Duponnois
Received: 2 October 2006 /Accepted: 30 November 2006 / Published online: 13 January 2007# Springer-Verlag 2007
Abstract The main objectives of this study were (1) todescribe the diversity of mycorrhizal fungal communitiesassociated withUapaca bojeri, an endemic Euphorbiaceae ofMadagascar, and (2) to determine the potential benefits ofinoculation with mycorrhizal fungi [ectomycorrhizal and/orarbuscular mycorrhizal (AM) fungi] on the growth of thistree species and on the functional diversity of soil microflora.Ninety-four sporophores were collected from three survey
sites. They were identified as belonging to the ectomycor-rhizal genera Afroboletus, Amanita, Boletus, Cantharellus,Lactarius, Leccinum, Rubinoboletus, Scleroderma, Tricho-loma, and Xerocomus. Russula was the most frequentectomycorrhizal genus recorded under U. bojeri. AMstructures (vesicles and hyphae) were detected from theroots in all surveyed sites. In addition, this study showed thatthis tree species is highly dependent on both types ofmycorrhiza, and controlled ectomycorrhization of thisUapaca species strongly influences soil microbial catabolicdiversity. These results showed that the complex symbioticstatus of U. bojeri could be managed to optimize itsdevelopment in degraded areas. The use of selectedmycorrhizal fungi such the Scleroderma Sc1 isolate innursery conditions could be of great interest as (1) thisfungal strain is very competitive against native symbioticmicroflora, and (2) the fungal inoculation improves thecatabolic potentialities of the soil microflora.
A high botanical diversity and a high degree of endemismcharacterize Madagascarian forests (Lowry et al. 1997), butthey are often deforested for their conversion to agriculture.Deforestation rates were estimated to be 0.11 Mha year−1
between 1950 (7.6 Mha) and 1985 (3.8 Mha; Green and
N. Ramanankierana :N. Rakotoarimanga : E. Randrianjohany :L. RamarosonLaboratoire de Microbiologie de l’Environnement,Centre National de Recherches sur l’Environnement,P.O. Box 1739, Antananarivo, Madagascar
M. Ducousso :Y. Prin :A. GalianaCIRAD, UMR 113 CIRAD/INRA/IRD/AGRO-M/UM2,Laboratoire des Symbioses Tropicales et Méditerranéennes(LSTM), TA10/J, Campus International de Baillarguet,34398 Montpellier Cedex 5, France
J. ThioulouseCNRS, Laboratoire de Biométrie et Biologie Evolutive,UMR 5558, Université Lyon 1,69622 Villeurbanne Cedex, France
M. Kisa :R. DuponnoisIRD, UMR 113 CIRAD/INRA/IRD/AGRO-M/UM2, Laboratoiredes Symbioses Tropicales et Méditerranéennes (LSTM), TA10/J,Campus International de Baillarguet,34398 Montpellier Cedex 5, France
R. Duponnois (*)IRD, Laboratoire Commun de Microbiologie IRD/ISRA/UCAD,Centre de Recherche de Bel Air,P.O. Box 1386, Dakar, Senegale-mail: [email protected]
Sussman 1990). Disturbances of the vegetation cover areoften accompanied by rapid erosion of surface soil thatinduces a loss or reduction of major physicochemical andbiological soil properties (Vagen et al. 2006a,b). Inparticular, it has been shown that mycorrhizal soil potentialwas drastically reduced (Marx 1991; Jasper et al. 1991;Herrera et al. 1993; Dickie and Reich 2005). Hence, anincrease of this fungal inoculum potential is needed in bothnatural and artificial revegetation processes (McGee 1989).However, the mycorrhizal status of the Madagascarian florais poorly known. Typical ectomycorrhizal fungi werereported more than 60 years ago (Heim 1970). Morerecently, mycological surveys show the large diversity ofthe associated ectomycorrhizal fungi (Buyck et al. 1996;Ducousso et al. 2004). The mycorrhizal inoculation ofplants is very efficient in establishing plants on disturbedsoils (Estaun et al. 1997; Duponnois et al. 2001, 2005). Themanagement of mycorrhizal symbiosis needs to investigatethe presence, abundance, and community composition ofmycorrhizal fungi associated with plants. Furthermore,efficient fungal strains have to be selected to help treeestablishment and also to improve soil quality (Fransonand Bethlenfalvay 1989; Duponnois and Plenchette 2003;Diédhiou et al. 2005; Duponnois et al. 2005).
The benefits of mycorrhizal symbiosis to the host planthave usually been considered a result from the closerelationship between fungal symbionts and plant species.However, it has been demonstrated that mycorrhizalsymbiosis has a great influence on the soil bacterial andfungal communities in natural conditions (Frey et al. 1997;Founoune et al. 2002a,b; Mansfeld-Giese et al. 2002; Frey-Klett et al. 2005). This microbial compartment is common-ly named “mycorrhizosphere” (Linderman 1988) and isusually divided in two different zones: one is subjected tothe dual influence of the root and the mycorrhizalsymbionts (the mycorrhizosphere) and, the other, underthe influence of mycorrhizal hyphae (the hyphosphere).The microbial activities that occur in the hyphosphere aredifferent from those recorded in the mycorrhizosphere(Andrade et al. 1998). Hyphosphere microorganisms mayinfluence mycorrhizal functions such as nutrient and wateruptake carried out by the external hyphae of the mycorrhi-zal fungi (Duponnois, unpublished data). Hence, theassociation between the fungus and the host plant has beenenlarged to the soil microflora to form a multitrophicmycorrhizal complex (Frey-Klett et al. 2005). The micro-bial functional diversity of each soil compartment includesa vast range of activities (nutrient transformations, decom-position, etc.) and can be characterized by the measurementof catabolic response profiles (CRPs; Degens and Harris1997; Degens et al. 2001). The measurement of CRPsdirectly assesses the catabolic diversity of microbialcommunities involved in decomposition activities by add-
ing a range of simple organic substrates directly to the soiland measuring the short-term catabolic responses (Degensand Harris 1997). Catabolic evenness, a component ofmicrobial functional diversity is defined as the uniformityof substrate use and can be calculated from the CRPs(Degens and Harris 1997).
Mycorrhizal fungi are ubiquitous components of mostecosystems throughout the world and are considered keyecological factors in governing the cycles of major plantnutrients and in sustaining the vegetation cover (van derHejden et al. 1998; Requena et al. 2001; Schreiner et al.2003). Two major forms of mycorrhizas are usuallyrecognised: the arbuscular mycorrhizas (AM) and theectomycorrhizas (ECMs). AM symbiosis is the mostwidespread mycorrhizal association type with plants thathave true roots, i.e. pteridophytes, gymnosperms andangiosperms (Read et al. 2000). They affect about 80–90% land plants in natural, agricultural, and forestecosystems (Brundrett 2002). ECMs affect trees andshrubs, gymnosperms (Pinaceae) and angiosperms, andare usually the result of the association of Homobasidio-mycetes with about 20 families of mainly woody plants(Smith and Read 1997). These woody species are associ-ated with a larger (compared to the AM symbiosis)diversity of fungi, comprising 4,000 to 6,000 species,mainly Basidiomycetes and Ascomycetes (Allen et al.1995; Valentine et al. 2004).
The main objectives of this study were (1) to describethe diversity of mycorrhizal fungal communities associatedwith Uapaca bojeri, an endemic Euphorbiaceae of Mada-gascar and (2) to determine the potential benefits ofinoculation with mycorrhizal fungi (ectomycorrhizal and/or AM fungi) on the growth of this tree species and on thefunctional diversity of soil microflora.
Materials and methods
Site description and sporophore sampling
Three forests in Madagascar were visited at 2- to 3-weekintervals during the sampling seasons, mid-November toearly February 1993, July–August 1994, and July to mid-September 1995, to collect ectomycorrhizal fungi fruitingunder U. bojeri. The forests were located 50 km to the westof Antananarivo (Arivonimamo site as site A), 20 km to thesouth of Antsirabe (Ambositra site as site B), and 100 kmto the east of Toliara (Isalo site as site C). The mean annualrainfall varied from 912.4 mm (site C), 1,428.8 mm (siteA), to 1,554.4 mm (site B). The vegetation sampledincluded savannas (sites A and B) and deciduous forests(site C). The main chemical characteristics of the upper soillayer (0–20 cm) of these sites are shown in Table 1.
196 Mycorrhiza (2007) 17:195–208
Sporophores of putative epigeous ectomycorrhizal fungiwere collected under U. bojeri, photographed, described asfresh material, preserved by oven-drying, and deposited atthe herbarium at Laboratoire de Microbiologie de l’Envi-ronnement (LME, Madagascar). In addition, roots of U.bojeri were collected in each site, and fine roots werestained for AM according to the procedure of Phillips andHayman (1970) and examined with light microscopy.
Time sequence of mycorrhizal colonization on U. bojeriin glasshouse conditions
Surface forest soil (0- to 20-cm depth) was collected fromthe native stand of U. bojeri in site A, crushed, passedthrough a 2-mm sieve, carefully mixed, and distributed in1-l pots. The seeds of U. bojeri collected in site A weresurface sterilized in hydrogen peroxide for 10 min, rinsedand soaked in sterile distilled water for 12 h, and ger-minated on 1% agar. After 1 week of incubation at 30°C inthe dark, one pre-germinated seed was planted per pot. Theseedlings were screened from the rain and grown undernatural light (daylight of approximately 12 h, average dailytemperature of 25°C). They were watered regularly withtap water without fertilizer.
During 5 months, four plants per month were randomlysampled, uprooted, and their root systems gently washed withtap water. About 30 lateral roots were randomly chosen alongthe tap root of each plant, cut into short pieces, and observedunder a stereomicroscope (magnification ×40). All ECMswere counted on each root fragment. Other root samples werecollected from each plant to detect AM structures using thesame procedure as before (Phillips and Hayman 1970).
Assessment of U. bojeri mycorrhizal dependency
A strain of Scleroderma sp. (strain Sc1) was isolated from asporocarp collected in site A. This fungal isolate waspreviously tested for its compatibility with U. bojeri inaxenic conditions (data not shown). The fungal strain wasmaintained in Petri dishes on modified Melin–Norkrans(MMN) agar medium at 25°C (Marx 1991). The fungal
inoculum was prepared according to Duponnois andGarbaye (1991).
The AM fungus Glomus intraradices (Schenk and Smith,DAOM 181602, Ottawa Agricultural Herbarium) wasmultiplied on leek (Allium porrum L.) on Terragreen (OilDri UK) in glasshouse conditions. The culture substrate wasan attapulgite (calcined clay; average particle size, 5 mm)from Georgia used for the propagation of AM fungi(Plenchette et al. 1996). After 12 weeks of culturing, theleek plants were uprooted and gently washed, and the rootswere cut into 0.5-cm pieces bearing around 250 vesicles percentimeter. Non-mycorrhizal leek roots prepared as abovewere used for the control treatment without AM inoculation.
The seeds of the U. bojeri were surface sterilized asdescribed above. The germinated seeds were individuallygrown in 1-l polythene bags filled with sterilized sandy soil(140°C, 40 min) in which G. intraradices and/or Scleroder-ma Sc1 were already inoculated. A control treatment withoutfungi was included. After autoclaving, the soil chemicalcharacteristics were as follows: pH 5.01 (H2O); total carbon,9.3%; total nitrogen, 0.06%; total phosphorus, 120.6 mgkg−1. For ectomycorrhizal inoculation, the soil was mixedwith fungal inoculum (10/1; v/v). The treatments withoutfungus received an autoclaved mixture of moistened (MMNmedium) vermiculite/peat moss at the same rate. Forendomycorrhizal inoculation, one hole (1×5 cm) was madein each pot and filled with 1-g fresh leek root (mycorrhizalfor the experimental treatment or non-mycorrhizal for thecontrol treatment without fungus). The holes were thencovered with the same autoclaved soil. They were wateredregularly with tap water without fertilizer. The pots werearranged in a randomized complete block design with 25replicates per treatment. The seedlings were screened fromthe rain and grown under natural light (daylight ofapproximately 12 h, average daily temperature of 25°C).
After 5 months of culture, the Uapaca plants wereuprooted, and the oven dry weight (1 week at 65°C) of theshoot was measured. The root systems were gently washed,cut into 1-cm root pieces, mixed, and the percentage ofectomycorrhizal short roots (number of ectomycorrhizalshort roots/total number of short roots) was determined ona random sample of at least 100 short roots under astereomicroscope (magnification ×40). Then these rootpieces were cleared and stained according to the method ofPhillips and Hayman (1970). The root pieces were placed ona slide for microscopic observation at 250× magnification(Brundrett et al. 1985). About 100 1-cm root pieces wereobserved per plant. The extent of mycorrhizal colonizationwas expressed in terms of the fraction of root length with theinternal fungal structures (vesicles and arbuscules). Therelative mycorrhizal dependency was determined by express-ing the difference between the shoot dry weight of themycorrhizal plant and the shoot dry weight of the non-
Table 1 Main-chemical characteristics of the upper soil layer (0–20 cm)
Site Site A Site B Site C
pH (H2O) 4.96 5.37 4.54pH (KCl) 4.75 5.23 4.45Total C (%) 1.12 3.09 1.33Total N (%) 0.07 0.15 0.91Total organic matter (%) 1.92 5.31 2.28C/N 16.0 21.0 14.6Total P (mg kg−1) 15.2 15.2 17.3Available P (mg g−1, Olsen et al. 1954) 3.42 7.01 5.24
Mycorrhiza (2007) 17:195–208 197
mycorrhizal plant as a percentage of the shoot dry weight ofthe mycorrhizal plant (Plenchette et al. 1983).
Influence of ectomycorrhizal inoculation on soil microbialcatabolic diversity
The Uapaca seedlings were grown in 1-l pots filled withnatural soil collected in site A. One part of the soil wasautoclaved (140°C, 40 min) and the other part was notdisinfected before use. After autoclaving, its chemicalcharacteristics were as follows: pH 5.2 (H2O); total C,1.01%; total N, 0.08%; organic matter, 1.55%; C/N, 13.2;total P, 11.9 mg kg−1. The native chemical characteristics ofthis soil are indicated in Table 1. The ectomycorrhizalinoculation with the Scleroderma isolate Sc1 was per-formed as described above, and the same treatment wasperformed for the control treatment. They were wateredregularly with tap water without fertilizer. The pots werearranged in a randomized complete block design with tenreplicates per treatment. The seedlings were screened fromthe rain and grown under natural light (daylight ofapproximately 12 h, average daily temperature of 25°C).
After 5 months of culture, Uapaca plants were uprooted,the shoot biomass and the ectomycorrhizal colonizationwere measured as described before. Most of the soil from 3randomly chosen pots in each treatment was carefullymixed and kept at 4°C for further analysis.
The microbial catabolic diversity was measured by addinga range of simple organic compounds to the soil anddetermining the short-term respiration responses (Degensand Harris 1997; Degens et al. 2001). Each of the 31substrates suspended in 2-ml sterile distilled water wasadded to 1 g of moist soil in 10-ml bottles (West andSparling 1986). The CO2 production from the basalrespiratory activity in the soil samples was measured byadding 2-ml sterile distilled water to 1 g of the equivalentdry weight of soil. After the addition of the substratesolutions to the soil samples, the bottles were immediatelysealed with a vacutainer stopper and incubated at 28°C for4 h in darkness. After 4 h, respired CO2 in the headspace ofeach bottle was determined by taking a 1-ml syringe sampleand analysing the CO2 concentration using an infrared gasanalyser (Polytron IR CO2, Dräger™) in combination with athermal flow meter (Heinemeyer et al. 1989). The resultswere expressed as μg CO2 g−1 soil h−1. There were 10amino acids (L-glutamine, L-serine, L-arginine, L-asparagine,L-cystein, L-histidine, L-lysine, L-glutamic acid, L-phenylala-nine, L-tyrosine), 3 carbohydrates (D-glucose, D-mannose,sucrose), 2 amides (D-glucosamine and succinamide), and 16carboxylic acids (ascorbic acid, citric acid, fumaric acid, glu-conic acid, quinic acid, malonic acid, α-ketoglutaric acid,α-ketobutyric acid, succinic acid, tartaric acid, uric acid,oxalic acid, malic acid, hydroxybutyric acid). The amines
and amino acids were added at 10 mM, whereas thecarbohydrates were added at 75 mM and the carboxylicacids at 100 mM (Degens and Vojvodic-Vukovic 1999).The catabolic richness and catabolic evenness werecalculated to evaluate the catabolic diversity of both soiltreatments. The catabolic richness, R, expressed thenumber of substrates used by the microorganisms in eachsoil treatment. The catabolic evenness, E, representing thevariability of used substrates amongst the range of thesubstrates tested was calculated using the Simpson–Yuleindex E ¼ 1
�p2i with pi=respiration as the response to
individual substrates/total respiration activity induced byall substrates for a soil treatment (Magurran 1988).
Statistical analysis
The data were treated with one-way analysis of variance. Themeans were compared using the Newman and Keuls test (p<0.05). The percentages of the mycorrhizal colonization weretransformed by arcsin(sqrt) before the statistical analysis.
The between-group analysis (BGA, Dolédec and Chessel1987; Culhane et al. 2002) was used to analyse the surfaceinsulation resistance (SIR) responses in soil samplesinoculated with Scleroderma Sc1 and samples withoutinoculation. BGA is a multivariate analysis techniquederived from principal components analysis (PCA). Theaim of PCA is to summarize a data table by searchingorthogonal axes on which the projection of the samplingpoints (rows of the table) has the highest possible variance.
From a theoretical point of view, BGA is the particularcase of PCAwith respect to instrumental variables (principalcomponent analysis with instrumental variables, Rao 1964;Lebreton et al. 1991) where the instrumental variables tableis reduced to just one qualitative variable. This variabledefines groups of rows in the data table, and BGA consistsof the PCA of the table of the means by groups. This tablehas a number of rows equal to the number of groups, andthe same number of columns as the original table. The aimof this analysis is to separate the groups. This is also theaim of discriminant analysis (also called canonical variatesanalysis), but whilst discriminant analysis is limited totables that have a high number of samples compared to thenumber of variables, BGA can be used even when thenumber of rows is less than the number of variables. BGAcan, thus, be considered as a robust alternative todiscriminant analysis when the number of samples is low.
A Monte Carlo test (permutation test) can be used to checkthe significance of the differences between groups. Thismethod consists, in performing many times, a randompermutation of the rows of the table (but not of the qualitativevariable defining the groups) followed by the recomputationof the between-class inertia. By comparing the between-classinertia obtained in the normal analysis with the between-class
198 Mycorrhiza (2007) 17:195–208
inertia obtained after randomization, we get an estimation ofthe probability of meeting a situation similar to the observedsituation without differences between groups (i.e. a signifi-cance test of the differences between groups).
The computations and graphical displays were made withthe free ADE-4 software (Thioulouse et al. 1997) available inthe Internet at http://www.pbil.univ-lyon1.fr/ADE-4/.
Results
Sporophore survey
We collected 94 sporophores in three survey sites (S 1).They were identified as belonging to the ectomycorrhizal
genera Afroboletus, Amanita, Boletus, Cantharellus, Lecci-num, Gyroporus, Rubinoboletus, Russula, Scleroderma,Suillus, Tricholoma, and Xerocomus (S 1). The highestfungal diversity of the above-ground sporophores wasrecorded in site A (40 species), whereas only 27 and 29fungal species were detected in sites B and C, respectively(S 1). Russula was the most frequent ectomycorrhizalgenus recorded under U. bojeri (32.9% of the above-ground sporophore diversity) followed by the generaAmanita (17.1%) and Cantharellus (Fig. 1a). Twenty-onedifferent species were recorded for Russula followed byAmanita (14 species) and the genera Cantharellus andBoletus (10 species; Fig. 1b). AM structures (vesicles andhyphae) were detected from the roots in all surveyed sites.
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Fig. 1 a Structure of theectomycorrhizal community(above-ground diversity)expressed as genus relativefrequency (b). Number ofspecies per genus
Time sequence of mycorrhizal colonization on U. bojeri
First, ECMs were recorded after 2 months (Fig. 2). Nativeectomycorrhizal fungi colonized approximately 50% of thelateral roots sampled after 5 months of culture (Fig. 2). AMstructures were also observed after 2 months of culturing(Fig. 2).
Mycorrhizal dependency of U. bojeri seedlings
The shoot dry weight of the plants inoculated with G.intraradices or Scleroderma sp. Sc1 was significantlyhigher than in the control treatment (Tables 2 and 3).Compared to the control treatment, the shoot growth ofectomycorrhized plants was stimulated 1.9 times, whereas itwas 1.7 times for plants inoculated with G. intraradices(Table 2). When both fungal symbionts were co-inoculated,the shoot dry weight significantly increased over the singleinoculation treatments (Table 2). The shoot dry weightincreased 2.1 times compared to the mean shoot dry weightof the single fungus treatments (G. intraradices alone orScleroderma sp. Sc1 alone). The dual fungal inoculationdid not significantly modify the establishment of ectomy-corrhizal and AM symbioses compared to the ectomycor-rhizal or AM colonization rates measured in the singleinoculation treatments (Table 2).
Influence of ectomycorrhizal inoculation on soil microbialcatabolic diversity
The growth of U. bojeri seedlings was significantly higherin the native soil than in the autoclaved soil (Table 3).Ectomycorrhizal fungal inoculation significantly increasedshoot biomass of U. bojeri seedlings. There were nosignificant interactions between the autoclaving and theinoculum treatments (Table 3).
Catabolic richness did not differ between the treatments(Table 3). However, catabolic evenness was significantlyinfluenced by the soil treatments (autoclaved or not) and bythe fungal inoculation (Table 4).
The BGA of the SIR responses for the four soiltreatments are presented in Fig. 3. The map of the soilsamples (Fig. 3b) shows that the four treatments (NDNI,NDI, DNI, and DI) were clearly separated. This resultindicates that the microbial communities were different (incomposition or at least in activity), according to the soiltreatment. The map of the substrates (Fig. 3a) shows that,on the first axis, the use of four organic acids was highest innon-autoclaved soil samples and in inoculated samples (leftpart of the figure: ketobutyric, ketoglutaric, oxalic, andcitric acids). The Monte Carlo test is significant (p=0.025).The soil autoclaving involved a lower rate of use of thesefour organic acids, whereas fungal inoculation led to ahigher rate. Moreover, the effect of inoculation seemedstronger in non-disinfected soil samples.
Discussion
The main results of this study show that (1) a largediversity of sporophores was recorded under U. bojeri, (2)U. bojeri formed AMs and ECMs in natural soils, (3) thistree species is highly dependent on both types of mycor-rhiza, and (4) controlled ectomycorrhization of U. bojeristrongly influences soil microbial catabolic diversity.
Our investigations show that forests dominated by U.bojeri contain a wide range of sporophores belonging to atleast four different fungal families: Russulaceae, Canthar-ellaceae, Boletaceae, and Amanitaceae. In tropical forests,these families of putative ectomycorrhizal fungi have been
0
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n (
% o
f sh
ort
ro
ots
) (%
)
Time (months)
Fig. 2 Sequence of mycorrhizal colonization on U. bojeri seedlings inexperiment 1 (square, AM colonization; diamond, total ectomycor-rhizal colonization)
Table 2 Shoot growth, mycorrhizal development, and relativemycorrhizal dependency of U. bojeri seedlings 5 months after G.intraradices and/or Scleroderma sp. Sc1 inoculation in pot culture
Treatments Shootbiomass (mgper plant)
Ectomycorrhizalcolonization(%)
AMcolonization(%)
RMD(%)a
Control 91.1ab 0a 0a –Sclerodermasp. Sc1
181.2b 8.7b 0a 47.6a
G. intraradices 160.1b 0a 77.5b 42.7aSclerodermasp. Sc1 + G.intraradices
360.3c 11.5b 82.5b 70.7b
aRMD Relative mycorrhizal dependencyb Data in the same column followed by the same letter are notsignificantly different according to the one-way analysis of variance(p<0.05).
200 Mycorrhiza (2007) 17:195–208
described under Afzelia africana, Monotes kerstingii,Uapaca guineensis, and U. somon in Africa (Thoen andBâ 1989; Sanon et al. 1997) and in Asia under dipterocarps(Lee 1998). It is also well known that Russulaceae are oftendominant in tropical rainforests of Africa, Asia, andMadagascar (Buyck et al. 1996; Lee et al. 1997; Watlingand Lee 1998; Riviere et al. 2006). The identification ofthis group in the tropics remains problematic as manyspecies are new and undescribed. A high diversity ofectomycorrhizal fungi was associated with U. bojeri. Withother tropical ectomycorrhizal tree species, Lee et al.(1997) recorded only 28 fungal species under Shorealeprosula, and Sanon et al. (1997) had identified 14 fungalspecies under U. guineensis and 11 species under U. somonin Burkina Faso. However, numerous studies in temperateareas indicate little correlation between above-ground(sporophores) and below-ground (ECMs) fungal diversity(Buscot et al. 2000; Horton and Bruns 2001). Furthermolecular-based studies are needed to determine the fungaldiversity of ECMs associated with U. bojeri in naturalconditions.
Most mycorrhizal species are generally associated withonly one type of mycorrhiza, usually either AMs or ECMs(Moyersoen and Fitter 1999). It has also been reported thatsome plant species formed both AM and ECM (Molina et al.1992). The dual symbiotic association is well documentedfor Populus (Lodge and Wentworth 1990), Salix (Dhillion1994), Eucalyptus (Lapeyrie and Chilvers 1985), Alnus(Molina et al. 1994), Acacia (Founoune et al. 2002a,b),Pinaceae (Cazares and Trappe 1993), Quercus (Egerton-Warburton and Allen 2001), and Casuarinaceae (Duponnoiset al. 2003), but it was unknown for U. bojeri, although itwas usually stated that this tree species was only colonizedby ectomycorrhizal fungi (Moyersoen and Fitter 1999). But
it has also been reported that roots of U. guineensis seedlingsgrowing in a forest soil were only colonized by AM fungi(Moyersoen and Fitter 1999). The results of the presentstudy confirmed the high occupancy of AM fungi recordedon young seedlings (3-month-old root systems) and that AMstructures appeared for the first time on the plant culturefollowed by ECM colonization (Chilvers et al. 1987).
A synergistic effect of dual AM/ECM inoculation wasdescribed for Acacia holosericea inoculated with G.fasciculatum and Pisolithus albus (Founoune et al. 2002a,b), but the involved mechanisms remained unknown. Incontrast, in 1-year-old field seedlings of Quercus agrifoliawith a high glomalean and ectomycorrhizal fungal load,coexistent mycorrhizal types constituted a cost during theestablishment of young oaks and potentially limited theirdevelopment (Egerton-Warburton and Allen 2001). Theseauthors suggested that the progressive shift to predomi-nantly ectomycorrhizal colonization with increasing plantage become beneficial over time as it has been recordedwith U. bojeri after AM/ECM inoculation in the presentstudy.
Pirozynski and Malloch (1975) hypothesised that theAM habitat was a prerequisite for the early development ofland flora. Soil nutrient distribution in natural environmentsis typically heterogenous (Farley and Fitter 1999), andmycorrhizas may allow plants growing in low nutrientpatches to access resources in adjacent rich nutrient patches(Casper and Cahill 1998). In addition, ectomycorrhizalfungi are not uniformly distributed in terms of theirpresence, abundance, or community composition (Dickieand Reich 2005), and a lack of ectomycorrhizal fungi mayslow the invasion of disturbed sites by ectomycorrhizalplants. Young seedlings of U. bojeri that form AM couldsurvive in sites with low availability of ectomycorrhizal
Table 3 Shoot growth, mycorrhizal development, and relative mycorrhizal dependency of U. bojeri seedlings 5 months after Scleroderma sp. Sc1inoculation in disinfected or nondisinfected soil
aRMD Relative mycorrhizal dependencyb Catabolic richnessc Catabolic evennessd Data in the same column followed by the same letter are not significantly different according to the one-way analysis of variance (p<0.05).e Significant (p<0.05)f Nonsignificant (p<0.05)
Mycorrhiza (2007) 17:195–208 201
Tab
le4
Descriptio
nof
putativ
eectomycorrhizal
fung
icollected
from
thethreestud
iedsitesbeneathU.bo
jeri
Species
Prominentfeatures
Habitat
Sites
Site
ASite
BSite
C
Amanitaceae
Aman
itarubescensGray
White
pink
ishcap(8-cm
diam
eter)coveredwith
white
powderedandflat
scales,remnant
veil
visibleat
themargin,
white
stem
redd
eningby
wou
nd,ofteneatenby
insect
larvae
Solitary,scarce
x
Aman
itavirosa
(Fr.)
Bertillon
White
yello
wishfruitin
gbo
dy(7-to
12-cm
diam
eter),white
andchinated
stem
(1.2-cm
diam
eter)with
ring
andcupat
thebase
Patch
of5to
6individu
als
xx
x
Aman
itaph
alloides
var.vernaBullWhite
fruitin
gbody
(5.5-to
11-cm
diam
eter),stem
(0.6
diam
eter
by9.5cm
high)with
alarge
pend
antring
andabu
lbou
scupat
thebase
Patch
of5to
7individu
als
xx
X
Aman
itastrobiliformisBertillon
White
andbigfruitin
gbo
dy(10to
12cm
diam
eter),fleecy
remnant
veilon
thecap,
club
-shapedstem
(2.2-cm
diam
eter)with
aring
Solitary,scarce
x
Aman
itacf.Baccata
(Fr.)
Gillet
Big
white
fruitin
gbo
dysimilarfeatures
than
previous
speciesbu
twith
noring
,stem
(2-cm
diam
eter
by7cm
high
)Solitary,scarce
x
Aman
itasp1
White
finely
scaled
fruitin
gbo
dy(4-to
6-cm
diam
eter)turningyello
wishwhenageing
orby
wou
nd,
concolou
redgills
andflesh
Solitary,scarce
x
Aman
itacf.Strobiloceovolvata
Beeli
White
fruitin
gbo
dy(8.5-to
11-cm
diam
eter),stem
(1.2-cm
diam
eter
by10
.5cm
high
)with
outring
,well-developedbu
lbou
scupat
thebase
Patch
of3to
4individu
als
xx
x
Aman
itasp2
White
andbigspecieswith
aconv
exscalycap(10-
to13
-cm
diam
eter
by9to
10cm
high
),strong
bulbou
sstem
(3-to
4-cm
diam
eter)with
apend
antring
Solitary,scarce
x
Aman
itasp3
Palegrey
cap(4.5-cm
diam
eter)with
few
veilremanenceson
surface,
bulbou
sstem
(0.7
to6cm
)with
grey
chinates
Solitary,scarce
x
Aman
itasp4
Yellow
conicalandmucronatedcap(2.5-to
3-cm
diam
eter),palerto
whitishgills
andstem
(0.5-cm
diam
eter
by12
cmhigh
),white
scalybasalcup
Solitary,scarce
x
Aman
itacf.cecilia
(Berk.
etBroom
e)Bas
Yellow
grey
cap(4-to
5-cm
diam
eter)with
risedscales,white
gills
andconcolou
redstem
(0.7-cm
diam
eter
to6cm
high
),bu
lbou
sbase
coveredby
grey
chinates
andveilremanences
Solitary,scarce
x
Aman
itasp5
Con
vexandgrey
purplish-blue
cap(4
to4.5cm
diam
eter)with
grey
flat
scales
atthecentre
andhairy
ones
atthemargin,
white
fleshandgills,white
bulbou
sstem
(0.9-cm
diam
eter
by6cm
high
)turning
togrey
bytouchwith
apend
antring
Solitary,scarce
x
Aman
itasp6
Smallwhite
species(2-to
3-cm
diam
eter)with
yello
wishscales,bu
lbou
sbasedstem
with
pend
ant
ring
Solitary,scarce
x
Aman
itasp7
Big
white
flat
capspecies(9-to
13-cm
diam
eter)with
veilremanencesat
themargin,
strong
bulbou
sstem
(3-to
4-cm
diam
eter)with
aring
Patch
of2to
3individu
als
xx
x
Boletaceae
Rub
inob
oletus
griseus
Big
red-pink
andgrey-brownish
dryandsm
ooth
cap(10-
to12
-cm
diam
eter
by8to
9cm
high
),white
flesh(1.8
cmthick)
partially
burnishing
aftersectioning
,pale
reticulated
hairyscaled
stem
,bu
rnishing
likepo
resby
touch
Patch
of5to
6individu
als
xx
x
Gyrop
orus
cf.cyan
escens
(Bulliard
Fr.)
Quélet
Big
white
yello
wishsm
ooth
cap(10-
to12
-diameter
by8–
9cm
high
),concolou
redtubesandstem
turningto
blue
bywou
ndPatch
of3to
4individu
als
xx
x
Boletus
sp1
Brownish
tobrow
ncap,
with
largedarker
flat
scales,cylin
drical
anddark
stem
,redreticulated,
becomingyello
wat
thebase
likerhizom
orph
,fleshandpo
resturningblue
bywou
ndPatch
of3to
4individu
als
x
202 Mycorrhiza (2007) 17:195–208
Leccinu
msp1
Smallgrey
boletus(1.8-to
3-cm
diam
eter
by3to
4cm
high
),yello
wpo
res,redhairyscales
onthe
stem
,base
ofthestem
yello
wlik
etherhizom
ophs
Patch
of3to
4individu
als
xx
x
Boletus
sp2
Big
brow
nish-brownwet
cap(7-to
8-cm
diam
eter
to12
to15
cmhigh
),white
andsm
ooth
flesh
Patch
of5to
6individu
als
xXerocom
ussp1
Brownscalycap(8.5-cm
diam
eter)show
ingwhite
fleshbetweenscales,white
stem
(1.4-cm
diam
eter
by5to
6cm
high
)with
someredzone
Solitary,scarce
x
Leccinu
msp2
Yellow
grey
scaled
boletus(4.5-to
6-cm
diam
eter
by6to
7cm
high
),stem
yello
wat
thebase
and
redin
itsup
perpart,yello
wblueishing
pores
Solitary,scarce
x
Boletus
sp3
Browncap(7.5-cm
diam
eter)with
red-pink
pigm
ents,yello
wandredpo
res,greenishingand
blueishing
tubes,yello
wishstem
with
someredpigm
ents
Solitary,scarce
x
Leccinu
msp3
Red
purplish-blue
wet
cap(7-cm
diam
eter),yello
wbu
rnishing
stem
(0.8-cm
diam
eter
by6cm
high
),concolou
redyello
wfleshandpo
res,blueishing
afterairexpo
sure
Solitary,scarce
x
Boletus
sp4
Big
smoo
thandshinyredbo
letus(8-to
12-cm
diam
eter
by7to
8cm
high
),yello
wishstem
with
somepink
pigm
ents,concolou
redflesh(1.6
cmthick)
Patch
of2to
3individu
als
xx
x
Xerocom
ussp2
Paleto
dark
brow
nscalydrycap(5-cm
diam
eter),white
dirtystem
(0.8-cm
diam
eter
by4cm
high
)with
awhite-yellowishflesh,
yello
wgreenish
andpink
pores
Solitary,scarce
x
Boletus
sp5
Yellowishbrow
ncap(8-cm
diam
eter)with
flat
partially
pink
scales,yello
wpo
resandstem
(1.2-cm
diam
eter
by6cm
high
),white
flesh(1.6
cmthick)
Solitary,scarce
x
Boletus
sp6
Darkbrow
nscalycapshow
ingyello
wflesh,
pale
concolou
redpo
resandstem
Solitary,scarce
xBoletus
sp7
Brownbo
letuswith
dryandsilkycap(4.5-cm
diam
eter),concolou
reddark
stem
(2.2-cm
diam
eter
by5.2cm
high
),white
flesh(1.6
cmthick)
rapidlyturningto
red,
then
blackafterairexpo
sure
Solitary,scarce
x
Boletus
sp8
Palebrow
nbo
letuswith
silkycap(5-cm
diam
eter),white
stem
(1.5-cm
diam
eter
by5.2cm
high
)andflesh(1.3
cmthick)
turningpu
rplish-blue
afterairexpo
sure
Solitary,scarce
x
Leccinu
msp4
Yellow
andwet
cap(3.5
cm)with
hairygrey
scales,yello
wpo
res,yello
wandredstem
(0.5-cm
diam
eter)with
dark
scales
andanarrow
base
Solitary,scarce
x
Leccinu
msp5
Yellowish-brow
ndrycap(3.5-cm
diam
eter),redpo
resandlig
hter
stem
(0.6-cm
diam
eter
by4cm
high
)turningto
dark-brownish
insection,
white
fleshturningbu
rnishafterairexpo
sure
Solitary,scarce
x
Suillus
sp2
Yellow
andgrey
scalycap(5-cm
diam
eter),yello
wpo
rescoveredby
ayello
wpartialveilwhen
youn
g,yello
wstem
(1.4-cm
diam
eter
by4.5cm
high
)with
greenish
grey
scales,becomingvery
slim
y
Patch
of2to
3individu
als
x
Boletus
sp9
Yellow
brow
nish
boletus(7-to
8-cm
diam
eter)with
asticky
surface,
yello
wpo
resandstem
,yello
wishflesh(1.7
cmthick)
Solitary,scarce
x
Leccinu
msp6
Palebrow
ncap(5-to
4-cm
diam
eter)with
redbrow
nish
scales
atthecentre,white
poresandwhite
fleshturningrapidlyto
red,
then
blackby
wou
ndSolitary,scarce
x
Boletus
sp10
Yellow
brow
nbo
letus(4.5-to
5.7-cm
diam
eter)with
wet
andsm
ooth
surface,
yello
wpo
res,yello
wstem
(1.2
diam
eter
by3cm
high
),white
flesh(1
cmthick)
Solitary,scarce
x
Cantharellaceae
Can
tharellussp1
Tallthickandlobedfasciculatebright
yello
wcaps
(4-to
6-cm
diam
eter)form
ingpatchesof
4to
5individu
als(12cm
),grainedgills,pale
yello
wstem
(1.8
cm),white
flesh
Patch
of8to
10individu
als
xx
x
Can
tharellussp2
Smallorange-brownish
cap(2-to
2.2-cm
diam
eter),white
pink
ishgills,pink
stem
andwhite
flesh
Solitary,scarce
xCan
tharellussp3
Yellowishto
pale
brow
ncap(3.5-to
3.2-cm
diam
eter),yello
wgrainedgills,pale
yello
wstem
(0.6
to2.5cm
)Solitary,scarce
xx
x
Mycorrhiza (2007) 17:195–208 203
Tab
le4
(con
tinued)
Species
Prominentfeatures
Habitat
Sites
Site
ASite
BSite
C
Can
tharellussp4
Red
orange
cap(3.2-to
3.5-cm
diam
eter),largelyspaced
yello
wishgrainedgills,pale
yello
wto
redd
ishstem
(0.9
cm)
Patch
of8to
10individu
als
xx
x
Can
tharellussp5
Palebrow
ncap(3.2-to
3.5-cm
diam
eter),pale
pink
grainedgills,white
stem
andflesh,
turningto
yello
wby
touchor
sectioning
Solitary,scarce
xx
x
Can
tharellussp6
Red
pink
ishfasciculatecaps
(2.5-cm
diam
eter)form
ingsm
allpatch(3.5
to4cm
),yello
wishgrained
gills,pink
orange
stem
andwhite
fibrou
sflesh
Patchy
x
Can
tharellussp7
Smallandfragile
bright
yello
wcap(2-to
3.2-cm
diam
eter),pale
yello
wgills,concolou
redshort
stem
(0.3
cm)
Solitary,scarce
x
Can
tharelluscf
decolorans
Eyss.
etBuy
ckSmallpink
orange
cap(0.7-to
1.5-cm
diam
eter,2.5to
3.5cm
high
),concolou
redgills
andshort
stem
(0.2
cm)
Patch
of5to
6individu
als
x
Can
tharelluscf.Cyano
xanthu
sR.Heim
Yellow
andpu
rple
cap(4-cm
diam
eter),pale
pink
grainedgills,pale
yello
wstem
(1.8
cm),fibrou
sflesh
Patch
of2to
3individu
als
x
Can
tharellusrubb
erR.Heim
Palepink
cap(3.5-to
4-cm
diam
eter),concolou
redstem
andgills
Patch
of2to
3individu
als
xRussulaceae
Russula
subfistulosa
Buy
ckWhite-greyish
(darkerat
thecentre)um
bilicated
cap(3-to
12-cm
diam
eter)
Solitary
topatchof
3individu
als
xx
x
Russula
ochraceorivulosa
Greyish
topu
rplish-blue
grey
cap(7-to
8-cm
diam
eter),conv
excapwith
anun
dulatin
gmargin
Solitary
xx
xRussula
patouiillardi
Paleyello
wandpu
rple
(darkerat
thecentre)dryscalycap,
white
andpu
rple
stem
Solitary
topatchof
5individu
als
xx
x
Russula
liberiensisBuy
ckWhite-greyish
fibrillosecap(3-to
12-cm
diam
eter)turningbrow
nwhenageing
,closelyspaced
decurrentgills
Solitary
topatchof
3individu
als
xx
x
Russula
cf.Cyano
xantha
Pinkto
purple-red
cap(5-to
15-cm
diam
eter),white
stem
Patch
of2to
3individu
als
xRussula
cellu
lata
Buy
ckBrownscalycap(3-to
9-cm
diam
eter),closelyspaced
decurrentgills
Patch
of2to
3individu
als
xRussula
cf.archae
R.Heim
White
smoo
thandflat
cap(4.5-to
6-cm
diam
eter)
Solitary
xRussula
cf.nigrican
sWhite-greyish
capturningto
brow
nwhenageing
,white
fleshturn
rapidlypink
toredby
air
expo
sure
Solitary
x
Russula
cf.subfistulosa
White-greyish
conv
excap(3-to
8-cm
diam
eter)
Solitary
topatchof
4individu
als
x
Russula
sp3
White
topale
yello
wglueyandconv
excap(3-to
13-cm
diam
eter)
Solitary
topatchof
3individu
als
x
Russula
sp5
Yellow
smoo
thum
bilicated
cap(6-to
12-cm
diam
eter),with
avery
regu
larmargin
Patch
of2to
3individu
als
xx
xRussula
sp6
White-yellowishflat
orslightly
umbilicated
cap(4-to
10-cm
diam
eter),white
fleshturningredd
ish
afterairexpo
sure
Patch
of3to
5individu
als
x
Russula
sp7
Darkgrey
tobrow
nconv
excap(3.5-to
8-cm
diam
eter),invo
lucrated
margin,
wet
surfacecovered
byorange
toyello
wlayers,white-yellowishflesh
Solitary,rarely
patchy
x
Russula
sp8
White
conv
exto
slightly
umbilicatecap(4-to
13-cm
diam
eter)turningbrow
nwhenageing
,sm
ooth
surfacewith
invo
lucrated
margin,
white
fleshturningredd
ishafterairexpo
sure
Solitary,scarce
x
204 Mycorrhiza (2007) 17:195–208
Russula
sp10
Darkgrey
tobrow
nwhenfully
matureconv
exto
flat
cap(4-to
9-cm
diam
eter),white
flesh
Patch
of2to
4individu
als
xRussula
sp11
Smallpu
rple
topu
rple-reddish
umbilicatewhenyo
ungto
flat
whenageing
cap(2-to
7-cm
diam
eter),sticky
surface,
regu
larmargin,
adnate
white
toyello
wishcloselyspaced
gills,white
flesh
Solitary
topatchof
3individu
als
x
Russula
sp13
Brown-redd
ishconv
exandsm
ooth
glutinou
scap(6-to
15-cm
diam
eter),decurrentgills,white
flesh
turninggreyishby
airexpo
sure
Solitary
x
Russula
sp14
Darkyello
wto
brow
nconv
exto
flat
sticky
cap(4-to
10-cm
diam
eter),adnate
closelyspaced
gills
Patch
of3to
5individu
als
xRussula
sp15
Yellow
toorange-yellow
flat
slightly
umbilicated
with
aninvo
lucrated
yello
wmargincap(2-to
8-cm
diam
eter)with
asm
ooth
surfacewith
smallstrias
Patch
of2to
5individu
als
x
Russula
sp16
Pinkto
redd
ish(darkerat
thecentre)fragile
conv
exglutinou
scap,
(2-to
6-cm
diam
eter)with
asm
ooth
ordu
stysurface,
white
flesh
Patch
of2to
4individu
als
x
Russula
sp17
Slig
htly
umbilicated
conv
exandglutinou
scap(4-to
10-cm
diam
eter),dark
yello
wtend
ingto
brow
n,yello
wto
pale
orange
closelyspaced
gills
Solitary
topatchof
4individu
als
x
Strob
ilomycetacea
Afrob
oletus
sp1
Brown-pu
rple
scalycap(3-to12
-cm
diam
eter),fibrou
sstem
,pale
yello
wfleshturningpu
rplishby
airexpo
sure
Patch
of3to
5individu
als
x
Afrob
oletus
sp2
Flat-conv
exdu
stycap(3
to10
cmdiam
eter)with
dark-brownto
blackscales,fibrou
sstem
inflated
atthebase,greyish-yello
wflesh
Patch
of2to
3individu
als
x
Sclerod
ermataceae
Scleroderm
asp1
Whitishto
yello
wishsm
allpy
riform
icfruitbo
dies,size
below
3cm
indiam
eter,dark
grey
gleba
Solitary
topatchof
5individu
als
x
Scleroderm
asp2
Whitishto
yello
wish3-
to7-cm
diam
eter
fruitbo
dies
with
grey
spotsat
thetop,
dark
grey
gleba
Solitary,rarely
patchy
xTricho
lomasp2
Yellow
cap(3-to
9-cm
diam
eter),drysurface,
invo
lucrated
margin,
thickwidelyspaced
gills,
yello
wfleshkeepingyello
weven
afterexpo
sure
toair
Solitary
topatchof
4individu
als
x
Tricho
lomasp3
Yellow-greyish
cap(3-to
12-cm
diam
eter),drysurface,
white
yello
wishstalk,
white
flesh
Solitary
topatchof
4individu
als
x
Tricho
lomasp4
Dark-grey
cap(3-to
15-cm
diam
eter),sm
ooth
drysurface,
thickgills,white
flesh
Solitary
x
Mycorrhiza (2007) 17:195–208 205
fungi and develop ectomycorrhizas later as roots contactresidual ECM communities. This mycorrhiza successionalprocess would promote the development of subsequent
ectomycorrhizal fungus communities and facilitate theestablishment or re-establishment of the seedlings ofectomycorrhizal tree species after the disturbance (Perry etal. 1989), thus, influencing plant succession from prairie orold field to savanna or woodland.
Scleroderma species are considered “early-stage” sym-bionts (Deacon et al. 1983; Bâ et al. 1991) and can formmycorrhizas with a wide range of tropical tree species suchas Afzelia africana (Bâ and Thoen 1990), A. quanzensis,Isoberlinia doka, I. dalziellii, and Brachystegia speciformis(Sanon et al. 1997). In the present study, Sclerodermaisolate Sc1 increased Uapaca growth in disinfected and innon-disinfected soil, suggesting that this fungal strain washighly competitive against the native ectomycorrhizalmycota at least under the conditions of this pot-basedexperiment. In addition, ectomycorrhizal inoculation in-duced strong modification of the soil microflora function-alities and increased its catabolic microbial diversity. Elliottand Lynch (1994) hypothesised that microbial communitieswith reduced catabolic evenness are less resistant to stressand disturbance. Microbial functional diversity is involvedin a large range of activities such as nutrient transforma-tion, decomposition, etc. (Wardle et al. 1999). In partic-ular, ectomycorrhizal fungi mobilize P and other essentialplant nutrients directly from minerals through the excre-tion of organic acids (Landeweert et al. 2001). Amongstthe total organic acids in the soil solution, low molecularweight organic acids are considered to be the mostimportant biological weathering agents (Ochs 1996).Oxalate, citrate, and malate produced by plant roots andsoil microorganisms are the strongest chelators of trivalentmetals (Landeweert et al. 2001). Oxalic acid, commonlyproduced by many different fungal species, has the highestacid strength (Dutton and Evans 1996). In the presentstudy, SIR responses with all oxalic and citric acidsincreased in the fungal inoculated soil, suggesting thatScleroderma Sc1 and its associated microflora excretedhigher amounts of such organic acids and induced amultiplication of microorganisms that utilize these avail-able organic resources than noninoculated soil.
In conclusion, this study showed that U. bojeri has acomplex symbiotic status that can be managed to optimizeits development in degraded areas. In addition, the use ofselected mycorrhizal fungi such the Scleroderma Sc1isolate in nursery conditions could be of great interest, as(1) this fungal strain appears competitive against nativesymbiotic microflora and (2) the fungal inoculationimproves the catabolic potentialities of the soil microflora.However, further studies are needed to describe the below-ground diversity of ectomycorrhizal fungi and to demon-strate the potential interest of controlled mycorrhization innatural conditions in afforestation programs with U. bojeriin Madagascar.
28 14 27 22 15 25 17 20 19
6
29
1
2
3
5
111021 24
16 12
9 26 23
17 13 18
8
7
4
-14
14 -26 2
DIDNI
NDI
NDNI
-90
60 -90 60
a
b
Fig. 3 BGA of the SIR responses with respect to the fungaltreatments and soil treatments (DNI disinfected soil without fungalinoculation, DI disinfected soil with fungal inoculation, NDNInondisinfected soil without fungal inoculation, NDI nondisinfectedsoil with fungal inoculation, NIND: 1 Ketobutyric acid, 2 ketoglutaricacid, 3 oxalic acid, 4 citric acid, 5 phenylalanine, 6 gluconic acid, 7glucose, 8 uric acid, 9 malic acid, 10 asparagine, 11 tartaric acid, 12malonic acid, 13 gallic acid, 14 formic acid, 15 cystein, 16 histidine,17 sucrose, 18 tyrosine, 19 glutamic acid, 20 succinic acid, 21glucosamine, 22 succinamide, 23 mannose, 24 glutamine, 25 quinicacid, 26 lysine, 27 ascorbic acid, 28 serine, 29 arginine, 30 fumaricacid, 31 hyroxybutyric acid
206 Mycorrhiza (2007) 17:195–208
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208 Mycorrhiza (2007) 17:195–208
ORIGINAL PAPER
Restoring native forest ecosystems after exotic treeplantation in Madagascar: combination of the localectotrophic species Leptolena bojeriana and Uapaca bojerimitigates the negative influence of the exotic speciesEucalyptus camaldulensis and Pinus patula
R. Baohanta • J. Thioulouse • H. Ramanankierana • Y. Prin • R. Rasolomampianina •
E. Baudoin • N. Rakotoarimanga • A. Galiana • H. Randriambanona •
M. Lebrun • R. Duponnois
Received: 5 July 2011 / Accepted: 28 April 2012
� Springer Science+Business Media B.V. 2012
Abstract The objectives of this study were to
determine the impact of two exotic tree species (pine
and eucalypts) on the early growth of Uapaca bojeri
(an endemic tree species from Madagascar) via their
influence on soil chemical, microbial characteristics,
on ectomycorrhizal fungal community structures in a
Madagascarian highland forest and to test the ability of
an early-successional ectomycorrhizal shrub, Lepto-
lena bojeriana, to mitigate the impacts of these exotic
species. Finally, we hypothesized that L. bojeriana
could act as a natural provider for ectomycorrhizal
propagules. Soil bioassays were conducted with
U. bojeri seedlings grown in soils collected under
the native tree species (U. bojeri and L. bojeriana) and
two exotic tree species (Eucalyptus camaldulensis and
Pinus patula) and in the same soils but previously
cultured by L. bojeriana seedlings. This study clearly
shows that (1) the introduction of exotic tree species
induces significant changes in soil biotic and abiotic
statistical analysis. A principal component analysis
(PCA) was applied to the soil, plant, and microbial
parameters. The software used was the ade4 package
(Dray and Dufour 2007) for the R software for
statistical computing (R Development Core Team
2010).
Results
Mycorrhizal status of trees and early-successional
plant species in the Arivonimamo forest
All tree and shrub species recorded in the Arivonim-
amo forest formed mycorrhizas. Among these, 8
presented AM infections and 5 were found with both
AM and ECM (Table 1).
Impact of targeted tree species on soil chemical
characteristics, ectomycorrhizal colonization,
and growth of U. bojeri seedlings
The highest soil acidity was recorded with the
E. camaldulensis origin followed by P. patula,
U. bojeri, and the bulk soil (Table 2). For N and P
soil contents, the opposite ranking was found with the
highest values recorded with E. camaldulensis soil
(Table 2). The total organic matter in soil was
significantly higher in U. bojeri and the lowest value
was found in the bare soil whereas P. patula and
E. camaldulensis soils had intermediate TOC contents
(Table 2).
The acid phosphatase and FDA activities were
significantly higher in the soils collected under the
targeted tree species compared to the bulk soil but
these activities were higher in the soils sampled under
exotic tree species than in the U. bojeri origins
(Table 2). With the alkaline phosphatase activity, an
opposite pattern was found with a higher activity in the
U. bojeri soil followed by the P. patula soil and finally
by the bulk and E. camaldulensis soils (Table 2).
After 5 months of culturing, shoot and root bio-
mass, total biomass of U. bojeri seedlings were
significantly lower in the soil collected under
E. camaldulensis than in the other soil origins,
whereas the highest root and total growth were found
in the U. bojeri soil (Table 3). Compared with the
control (bulk soil), no significant effect of P. patula
origin was recorded for the root and total biomass
except for the shoot biomass (Table 3). According to
the soil origins, root/shoot ratios ranged as follows:
U. bojeri [ P. patula [ bulk soil (control) [ E. cam-
aldulensis (Table 3). Nitrogen leaf contents were not
significantly different among soil origins, whereas
phosphorus foliar content of U. bojeri seedlings was
significantly higher in the soil originating from around
U. bojeri compared with P. patula soil (Table 3).
Compared with the bulk soil, the extent of
ectomycorrhizal colonization was significantly higher
in the soil collected under U. bojeri (73.7 %) and
significantly lower in the E. camaldulensis soil
(16.3 %) (Table 3). Structures of ectomycorrhizal
Table 1 Mycorrhizal
status of trees and early-
successional plant species
in the Arivonimamo forest
ECM ectomycorrhizas, AMarbuscular mycorrhizas,
ECM & AM co-existence of
arbuscular mycorrhizas and
ectomycorrhizas
Shrub and tree species Family Mycorrhizal status
Leptolaena pauciflora Baker. Sarcolaenaceae ECM & AM
Leptolaena bojeriana (Baill.) Cavaco. Sarcolaenaceae ECM & AM
Trema sp. Cannabaceae AM
Vaccinium emirnense Hook. Ericaceae AM
Aphloia theaeformis (Vahl.) Benn. Aphloiaceae AM
Rhus taratana (Baker.) H. Perrier Anacardiaceae AM
Helychrysum rusillonii Hochr. Asteraceae AM
Psiadia altissima (D.C.) Drake Asteraceae AM
Rubus apetalus Poir. Rosaceae AM
Erica sp. Ericaceae AM
Eucalyptus camaldulensis Dehn. Myrtacea ECM & AM
Pinus patula Schiede ex Schtdl. & Cham. Pinaceae ECM & AM
Uapaca bojeri L. Euphorbiaceae ECM & AM
Restoring native forest ecosystems
123
communities associated with U. bojeri root systems
in the different soil origins were significantly different
(Table 4; Fig. 1). The RFLP types UA1 (Russula
earlei), UA2 (Amanita sp.), UA3 (Thelephoroid
symbiont), and UA4 (uncultured ECM fungus)
were only recorded on U. bojeri seedlings grown in
U. bojeri soil, whereas in the soils collected under
exotic tree species, UD1 (Bondarcevomyces), UC3
(Russula exalbicans), and UB6 (Boletellus projectel-
lus) were found. In the bare soil, the RFLP type
UC3 was mainly detected and two other types,
UC2 (Boletus rubropunctus) and UB5 (Coltricia
perennis) at lower abundances (Fig. 1). The RFLP
type UB4 (Xerocomus chrysenteron) was only
recorded in the E. camaldulensis soil treatment
(Fig. 1).
Responses of soil characteristics and U. bojeri
growth to the L. bojeriana cultivation
A data table with 36 rows and 12 columns was
constructed with the soil, plant, and microbial activity
parameters. The 12 variables were: pH, soluble
phosphorus, total nitrogen and total organic matter,
total microbial activity, acid and alkaline phosphatase
activities, shoot and root biomass of U. bojeri
seedlings, ectomycorrhizal rate, leaf nitrogen and
phosphorus contents, and the Shannon diversity index
of the ectomycorrhizal fungal morphotypes. The 36
rows corresponded to three samples of the four soil
origins: soil collected under E. camaldulensis,
P. patula, U. bojeri, or bare soil. For each soil origin,
three treatments were considered: U. bojeri seedling
Table 2 Chemical and biochemical characteristics of rhizosphere soils collected under a native tree species (Uapaca bojeri), two
exotic tree species (Pinus patula and Eucalyptus camaldulensis) and from the bare soil (control) in the Arivonimamo forest
Soil origins
Control U. bojeri P. patula E. camaldulensis
pH (H2O) 5.26 (0.03)1 d2 4.94 (0.01) c 4.78 (0.01) b 4.52 (0.01) a
Total nitrogen (%) 0.09 (0.006) a 0.19 (0.003) c 0.15 (0.006) b 0.22 (0.006) d
Soluble P (mg kg-1) 1.45 (0.02) a 2.85 (0.02) c 2.14 (0.07) b 3.09 (0.02) d
Total organic matter (%) 1.76 (0.009) a 4.26 (0.038) d 3.23 (0.041) b 3.53 (0.026) c
Total microbial activity
(lg of hydrolyzed FDA h-1 g-1 of soil)
5.61 (0.05) a 6.69 (0.25) b 11.54 (0.65) c 15.33 (2.05) c
Acid phosphatase activity
(lg p-nitrophenol g-1 of soil h-1)
130.56 (31.8) a 314.01 (11.7) b 867.06 (50.7) c 586.51 (104.9) c
Alkaline phosphatase activity
(lg p-nitrophenol g-1 of soil h-1)
166.51 (6.91) a 302.54 (7.44) c 170.95 (8.47) b 82.54 (5.59) a
1 Standard error of the mean. 2 Data in the same line followed by the same letter are not significantly different according to the
Newman–Keuls test (p \ 0.05
Table 3 Response of U. bojeri seedling growth and ectomycorrhizal colonization in soils from different tree species (Uapaca bojeri,Pinus patula and Eucalyptus camaldulensis) and from the bare soil (control) after 5 months culturing in glasshouse conditions
Soil origins
Control U. bojeri P. patula E. camaldulensis
Shoot biomass (mg dry weight) 131 (11)1 b2 125 (15) b 85 (12) a 83 (9) a
Root biomass (mg dry weight) 113 (12) b 295 (35) c 119 (10) b 27 (4) a
Total biomass (mg dry weight) 244 (12) b 419 (48) c 205 (22) b 110 (8) a
Root:shoot ratio 0.88 (0.15) b 2.37 (0.16) d 1.42 (0.12) c 0.34 (0.08) a
N leaf mineral content (mg per plant) 0.89 (0.06) a 0.85 (0.1) a 0.65 (0.09) a 0.65 (0.07) a
P leaf mineral content (mg per plant) 71.1 (7.3) ab 94.1 (9.9) b 58.9 (8.7) a 62.3 (7.3) ab
Ectomycorrhizal colonization (%) 36.1 (2.08) b 73.7 (3.18) c 29.3 (5.55) ab 16.3 (2.40) a
1 Standard error of the mean. 2 Data in the same line followed by the same letter are not significantly different according to the
Newman–Keuls test (p \ 0.05)
R. Baohanta et al.
123
was planted alone, with a L. bojeriana seedling, or
with a L. bojeriana seedling that aerial part was cut
after 4 months of cultivation, but keeping intact its
root system. The resulting data table was submitted to
a principal component analysis (PCA) to describe the
main structures of this data set.
The Fig. 2 showed the results of this PCA. The
upper part (Fig. 2a) graphic was the correlation circle
of all the parameters, and the lower part graphic
(Fig. 2b) was the map of sample scores on the first two
principal components. The correlation circle (Fig. 2a)
showed that the first principal component (PC1) was
well correlated to plant growth, with better growth
toward the right of the graphic (shoot biomass, leaf
phosphorus and leaf nitrogen contents) and also to the
microbial activities (total microbial activity, acid and
alkaline phosphatase activity), to the ectomycorrhizal
rate, and to the Shannon diversity index of ectomy-
corrhizal fungi. The second principal component
(PC2) was negatively correlated to root biomass
increase and soil total nitrogen (downward arrows)
and positively to organic matter and pH (upward
arrows).
The map of sample scores (Fig. 2b) showed on the
PC1 the very strong effect of the L. bojeriana plant
(solid arrows pointing right). This effect was positive,
as it corresponded to an increase of U. bojeri seedling
growth, of microbial activities, and of ectomycorrhizal
fungal diversity. This effect was highest when the
Table 4 Identification by ITS sequence of RFLP types for
ectomycorrhizas collected on U. bojeri seedling after 5 month
culturing in glasshouse conditions on soils collected under a
native tree species (Uapaca bojeri), two exotic tree species
(Pinus patula and Eucalyptus camaldulensis) and from the bare
soil (control) in the Arivonimamo forest
RFLP
types
GenBank
accession
number
Closest GenBank
species
BLAST
expected
value
UA1 AF518722 Russula earlei 2e-144
UD1 DQ534583 Bondarcevomyces taxi 3e-138
UA2 AM117659 Amanita sp. 0.0
UA3 AJ509798 Telephoroid
mycorrhizal sp.
1e-154
UC3 AY293269 Russula exalbicans 2e-170
UA4 AY157720 Uncultured ECM
homobasidiomycete
Clone E2
0.0
UB6 DQ534582 Boletellus projectellus 0.0
UC2 FJ480421 Boletus rubropunctus 2e-171
UB5 None Coltricia perennis 2e-141
UB4 AD001659 Xerocomuschrysenteron
4e-173
Fig. 1 Similarities in ectomycorrhizal communities between
U. bojeri seedlings growing in soils collected under Uapacabojeri, Eucalyptus camaldulensis, Pinus patula and from a bulk
soil (d). Values are expressed by RFLP type percentages with
regards to the soil treatments. UA1: Russula earlei, UD1: