Embed Size (px)
Citation preview
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
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
1
CURRICULUM VITAE
Dr RAMANANKIERANA Heriniaina
IM 321 637
Maître de recherches
Né le 16 octobre 1974 à Andramasina
Marié – deux enfants
-----------------------------------------------------------------------------------------------------------------
Adresse professionnelle :
Laboratoire de Microbiologie de l’Environnement du Centre National de Recherches sur
l’Environnement (CNRE)
BP 1739 Fiadanana – Antananarivo Madagascar
E-mail : [email protected]
Tél : +261 32 40 614 57
FORMATIONS :
25 – 26 janvier 2012 : Formation sur le format des enregistrements du BCH
(Biosecurity Clearing House) et les procédures d’enregistrement et de publication des
décisions liées à la biosécurité. ONE (Office National de l’Environnement)
Antananarivo-Madagascar.
Janvier – Mai 2009 : Formation sur l’utilisation des outils biomoléculaires modernes
dans l’identification et caractérisation de souches fongiques et dans le conditionnement
et suivi des inocula fongiques et leurs microorganismes associés (Bourse d’Echange
Scientifique de Courte Durée – IRD).
- Laboratoire des Symbioses Tropicales et Méditerranéennes – Montpellier
France
2006 – 2008 : Perfectionnement post doctoral
Sujet : Gestion des communautés de champignons ectomycorhiziens par les espèces
arbustives pionnières et ectotrophes des formations forestières Malagasy : impact sur la
succession végétale et sur le développement des arbres endémiques de Madagascar
(Bourse post doctorale AUF et Bourse d’Echange Scientifique de Courte Durée – IRD)
- Laboratoire des Symbioses Tropicales et Méditerranéennes – Montpellier
France
2
- Laboratoire Commun de Microbiologie : IRD/ISRA/UCAD Dakar Sénégal
- Laboratoire de Microbiologie de l’Environnement – CNRE Antananarivo
Madagascar
Novembre – Décembre 2007 : Ecole Thématique en Ecologie Tropicale « Insularité et
Biodiversité » - Morondava Madagascar
2005 : Doctorat de 3e cycle en Biochimie
Sujet de thèse : La symbiose mycorhizienne dans la domestication d’Uapaca bojeri,
une plante ligneuse endémique de Madagascar (Bourse de formation à la recherche –
AUF)
- Faculté des Sciences, Université d’Antananarivo Madagascar
- Laboratoire de Biologie du sol – IRD Burkina Faso
28 avril - 29 Mai 2005 : Ecole Thématique « Ecologie microbienne des sols tropicaux :
biodiversité microbienne et dérèglements environnementaux » - Dakar Sénégal
Juin 2000 : Diplôme d’Etude Approfondie (DES) en Sciences Biologique Appliquée,
Option Biotechnologie – Microbiologie
- Faculté des Sciences, Université d’Antananarivo Madagascar
1997 : Maîtrise de recherche en Sciences Biologiques Appliquées, Option
Biotechnologie – Microbiologie
1996 : Licence ès-Sciences, Université d’Antananarivo Madagascar
1992 : Baccalauréat Série D, délivré par l’Université d’Antananarivo Madagascar
Connaissances diverses :
Ayant une maîtrise importante de l’outil microinformatique (WinWord, Microsoft
Excel, Power Point) et de l’analyse statistique (STATISTICA, ADE 4, SPAD, SPSS,
Logiciel R)
Ayant une bonne connaissance de la langue Malagasy, Française et Anglaise
o Intermediate level in English language (London Business Academy)
Ayant une forte motivation pour le travail d’équipe
PARTICIPATION A DES RESEAUX DE RECHERCHE :
Depuis février 2011 : Point focal du réseau AFRINOM pour Madagascar et la région de
l’océan indien
Depuis décembre 2009 : Membre fondateur du réseau SYMETROP associant des
scientifiques africains francophones travaillant dans le domaine de champignons
mycorhiziens
3
Depuis 2008 : Membre de l’Académie des Sciences du Tiers monde
Depuis avril 2006 : Membre du réseau Biotechnologie végétale, amélioration des
plantes et sécurité alimentaire (BIOVEG) – Agence Universitaire de la Francophonie
Depuis mars 2007 : Membre de l’association « African Mycology Association »
Depuis décembre 2004 : Président fondateur de l’association « Jeunes Chercheurs
Associés » à Madagascar
Depuis août 2002 : Membre du Collège des chercheurs Associés UNU/INRA
(Université des Nations Unies/Institut de Recherche sur les Ressources Naturelles en
Afrique)
ACTIVITES PROFESSIONNELLES :
De juin 2009 à ce jour : Maître de recherches au Laboratoire de Microbiologie de
l’Environnement (LME) du Centre National de Recherches sur l’Environnement
(CNRE), Antananarivo Madagascar
Chercheur Enseignant et Responsable de l’Unité de Recherche
« Microbiologie en milieux naturels » au sein du LME/CNRE
Encadreur d’étudiants préparant des mémoires de Diplôme d’Etudes
Approfondies en microbiologie et en écologie microbienne
De 2006 à ce jour : Enseignant vacataire au Département de Biochimie Fondamentale et
Appliquée de la Faculté des Sciences, Université d’Antananarivo
Enseignant de la matière « Valorisation de la biomasse » pour les
étudiants en M2, Option Biotechnologie – Microbiologie
De 2009 à ce jour : Enseignant à la Formation GRENE de l’Université de Toamasina
Enseignant de la matière « Ecologie générale et Ecologie microbienne »
pour les étudiants de la première année
Encadreur d’étudiants préparant des mémoires de Maîtrise Spécialisée et
de Diplôme d’Etudes Supérieurs Spécialisées
EXPERIENCES D’ENCADREMENT :
Mémoires de DEA :
M. ANDRIANANDRASANA Doret Martial. Effets mycorhizosphériques d’Acacia
mangium : impacts sur la structure et l’activité de la population microbienne du sol et sur le
développement d’une essence ligneuse autochtone, Intsia bijuga. Mémoire de Diplôme
d’Etudes Approfondies, Département de Biochimie Fondamentale et Appliquée - Faculté des
4
Sciences - Université d’Antananarivo, Madagascar. Soutenu le 20 novembre 2009.
(Rapporteur)
M. RAZAKATIANA Tsoushima. Algues marines et microorganismes du sol
termitière : source potentielle de fertilisant biologique. Mémoire de Diplôme d’Etudes
Approfondies, Département de Biochimie Fondamentale et Appliquée - Faculté des Sciences -
Université d’Antananarivo, Madagascar. Soutenu le 15 décembre 2010. (Rapporteur)
Mlle RAKOTONIAINA Henintsoa Volatiana. Caractère invasif de Grevillea banksii et
ses impacts sur la régénération de Dalbergia trichocarpa : implication de la composante
microbienne du sol. Mémoire de Diplôme d’Etudes Approfondies, Département de Biochimie
Fondamentale et Appliquée - Faculté des Sciences - Université d’Antananarivo, Madagascar.
Soutenu le 08 avril 2010. (Rapporteur)
Mlle ANDRIAMBOAVONJISOA Harimino. Performance de la roche volcanique en
tant que substrat dans la production d’inoculum de champignons ectomycorhiziens. Mémoire
de Diplôme d’Etudes Approfondies, Département de Biochimie Fondamentale et Appliquée -
Faculté des Sciences - Université d’Antananarivo, Madagascar. Soutenu le 05 Août 2011.
(Rapporteur)
M. RANAIVORADO Tojo Heritiana. 2012. Activité antimicrobienne des
actinomycètes du sol forestier d’Ibity. Mémoire de Diplôme d’Etudes Approfondies,
Département de Biochimie Fondamentale et Appliquée - Faculté des Sciences - Université
d’Antananarivo, Madagascar. Soutenu le 13 mars 2012. (Rapporteur)
Mémoires de Maîtrise Spécialisée :
M. TODISOA Edmond Mamonjy. 2009. Etude de la composition de la communauté de
poisson du canal des Pangalanes (région Atsinanana). Maîtrise spécialisée en Gestion de
Ressources Naturelles et de l’Environnement Université de Toamasina. Soutenu au mois de
mai 2009. (Encadreur pédagogique et Rapporteur)
M. RASOLOFOMANANA Robert. 2011. Implication de la symbiose mycorhizienne
sur le développement de trois essences (Intsia bijuga, Uapaca louvelii et Harunga
madagascariensis) natives de la station forestière d’Ivoloina. Maîtrise spécialisée en Gestion
de Ressources Naturelles et de l’Environnement Université de Toamasina. Soutenu au mois
d’octobre 2011. (Encadreur pédagogique et Rapporteur)
Mémoire de Licence professionnelle :
Mlle MICHEL BENANGO Anne marie. 2010. Analyse de peuplements aquatiques au
large de Fénérive-Est : cas observé de Nosin-dRatsimilaho. Mémoire de fin d’étude
5
pour l’obtention du diplôme de Licence professionnelle en Gestion de Ressources
Naturelles et de l’Environnement, Université de Toamasina. Soutenu au mois de
juillet 2010. (Encadreur pédagogique et Rapporteur)
PARTICIPATION AU JURY DE SOUTENANCE
A la fois enseignant à l’Université et chercheur au sein du Laboratoire de Microbiologie de
l’Environnement (CNRE), j’ai eu la chance de participer au jury de la soutenance de plusieurs
mémoires dont cinq (5) mémoires de DEA, cinq (5) mémoires de DESS, quatre (4) mémoires
de Maîtrise spécialisée et deux (2) mémoires de Licence professionnelle.
Mémoire de DEA
1. M. ANDRIANANDRASANA Doret Martial. 2009. Effets
mycorhizosphériques d’Acacia mangium : impacts sur la structure et l’activité de la population
microbienne du sol et sur le développement d’une essence ligneuse autochtone, Intsia bijuga.
Mémoire de Diplôme d’Etudes Approfondies, Département de Biochimie Fondamentale et
Appliquée - Faculté des Sciences - Université d’Antananarivo, Madagascar : Rapporteur
2. M. RAZAKATIANA Tsoushima. 2010. Algues marines et microorganismes du
sol termitière : source potentielle de fertilisant biologique. Mémoire de Diplôme d’Etudes
Approfondies, Département de Biochimie Fondamentale et Appliquée - Faculté des Sciences -
Université d’Antananarivo, Madagascar : Rapporteur
3. Mlle RAKOTONIAINA Henintsoa Volatiana. 2011. Caractère invasif de
Grevillea banksii et ses impacts sur la régénération de Dalbergia trichocarpa : implication de
la composante microbienne du sol. Mémoire de Diplôme d’Etudes Approfondies, Département
de Biochimie Fondamentale et Appliquée - Faculté des Sciences - Université d’Antananarivo,
Madagascar : Rapporteur
4. Mlle ANDRIAMBOAVONJISOA Harimino. 2011. Performance de la roche
volcanique en tant que substrat dans la production d’inoculum de champignons
ectomycorhiziens. Mémoire de Diplôme d’Etudes Approfondies, Département de Biochimie
Fondamentale et Appliquée - Faculté des Sciences - Université d’Antananarivo, Madagascar :
Rapporteur
5. M. RANAIVORADO Tojo Heritiana. 2012. Activité antimicrobienne des
actinomycètes du sol forestier d’Ibity. Mémoire de Diplôme d’Etudes Approfondies,
Département de Biochimie Fondamentale et Appliquée - Faculté des Sciences - Université
d’Antananarivo, Madagascar : Rapporteur
6
Mémoire de DESS
1. M. MAHEFA Robert. 2010. Analyse de l’importance des usages coutumiers des
plantes en relation avec la conservation des ressources naturelles d’Analalava (Foulpointe).
Mémoire de fin d’étude pour l’obtention du Diplôme d’Etude Supérieure Spécialisée (DESS)
en Gestion des Ressources Naturelles et de l’Environnement. Université de Toamasina,
Madagascar. Examinateur
2. Mlle RAZANAKOLONA Antinone. 2010. Plan de gestion et de conservation de
l’espèce : Dioscorea orangeana dans la forêt de la nouvelle Aire Protégée (NAP) Oronjia
Commune Rurale de Ramena, District d’Antsiranana II. Mémoire de fin d’étude pour
l’obtention du Diplôme d’Etude Supérieure Spécialisée (DESS) en Gestion des Ressources
Naturelles et de l’Environnement. Université de Toamasina, Madagascar. Examinateur.
3. M. TODISOA Edmond Mamonjy. 2010. Etude de l’écologie et de la
reproduction de trois espèces de poissons endémiques de Madagascar à la station piscicole et
au Parc Ivoloina : cas de Paretroplus polyactis, Paratilapia sp et Ptychochromis grandidierie
(région Atsinanana). Mémoire de fin d’étude pour l’obtention du Diplôme d’Etude Supérieure
Spécialisée (DESS) en Gestion des Ressources Naturelles et de l’Environnement. Université
de Toamasina, Madagascar. Examinateur.
4. M. ANDRIAMALALA Heritiana. 2010. Pratique d’agroforesterie (aspect socio-
économique), cas du village d’Ambonivato, Commune Rurale d’Antetezambaro, Région
Atsinanana. Mémoire de fin d’étude pour l’obtention du Diplôme d’Etude Supérieure
Spécialisée (DESS) en Gestion des Ressources Naturelles et de l’Environnement. Université
de Toamasina, Madagascar. Examinateur
5. M. MAHEFA Christian Olivier. 2010. Promotion et développement des activités
écotouristiques du Parc marin Masoala (03 parcelles marines : Tampolo, Ambodilaitry,
Tanjona). Mémoire de fin d’étude pour l’obtention du Diplôme d’Etude Supérieure Spécialisée
(DESS) en Gestion des Ressources Naturelles et de l’Environnement. Université de
Toamasina, Madagascar. Examinateur
Mémoire de Maîtrise spécialisée
(1) M. TODISOA Edmond Mamonjy. 2009. Etude de la composition de la
communauté de poisson du canal des Pangalanes (région Atsinanana). Maîtrise spécialisée en
Gestion de Ressources Naturelles et de l’Environnement Université de Toamasina :
Rapporteur
7
(2) M. BESIRY Martino. 2011. Considérations générales sur l’exploitation des
crevettes dans la zone de Sahamalaza : niveau d’exploitation et dynamique de la population de
crevettes à Antafiantambalaka et Antsiraka. Maîtrise spécialisée en Gestion de Ressources
Naturelles et de l’Environnement Université de Toamasina : Président
(3) M. RASOLOFOMANANA Robert. 2011. Implication de la symbiose
mycorhizienne sur le développement de trois essences (Intsia bijuga, Uapaca louvelii et
Harunga madagascariensis) natives de la station forestière d’Ivoloina. Maîtrise spécialisée en
Gestion de Ressources Naturelles et de l’Environnement Université de Toamasina :
Rapporteur
(4) M. RANDRIANASOLO Arcatia. 2011. Contribution à l’élaboration d’un
schéma d’aménagement pour la pérennisation de la gestion d’une forêt artificielle : cas de la
forêt de Fanalamanga (Moramanga), Région alaotra Mangoro. Maîtrise spécialisée en Gestion
de Ressources Naturelles et de l’Environnement Université de Toamasina : Examinateur
Mémoire de Licence
(1) Mlle MICHEL BENANGO Anne marie. 2010. Analyse de peuplements
aquatiques au large de Fénérive-Est : cas observé de Nosin-dRatsimilaho. Mémoire de fin
d’étude pour l’obtention du diplôme de Licence professionnelle en Gestion de Ressources
Naturelles et de l’Environnement, Université de Toamasina. Rapporteur
(2) M. ANDRIANAVONJIHASINA Nirina Zo Michel. 2010. Essai d’utilisation
des produits locaux pour l’alimentation des poissons (Oreochromis niloticus, Cichlidae) à
Ambila Lemaitso. Mémoire de fin d’étude pour l’obtention du diplôme de Licence
professionnelle en Gestion de Ressources Naturelles et de l’Environnement, Université de
Toamasina, Madagascar. Président
GESTION DE PROJET ET/OU CONTRIBUTION A LA REALISATION DE PROJET :
2000 - 2002 : Projet de valorisation des plantes médicinales et aromatiques de
Madagascar, Projet financé par le Gouvernement Malagasy
Responsabilité : Responsable du volet « Micropropagation des espèces d’orchidées
aromatiques »
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
8
Responsabilité : Responsable du volet « Etablissement d’une collection de souches
ectomycorhiziennes »
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
Responsabilité : Porteur du projet
2006 - 2009: Ectomycorrhizal host shrubs as an important nurse plant to tree
successional processes and ecology restoration in highland of Madagascar, Projet
financé par British Ecological Society (BES).
Responsabilité : Porteur du projet
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 »
Responsabilité : Porteur du projet
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 et par le programme « Bond’innov »
ORGANISATION DES MANIFESTATIONS SCIENTIFIQUES :
Décembre 2009 : Atelier SYMETROP « La symbiose mycorhizienne et les
champignons comestibles en Afrique francophone », décembre 2009, Dakar Sénégal
Responsabilité : Membre du comité d’organisation et participant
Décembre 2010 : Atelier de restitution à mi-parcours du programme « Madasym -
Fonctionnement symbiotiques des écosystèmes forestiers de Madagascar » le 09
décembre 2010 à la Résidence d’Ankerana, Antananarivo Madagascar
Responsabilité : Coordinateur de l’atelier
Novembre 2011 : Premier congrès international sur les mycorhizes organisé dans la
région de l’océan indien « Symbioses mycorhiziennes : écosystèmes et environnement
des Etats insulaires de l’Océan Indien » 21 – 23 novembre 2011 Antananarivo
Madagascar
Responsabilité : Coordinateur du congrès.
9
PARTICIPATION A DES MANIFESTATIONS SCIENTIFIQUES :
Au niveau national et régional:
19 – 21 octobre 2011 : Atelier régional sur le thème « Exploitation des acquis de la
recherche pour améliorer la gestion des forêts ». 25e Anniversaire du SNGF. Ecole
Supérieur des Sciences Agronomiques. Université d’Antananarivo – Madagascar.
Octobre 2011 : Atelier de validation du rapport national sur les ressources
phylogénétiques forestières de Madagascar. CNEAGR Nanisana. Antananarivo
Madagascar
22 – 23 juillet 2010 : Atelier d’évaluation « fin phase de construction du projet
Ambatovy ». Ankorondrano Antananarivo – Madagascar
13 – 15 octobre 2009 : Symposium « Biodiversité et substances naturelles –
BIOMAD ». Antananarivo – Madagascar
Décembre 2008 : Forum de la Recherche Nationale. MESupRES. Université
d’Antsiranana – Madagascar
Mai 2007 : International Foundation for Sciences Workshop. Pretoria – South Africa
Octobre 2007 : Célébration du XXe Anniversaire du SNGF. Antananarivo –
Madagascar
Au niveau international :
14 – 15 décembre 2011 : Atelier de restitution du programme « La biodiversité des îles
de l’Océan indien ». Paris – France
21 – 23 février 2011 : International Workshop « Mycorrhizae : a biological tool for
sustainable development in Africa ». Dakar – Sénégal.
11 – 13 octobre 2010 : International congress on Mycorrhizal symbiosis, Ecosystems
and Environment of Mediterranean area. Marrakech – Maroc.
7 – 10 décembre 2009 : Atelier de création du réseau « Symbioses mycorhiziennes en
Afriques francophones ». Dakar – Sénégal.
28 – 30 octobre 2009 : Atelier-rencontre du programme Jeunes Equipes Associées à
l’IRD. Marseille – France.
14 – 18 septembre 2009 : International Symposia on Environmental Biochemestry.
University of Hamburg – Germany.
3 – 6 novembre 2008 : Atelier de restitution « Groupement de Recherches
Internationales - Madagascar, South Africa, France ». Montpellier – France
10
PUBLICATIONS DANS DES REVUES A COMITE DE LECTURE :
Baohanta R.H., Thioulouse J, Ramanankierana H., Prin Y, Rasolomampianina R,
Baudoin E, Rakotoarimanga N, Galiana A, Randriambanona H & Lebrun M. (2012). Restoring
native forest ecosystems after exotic tree plantation in Madagascar: combination of the local
ectotrophic species Leptolaena bojeriana and Uapaca bojeri mitigates the negative influence
of the exotic speciea Eucalyptus camaldulensis and Pinus patula. Biological Invasions, In
press. DOI 10.1007/s10530-012-0238-5
Andrianandrasana M.D., Rakotoniaina H.V., Raherimandimby M, Ramanankierana H,
Baohanta R.H. & Duponnois R. (2011). Propagation of Grevillea banksii, an invasive exotic
plant species: impacts on structure and functioning of mycorrhizal community associated with
natives tree species in eastern part of Madagascar. Procceding of 3rd International
Symposium on Weeds and Invasive Plants. Ascona Switzerland.
Ducousso, M., Ramanankierana, H., Duponnois, R., Rabevohitra, R., Randriahasipara,
L., Vincelette, M. Dreyfus, B. & Prin, Y. (2008). The 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.
Ramanankierana, H. Prin, Y., Rakotoarimanga, N., Thioulouse, J. Randrianjohany, E.,
Ramaroson, L.& Duponnois, R. (2007). Arbuscular mycorrhizas and ectomycorrhizas in
Uapaca nojeri (Euphorbiaceae) : patterns of root colonization and effects on seedling growth
and soil microbial functionalities. Mycorrhiza. 17 : 195 – 208.
Ramanankierana, H., Rakotoarimanga, N., Thioulouse, J., Kisa, M., Randrianjohany,
E., Ramaroson, L. & Duponnois, R. (2006). The ectomycorrhizosphere effect influences
functional diversity of soil microflora. International Journal of Soil Science. 1. (1) : 8 – 19
Duponnois, R., Assiegbetse, 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. Microbiel Ecology
Ramanankierana, H. (2005). La symbiose mycorhizienne dans la domestication de
Uapaca bojeri (Euphorbiaceae) plante ligneuse endémique de Madagascar. Doctorat en
Biochimie. Université d’Antananarivo - Madagascar
11
AUTRES PUBLICATIONS :
Articles scientifiques :
Ramanakierana H., Baohanta R. H, Razafimiaramanana H., Raherimandimby M. &
Duponnois R. (2011). Amélioration de la régénération d’Uapaca bojeri par la gestion des
communautés arbustives ectotrophes et la symbiose ectomycorhizienne . Acte de l’Atelier
régional. 25eme anniversaire du SNGF. Antananarivo – Madagascar.
Ramanankierana, H., Baohanta, R., H., Rakotoarimanga N., Rasolomampianina, R.,
Randriambanona H., Duponnois, R.. (2011). La communauté mycorhizienne associée aux
plantes cibles du projet d’exploitation minière Ambatovy. Monographie d’Ambatovy. Edition
Recherches et Développement, CIDST. Madagascar (Accepté pour publication).
Ramanankierana, H., Rasolomampianina, R., Rakotoarimanga, N., Randrianjohany, E.,
Ramaroson, L. & Duponnois, R. (2010). Des plantules munies de leurs partenaires
symbiotiques : Une technologie nouvelle pour la bonne réussite de reboisement et de
restauration écologique à Madagascar. Acte du forum de la Recherche Nationale 2010.
MESupRES. Madagascar
Chapitre de livre :
Ramanankierana H., Baohanta R.H., Thioulouse J., Prin Y., Baudoin E.,
Rakotoarimanga N., Galiana A., Randriambanona H., Lebrun M. & Duponnois R. (2012).
Improvement of the early growth of endemic tree species by soil mycorrhizal management in
Madagascar. In : Seedlings : growth, ecology and environmental influence. Eds Nova Science
Publisher Inc. Enfield, Hampshire 03748 USA
Ramanankierana H., Randriambanona H., Baohanta R.H., Sanon A.,
Andrianandrasana D.M., Rajaonarimamy E. & Duponnois R. (2012). Structure et
fonctionnement de la symbiose mycorhizienne au sein des écosystèmes forestiers du haut
plateau et de la région Est de Madagascar. In Les acquis du SYMETROP. Eds IRD
Baohanta R.H., Ramanankierana H., Thioulouse J., Prin Y., Rasolomampianina
R., Baudoin E., Rakotoarimanga N., Galiana A., Randriambanona H., Lebrun M. & Duponnois
R. (2012). Mycorrhizal fungi diversity and their importance on the establishment of native
species seedlings within Madagascarian degraded sclerophyllous forest”. (2012). In:
Ectomycorrhizal Symbioses in Tropical and Neotropical forests. Eds Nova Science Publisher
Inc. Enfield, Hampshire 03748 USA (Soumis)
12
Sanon A., Ndoye F., Ramanankierana H., Duponnois R. (2012). Implication of
mycoprrhizal symbiosis in the trajectory of plant invasion process: How do they matter? In
Mycomed Book. Eds Nova Science Publisher Inc. Enfield, Hampshire 03748 USA (Soumis).
Ramanankierana H., Baohanta R., Rakotoarimanga N., Rasolomampianina R.,
Randriambanona H. & Duponnois R. (2012). La communauté mycorhizienne associée aux
plantes cibles du projet d’exploitation minière Ambatovy. In : Monographie d’Ambatovy.
Eds : Recherches et Développement CIDST. Antananarivo, Madagascar. (Accepté pour
publication).
Communication orales :
Ramanankierana H. & Duponnois R. (2011). Lutte biologique intégrée contre Striga
asiatica à Madagascar par la valorisation de la biodiversité microbienne et de la diversité de
semis direct sur couverture végétale permanente. Communication orale. Atelier de
restitution du programme « La biodiversité des Îles de l’Océan Indien », 14 et 15
décembre 2011. Paris, France.
Ramanakierana H., Baohanta R. H, Razafimiaramanana H., Raherimandimby M. &
Duponnois R. (2011). Amélioration de la régénération d’Uapaca bojeri par la gestion des
communautés arbustives ectotrophes et la symbiose ectomycorhizienne . Communication
orale. Atelier régional. 25eme anniversaire du SNGF. Antananarivo – Madagascar
Ramanankierana H., Baohanta R., Razafimiaramanana H., Raherimandimby M. &
Duponnois R. (2011). Impact of two shrub species (Sarcolaena oblongifolia, Leptolaena
baujeriana) on soil microbial functioning and on seedling growth of Uapaca bojeri in
Madagascarian sclerophyllous forest. Communication orale. International Worshop
“Mycorrhizae: a biological tool for sustainable development in Africa”, 21 – 23 février
2011. Dakar, Senegal
Ramanankierana H., Ouhamane L., Baohanta R. H., Raherimandimby M., Mouhamed
H. & Duponnois R. (2010). Some established shrub species facilitate the early growth of tree
species in Madagascarian highland and in high Atlas of Morocco. Communication orale.
International congress on Mycorrhizal symbiosis, Ecosystems and Environment of
Mediterranean area. October 11 – 13, 2010.Marrakech, Maroc
Ramanankierana H., Rasolomampianina R. Baohanta R. & Rakotoarimanga N. (2010).
Les aspects microbiologiques de la régénération et conservation des espèces sensibles du projet
13
Ambatovy. Communication orale. Atelier d’évaluation fin phase de construction. 22 – 23
juillet 2010. Antananarivo – Madagascar
Ramanankierana H., Rasolomampianina R., Rakotoarimanga N., Baohanta R.H.,
Ramamonjisoa D., Ramaroson L. & Duponnois R. (2009). Connaissances et valorisation de la
diversité microbienne du sol : quel avenir pour Madagascar. Communication orale.
Symposium Biodiversité et Substances Naturelles – BIOMAD. 13 au 15 octobre 2009.
Antananarivo/Madagascar
Ramanankierana H., Baohanta R.H., Raherimandimby M. & Duponnois R., (2009).
Impact of ectomycorrhizal inoculation on soil microbial activity and seedling growth of
Leptolaena bojeriana, an early established shrub species at forest edge. Oral communication.
International Symposia on Environmental Biochemestry. 14 – 18 September 2009.
University of Hamburg – Germany
Ramanankierana H. (2009). Fonctionnement symbiotique des écosystèmes forestiers à
Madagascar. Communication orale. Atelier-rencontre du programme Jeunes Equipes Associées
à l’IRD. 28 – 30 octobre 2009. Marseille – France.
Ramanankierana H. (2009). Production de champignons comestibles à Madagascar.
Communication orale. Atelier sur la création du réseau « Symbioses mycorhiziennes en
Afriques ». 7 au 10 décembre 2009. Dakar – Sénégal
Ramanankierana H., Rasolomampianina R. & Rakotoarimanga N., (2008). Mycorrhizal
symbiosis as a key strategy in the regeneration and conservation of Madagascarian endemic
trees. Communication. Communication orale. Colloque “Groupement de Recherches
Internationales” Madagascar – South Africa – France. Montpellier 3 – 6 novembre 2008.
Ramanankierana H. , Raherimandimby M. & Duponnois R. (2007). The
ectomycorrhizal symbiosis as a key factor in regeneration strategies of Madagascarian highland
sclerophyllous forest. Oral communication. IFS Workshop. University of Pretoria – South
Africa
Ramanankierana H. & Raherimandimby M. (2007). La symbiose mycorhizienne dans la
conservation et valorisation d’essences ligneuses endémiques de Madagascar. Communication
orale. XXth Anniversary of SNGF Workshop. Antananarivo – Madagascar.
14
Brevet :
European Patent. Reforestation of a soil area with co-culture of tree species and nurse plants.
Patent n° 12305223. 5 – 2313 Février 2012
Article dans la presse :
Autour de la biodiversité : portrait d’une jeune équipe « MADASYM » par
Ramanankierana H. Sciences au Sud n° 55- juin – juillet – août 2010
Autour de la biodiversité : portrait d’une jeune équipe « MADASYM » par
Ramanankierana H. La Gazette de la Grande île. Mercredi 25 Août 2010
Les microorganismes au service des grands arbres : la preuve de l’ingéniosité de la
nature par les chercheurs par Ramanankierana H. et Randriambanona H. Journal de
l’économie du 23 au 29 août 2010
Plantes forestières : l’absence de bactérie affecte leur croissance par Ramanankierana
H. La Gazette de la Grande île. Jeudi 09 décembre 2010
15
SYNTHESE DES ENSEIGNEMENTS DISPENSES, DES PROJETS
de recherche MENES ET PRODUCTIONS SCIENTIFIQUES
16
INTRODUCTION
Cette partie développe les responsabilités techniques et scientifiques que j’ai prises après avoir
soutenu ma thèse de doctorat en novembre 2005 ainsi que les activités de valorisation des
résultats obtenus. La grande partie de ces activités ont été menées au Laboratoire de
Microbiologie de l’Environnement du CNRE où je travaille en étroite collaboration aussi bien
avec des collègues Malagasy qu’étrangers. Que ce soit l’enseignement, l’organisation de
rencontre scientifique ou l’encadrement des étudiants préparant des mémoires de fin d’études,
les thèmes abordés tournent toujours autour de la symbiose mycorhizienne et ses applications
pour la gestion durable des ressources naturelles et de la fertilité des sols cultivés. Ces activités
d’enseignement et d’encadrement ont été menées en collaboration avec plusieurs partenaires
dont entre autre la Faculté des Sciences, l’Ecole Supérieure des Sciences Agronomiques
(Département Forêt) et l’Ecole Supérieur Polytechnique de l’Université d’Antananarivo, la
Formation GRENE de l’Université de Toamasina (Madagascar), l’Ecole doctorale
« Biotechnologie végétale et microbienne » de l’Université Cheik Anta Diop de Dakar
(Sénégal), l’Institut de Biologie Intégrative et des Systèmes de l’Université de Laval (Canada),
la Faculté des Sciences de l’Université de Marrakech (Maroc) et le Laboratoire des Symbioses
Tropicales et Méditerranéennes de Montpellier (France). Les activités de formations menées
avec ces partenaires ont permis de créer différentes plates formes regroupant les scientifiques
selon leur domaine de recherche (Réseau SYMETROP, AFRINOM…) et d’intégrer certains
étudiants Malagasy dans des équipes scientifiques reconnues au niveau mondial (Université de
Laval, LMI-Laboratoire de Biotechnologie Microbienne et Végétale à Rabat, Maroc…). Au
niveau national, je dirige actuellement une équipe d’une dizaine de jeunes scientifiques en
début de leur carrière scientifique ou en phase finale de leur étude doctorale. Les membres de
cette équipe sont principalement issus de la Faculté des Sciences de l’Université
d’Antananarivo et ont été formés dans le cadre de partenariat avec les partenaires étrangers
cités ci-dessus.
ENSEIGNEMENT
Depuis l’année universitaire 2006 – 2007, j’ai dispensé des cours théoriques et/ou des
travaux pratiques dans deux universités publiques (Université d’Antananarivo et Université de
Toamasina) et un institut privé de formation supérieur (EPSA Bevalala). En décembre 2009,
j’ai participé à l’animation de l’école doctorale « Biotechnologie végétale et microbienne » à
l’Université Cheick Anta Diop de Dakar, Sénégal.
17
II.1. Enseignant du cours de « Valorisation de la biomasse » pour la deuxième année
de maîtrise
Option Biotechnologie – Microbiologie du Département de Biochimie Fondamentale
et Appliquée de la Faculté des Sciences Antananarivo, Madagascar (Depuis l’année
universitaire 2006 – 2007).
Résumé et grandes lignes du cours
L’U.E. biomasse vise à fournir aux étudiants des connaissances plus approfondies relatives aux
différentes sources pérennes et renouvelables de production alimentaire, de matériaux et
d’énergie. Elle permettra, par la suite, aux étudiants de se familiariser aux caractéristiques de
ces sources ainsi que d’évaluer l’importance de ces dernières par rapport aux autres. Toutes ces
connaissances constitueront une base solide de développement durable
Enseignement théorique Les différents types de biomasse
- Biomasse végétale
- Biomasse animale
- Biomasse microbienne
Les biomasses valorisables - Caractéristiques
- Disponibilité en quantité et en qualité
- Importance socio-économique et environnementale
- Stratégies de valorisation et externalités
- Intérêts et limites de la valorisation
La biomasse et les filières de transformation - Adaptabilité de la source à la filière de transformation
- Compétitivité et concurrence
Notion d’agriculture biologique Enseignement dirigé et Enseignement pratique
Technique d’évaluation de la qualité de la biomasse
Analyse et commentaire des caractéristiques de la biomasse et ses produits
de valorisation
Compétences acquises Aptitude à identifier des sources durables de production et à décrire des
approches de valorisation
Capacité à établir des approches de gestion durable des ressources
Secteur d'activité concerné : Energies renouvelables
Dépollution de l’environnement
18
Bio séquestration du carbone
Production alimentaire et de matériaux
II.2. Responsable du cours d’ « Ecologie Générale » pour les étudiants en premier
année au sein de la formation en Gestion des Ressources Naturelles et de l’Environnement
Université de Toamasina, Madagascar (Depuis l’année universitaire 2006 – 2007)
Résumé et grandes lignes du cours
Ce cours est articulé au tour de trois axes : (1) les notions de base en écologie permettant de
mettre à la disposition des étudiants les éléments fondamentaux constituant les écosystèmes,
(2) les interactions, dans un premier temps, entre ces différents éléments et puis entre ces
éléments et les différents facteurs du milieu et (3) les apports de connaissances sur l’écologie
en matière de conservation et de valorisation de la biodiversité et des ressources naturelles.
L’objectif étant d’apporter aux étudiants des connaissances élémentaires mais largement
suffisantes pour qu’ils puissent comprendre facilement, à la fin de la première année, le
fonctionnement écologique de différents types d’écosystème. Ces connaissances seront par la
suite renforcées par des séries de travaux dirigés et d’exposé dont les sujets visent à étudier
différents types d’écosystème Malagasy ou à comprendre l’importance des connaissances
écologiques sur la gestion durable des ressources naturelles. Au terme de cet enseignement, les
étudiants devront avoir une vue d’ensemble des différents éléments des écosystèmes, leur
interrelations et leur importance et être capables d’entamer des études plus approfondies
relatives aux aspects fonctionnels et analytique de l’écologie.
II.3. Responsable du cours d’ « Ecologie microbienne et fonctionnement des
écosystèmes » pour les étudiants en second cycle au sein de la formation en Gestion des
Ressources Naturelles et de l’Environnement
Université de Toamasina, Madagascar (Depuis l’année universitaire 2010 – 2011)
Résumé et grandes lignes du cours
L’écologie microbienne aborde globalement la dynamique et la place des
microorganismes dans leurs habitats ainsi que les différentes voies de valorisation des
microorganismes pour le bien être de l’Homme. L’objectif principal de ce cours est d’inculquer
aux étudiants la diversité des microorganismes au sein des différents écosystèmes (marin,
aquatique et terrestre), notamment ceux des écosystèmes humides de Madagascar, et leur
interaction avec les autres composantes du milieu. Ce cours qui sera composé de séances de
cours théorique ainsi que des travaux dirigés sera divisé en 3 parties comprenant i)
19
l’introduction à l’écologie microbienne, ii) les différents types d’interaction microbienne au
sein d’un écosystème et iii) les principaux secteurs d’exploitation des microorganismes. A la
fin de ce cours, les étudiants devraient être capables, d’une part, de décrire la composante
microbienne d’un écosystème comme étant une biodiversité toute entière, de comprendre le
fonctionnement microbiologique d’un écosystème et ses importances pour la conservation de
celui-ci et d’autre part d’identifier les différentes approches de valorisation des
microorganismes et de mettre en place des stratégies écologiques visant à mieux gérer les
ressources naturelles (stratégies de restauration écologique et/ou de valorisation de la biomasse,
traitement des déchets ou des eaux usées…).
II.4. Responsable du cours de « Microbiologie et qualité de l’environnement » pour
les étudiants en second cycle au sein de la formation en Gestion des Ressources Naturelles et
de l’Environnement
Université de Toamasina, Madagascar (Depuis l’année universitaire 2010 – 2011)
Résumé et grandes lignes du cours
Ce cours concernera l’importance de la microbiologie dans la gestion de la qualité de
l’environnement (l’air, l’eau, le sol, les produits alimentaires ou biologiques, les surfaces et les
matériaux etc.). Les objectifs principaux seront d’inculquer aux étudiants les méthodes de
recherche et d’analyse de la qualité microbiologique de l’environnement ainsi que les
approches descriptives des principaux microorganismes impliqués directement dans les
phénomènes conduisant à la modification de la qualité de l’environnement. A la fin de ce
cours, les étudiants devraient avoir la capacité de décrire la qualité microbiologique de
différents produits (aliments, eaux…) ou des milieux (milieux de préparation, de
transformation, de prélèvement…), d’interpréter les résultats d’analyses microbiologiques ainsi
que d’établir des stratégies de gestion de la qualité des produits ou des milieux. Au centre du
cours est situé le système HACCP (« Hazard Analysis Critical Control Point » ou méthode et
principes de gestion de la sécurité sanitaire des produits finis) et les mécanismes de sa mise en
place et de suivi dans les différentes chaines de production. Le cours sera divisé en quatre
parties : (i) les risques liés à la contamination de l’environnement et les approches de leur
identification, (ii) les différentes sources de contamination, (iii) les stratégies et méthodes de
suivi de la qualité de l’environnement et (iv) les approches pour limiter la propagation des
contaminants.
20
II.5. Responsable du cours théorique sur la « Microbiologie du sol et ses
applications en agriculture » pour la troisième année Filière Agriculture
Ecole Supérieure Professionnelle Bevalala, Antananarivo, Madagascar (2007 – 2010)
Résumé et grandes lignes du cours
Ce cours est articulé autour de trois axes : (1) les notions fondamentales de la microbiologie
du sol permettant de situer l’importance des microorganismes du sol au sein de l’écosystème
terrestre, (2) les approches d’étude des microorganismes du sol avec une attention particulière
sur les microorganismes connus pour leur importance en agriculture, en élevage et en
conservation de l’environnement et (3) les différentes applications de la microbiologie du sol
pour les besoins socio-économiques et environnementaux de l’humanité. L’objectif étant
d’apporter aux étudiants des connaissances élémentaires suffisantes du monde des
microorganismes du sol et leur relation avec les facteurs environnants. Ces connaissances
seront, par la suite, renforcées par la deuxième et troisième partie du cours consacrées à
l’exploitation rationnelle de ces microorganismes et leur importance. L’étude de ces
exploitations, depuis l’identification et la mise en culture de ces microorganismes jusqu’à la
maîtrise de leur utilisation, sera appuyée par des exemples étudiés en cours et pratiqués au
laboratoire et en milieu naturel. Les technologies de pointe utilisées pour l’étude des
microorganismes du sol seront largement exploitées en cours pour donner aux étudiants une
vue plus développée de la microbiologie. Au terme de cet enseignement, les étudiants devront
avoir une vue d’ensemble de la population microbienne du sol et ses fonctionnements, être
capables d’identifier les aspects positifs et négatifs de l’exploitation des microorganismes du
sol et devront avoir la capacité de mener des études prospectives et préliminaires sur terrain
relatives à l’analyse microbiologique d’un type de sol.
II.6. Mission d’enseignement
Animation de conférence scientifique pour les étudiants en master en Biotechnologie végétale
et microbienne et pour les thésards à l’Ecole doctorale de la Faculté des Sciences de
l’Université Cheik Anta Diop Dakar – Sénégal (Décembre 2009 et 2010)
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
Correspondence: Robin Duponnois, Institut
de Recherche pour le Developpement, UMR
113 CIRAD/INRA/IRD/AGRO-M/UM2,
Laboratoire des Symbioses Tropicales
et Mediterraneennes (LSTM), 34398
Montpellier, France. Tel.: (33) (0)4 67 59 38
82; fax: (33) (0)4 67 59 38 02;
e-mail: [email protected]
Received 31 August 2005; revised 14 October
2005; accepted 17 October 2005.
First published online 8 February 2006.
doi:10.1111/j.1574-6941.2006.00089.x
Editor: Ralf Conrad
Keywords
termitaria; fluorescent pseudomonads;
ectomycorrhizal symbiosis; Acacia holosericea.
Abstract
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
293Termite mounds enhance ectomycorrhizal symbiosis
was performed at 72 1C for 5 min. PCR products (7mL) were
digested in a total volume of 20mL at 37 1C for 2 h using
10 U of the endonucleases HaeIII and MspI (Promega,
Charbonnieres, France), as described by the manufacturer.
Restriction fragments were separated by horizontal electro-
phoresis in a 2.5% (weight in volume, w/v) Metaphor gel
(FMC, Rockland, ME). After 2 h of running at 80 V, the gel
was stained for 30 min with ethidium bromide (1 mg L�1)
and integrated with the Image Analysis software BIOCAPT
(Vilbert Lourmat, Paris, France) under UV light.
The amplified DNA fragments were purified using a
Qiaquick PCR purification kit (Qiagen, Courtaboeuf, France),
and then ligated into the pGEM-T vector and transformed into
cells (Escherichia coli DH5a) according to the instructions of
the manufacturer (pGEM-T easy vector system, Promega).
PCR amplification with the primers T7 and Sp6 was per-
formed directly from the selected white colonies (presumed
transformants) to confirm the presence of the insert of the
correct size. The plasmid insert from a clone representing each
isolate was sent for sequencing (Genome Express, Montreuil,
France). The sequence data were compared with gene libraries
[GenBank and European Molecular Biology Laboratory
(EMBL)] using BLAST (Heinemeyer et al., 1989) and FASTA
(Pearson & Lipman, 1988) programs.
Twenty-eight different Pseudomonas species were re-
trieved from the Ribosome Database Project (RDP) (http://
www.cme.m-su.edu/RDP) for phylogenetic comparison
with our Pseudomonas isolates. The phylogenetic analysis
was performed using the MEGA (Molecular Evolutionary
Genetics Analysis) version 2.1 package (Kumar et al., 2001).
Multiple sequence alignments were carried out using the
CLUSTALW program (Thompson et al., 1994). Phylogenetic
analysis was performed by the neighbour-joining method,
and the relative support for groups was determined on the
basis of 1000 bootstrap trees.
The nucleotide sequence obtained in this study has been
deposited in the GenBank database and assigned Accession
number AY327816.
Glasshouse experiment
Fungal and bacterial inoculum
The ectomycorrhizal fungi, strains IR408 and IR412, have
been identified as Scleroderma dictyosporum on the basis of
rDNA internal transcribed spacer phylogeny (Sanon, 1999).
They were isolated from sporocarps under Uapaca guineen-
sis in the province of Houet (Burkina Faso). The fungal
isolates were maintained on modified Melin–Norkrans
(MMN) agar (Marx, 1969) at 25 1C. The ectomycorrhizal
fungal inoculum was prepared according to Duponnois &
Garbaye (1991). Glass jars were filled with 600 mL of a
vermiculite–peat moss mixture (4/1, v/v) and autoclaved
(120 1C, 20 min). This substrate was moistened to field
capacity with 300 mL of liquid MMN medium. The jars
were sealed with a cotton float and autoclaved (120 1C,
20 min). Finally, 10 fungal plugs were placed aseptically into
each glass jar and incubated for 6 weeks at 28 1C in the dark.
One strain of fluorescent pseudomonad (Pseudomonas sp.
KR9) was randomly chosen from the selected bacterial
isolates. It was grown in 0.3% TSB (Difco Laboratories)
liquid medium for 3 days at 28 1C on a rotary shaker,
centrifuged (2400 g, 20 min) and suspended in 0.1 M
MgSO4. The final concentration of the bacterial suspension
was about 108 CFU mL�1, estimated by enumeration on a
plate count agar medium (King’s B medium) (King et al.,
1954). This suspension was used as inoculum.
Effect of the mound powder on IR408 and IR412ectomycorrhizal development
Seeds of Acacia holosericea, originating in Ndioum/Podor
(Senegal), were surface sterilized with concentrated 18 M
sulphuric acid for 60 min. The acid solution was decanted
off and the seeds were washed for 12 h in four rinses of
sterile distilled water. The seeds were then transferred
aseptically to Petri dishes filled with 1% (w/v) agar–water
medium. These plates were incubated at 25 1C in the dark.
The germinating seeds were used when the rootlets were
1–2 cm in length.
Acacia holosericea seedlings were grown in 1 L pots filled
with soil collected from a millet field near Ouagadougou
(Burkina Faso). Before use, the soil was crushed, passed
through a 2 mm sieve and autoclaved for 40 min at 140 1C.
One week after autoclaving, its chemical and physical
characteristics were as follows: pH (H2O) 5.6; clay, 4.6%;
fine silt, 0.0%; coarse silt, 0.8%; fine sand, 25.5%; coarse
sand, 69.1%; carbon, 0.204%; nitrogen, 0.04%; carbon/
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
responses. Co-inertia analysis (Chessel & Mercier, 1993;
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
295Termite mounds enhance ectomycorrhizal symbiosis
technique that describes the relationships between two data
tables. Numerous methods have been suggested for this [e.g.
canonical analysis (Gittins, 1985), canonical correspondence
analysis (Ter Braak, 1986) and partial least squares regres-
sion Hoskuldsson, 1988], but one of the simplest, from a
theoretical point of view, is co-inertia analysis. Computa-
tions and graphical displays were prepared with free ADE-4
software (Thioulouse et al., 1997), available at http://pbil.
univ-lyon1.fr/ADE-4/.
Results
Genotypic fingerprinting of fluorescentpseudomonad strains isolated from termitemounds of Macrotermes subhyalinus
Eighteen randomly chosen fluorescent pseudomonad strains
were subjected to PCR/restriction fragment length poly-
morphism (RFLP) analysis. PCR conditions allowed the
amplification of a single DNA fragment of the 16S rDNA
gene with the same size of 1000 bp for all 18 Pseudomonas
isolates studied. Digestion of the PCR products with two
restriction enzymes (HaeIII and MspI) did not show any
polymorphism in the patterns of the 16S rDNA fragments
(Fig. 1).
Sequence analysis of the 16S rDNA of all the Pseudomonas
sp. isolates studied showed 100% identity, signifying that
they were all identical. Only one isolate (Pseudomonas sp.
KR9) was chosen for phylogenetic analysis. The rDNA
sequence demonstrated high identity (99.7%) with 16S
rDNA sequences of Pseudomonas monteillii HR13 (Acces-
sion no. AY032725), P. mosselii (Accession no. AF072688),
P. putida (Accession no. AB029257), P. plecoglossicida (Ac-
cession no. AB09457) and P. monteillii (Accession no.
AF064458).
Phylogenetic analysis with other selected Pseudomonas
species from RDP was performed with the neighbour-join-
ing method using Escherichia coli as outgroup. The sequence
of the Pseudomonas isolate KR9 clustered highly with the
sequences of P. monteillii HR13, P. mosselii, P. putida,
P. plecoglossicida, P. monteillii and P. mevalonii (Fig. 2).
Effect of termite mound amendment on IR408and IR412 ectomycorrhiza formation
After 4 months of culture, the shoot growth of Acacia
holosericea seedlings was significantly stimulated by both
ectomycorrhizal fungal strains in comparison with the
noninoculated treatment (control) (Table 3). The termite
mound amendment also significantly improved the shoot
biomass (Table 3). No significant differences were recorded
between the M. subhyalinus treatment and the termite
mound amendment/ectomycorrhizal fungal inoculation
(Table 3). Compared with the control, Scleroderma dictyos-
porum IR408 significantly enhanced the root growth of
A. holosericea seedlings, whereas no significant effects were
recorded with S. dictyosporum IR412 (Table 3). In the soil
amended with the termite mound, the root growth was
significantly higher than that recorded in the control (Table
3). This termite mound effect was significantly enhanced
when ectomycorrhizal fungi were inoculated (Table 3). No
significant differences were recorded between the treatments
with regard to the total number of nodules per plant. The
arbuscular mycorrhizal colonization indices were very low
and not significantly different between the soil treatments
(Table 3). No ectomycorrhizal short roots were detected in
the soil amended with the termite mound (Fig. 3). The
ectomycorrhizal colonization indices of A. holosericea seed-
lings inoculated with S. dictyosporum IR408 and IR412 were
not significantly different (13.5%). The termite mound
amendment significantly increased ectomycorrhizal forma-
tion, which reached around 25% (Fig. 3).
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
297Termite mounds enhance ectomycorrhizal symbiosis
MS and IR412 1 MS are also shifted towards the right of the
figure. This positive effect seems to be stronger for the IR412
strain than for the IR408 strain. With regard to SIR
responses, Figs 4b and 4d clearly show that the positive
effect of M. subhyalinus mound powder amendment corre-
sponds to a strong modification of the functional microbial
diversity for the three treatments.
One-way analysis of variance confirmed these conclu-
sions (Table 4). Ectomycorrhizal establishment was mainly
characterized by higher SIR responses with L-arginine,
whereas termite mound amendment was indicated by high-
er SIR responses with sucrose, D-glucosamine, keto-glutaric,
hydroxy-butyric, ascorbic, quinic, gluconic, keto-butyric,
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
299Termite mounds enhance ectomycorrhizal symbiosis
and a better supply of nitrogen (Meiklejohn, 1965; Mohin-
dra & Mukerji, 1982), and to higher moisture levels and
higher substrate availability (Holt, 1987; Abbadie & Lepage,
1989).
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,
L-glutamine; 2, L-arginine; 3, L-serine; 4, L-histidine; 5, phenylalanine; 6, L-asparagine; 7, L-tyrosine; 8, L-glutamic acid; 9, L-lysine; 10, L-cysteine; 11,
D-glucose; 12, D-mannose; 13, sucrose; 14, D-glucosamine; 15, N-methyl-D-glucamine; 16, succinamide; 17, 2-keto-glutaric acid; 18, 3-hydroxy-butyric
acid; 19, ascorbic acid; 20, D-quinic acid; 21, D,L-malic acid; 22, formic acid; 23, fumaric acid; 24, gallic acid; 25, gluconic acid; 26, keto-butyric acid; 27,
malonic acid; 28, oxalic acid; 29, succinic acid; 30, tartaric acid; 31, trisodium citrate; 32, uric acid; 33, cyclohexane. (c) Factor map of plant growth.
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;
Thomas, 1987b; Wood & Thomas, 1989; Rouland-Lefevre,
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.
References
Abbadie L & Lepage M (1989) The role of subterranean fungus
comb chambers (Isoptera, Macrotermitinae) in soil nitrogen
cycling in a preforest savanna (Cote d’Ivoire). Soil Biol Biochem
8: 1067–1071.
Amato M & Ladd JM (1988) Assay for microbial biomass based
on ninhydrin-reactive nitrogen in extracts of fumigated soils.
Soil Biol Biochem 20: 107–114.
Bethlenfalvay GJ & Linderman RG (1992) Mycorrhizae in
Sustainable Agriculture, ASA Special Publication No. 54.
Agronomy Society of America, Madison, WI.
Black HIJ & Okwakol MJN (1997) Agricultural intensification,
soil biodiversity and agrosystem function in the tropics: the
role of termites. Appl Soil Ecol 6: 37–53.
Brauman A (2000) Effect of gut transit and mound deposit on
soil organic matter transformations in the soil feeding termite:
a review. Eur J Soil Biol 36: 117–125.
Bremner JM (1965) Inorganic forms of nitrogen. Methods of Soil
Analysis, Part 2. Agronomy Monographs, Vol. 9, (Black CA, ed.),
pp. 1179–1237. Agronomy Society of America and Soil Science
Society of America, Madison, WI.
Brundrett MC, Piche Y & Peterson RL (1985) A developmental
study of the early stages in vesicular–arbuscular mycorrhizal
formation. Can J Bot 63: 184–194.
Chessel D & Mercier P (1993) Couplage de triplets statistiques et
liaison espece-environnement. Biometrie et Environnement,
(Lebreton JD & Asselain D, eds), pp. 15–44. Masson, Paris.
Degens BP & Harris JA (1997) Development of a physiological
approach to measuring the catabolic diversity of soil microbial
communities. Soil Biol Biochem 29: 1309–1320.
Dickie IA & Reich PB (2005) Ectomycorrhizal fungal
communities at forest edges. J Ecol 93: 244–255.
Doledec S & Chessel D (1994) Co-inertia analysis: an alternative
method for studying species–environment relationships.
Freshwater Biol 31: 277–294.
Drever JL & Vance GF (1994) Role of soil organic acids in mineral
weathering processes. The Role of Organic Acids in Geological
Processes, (Lewan MD & Pittman ED, eds), pp. 138–161.
Springer-Verlag, New York.
Dunstan WA, Malajczuk N & Dell B (1998) Effects of bacteria on
mycorrhizal development and growth of container grown
Eucalyptus diversicolor F. Muell. seedlings. Plant Soil 201:
241–249.
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
301Termite mounds enhance ectomycorrhizal symbiosis
Duponnois R & Garbaye J (1991) Techniques for controlled
synthesis of the Douglas fir–Laccaria laccata ectomycorrhizal
symbiosis. Ann Sci For 48: 239–251.
Duponnois R & Lesueur D (2005) Sporocarps of Pisolithus albus
as an ecological niche for fluorescent pseudomonads involved
in Acacia mangium Wild–Pisolithus albus ectomycorrhizal
symbiosis. Can J Microbiol 50: 691–696.
Duponnois R & Plenchette C (2003) A mycorrhiza helper
bacterium enhances ectomycorrhizal and endomycorrhizal
symbiosis of Australian Acacia species. Mycorrhiza 13: 85–91.
Duponnois R, Plenchette C, Thioulouse J & Cadet P (2001) The
mycorrhizal soil infectivity and arbuscular mycorrhizal fungal
spore communities in soils of different aged fallows in Senegal.
Appl Soil Ecol 17: 239–251.
Duponnois R, Paugy M, Thioulouse J, Masse D & Lepage M
(2005) Functional diversity of soil microbial community, rock
phosphate dissolution and growth of Acacia seyal as influenced
by grass-, litter- and soil-feeding termite nest structure
amendments. Geoderma 124: 349–361.
Dutton MV & Evans CS (1996) Oxalate production by fungi: its
role in pathogenicity and ecology in the soil environment.
Can J Microbiol 42: 881–895.
Founoune H, Duponnois R, Ba AM, Sall S, Branget I, Lorquin J,
Neyra M & Chotte JL (2002a) Mycorrhiza helper bacteria
stimulate ectomycorrhizal symbiosis of Acacia holosericea with
Pisolithus alba. New Phytol 153: 81–89.
Founoune H, Duponnois R, Meyer JM, Thioulouse J, Masse D,
Chotte JL & Neyra M (2002b) Interactions between
ectomycorrhizal symbiosis and fluorescent pseudomonads on
Acacia holosericea: isolation of mycorrhiza helper bacteria
(MHB) from a Soudano-Sahelian soil. FEMS Microbiol Ecol
41: 37–46.
Frey P, Frey-Klett P, Garbaye J, Berge O & Heulin T (1997)
Metabolic and genotypic fingerprinting of fluorescent
pseudomonads associated with the Douglas fir Laccaria bicolor
Symbiosis. Appl Environ Microbiol 63: 1852–1860.
Frey-Klett P, Pierrat JC & Garbaye J (1997) Location and survival
of mycorrhiza helper Pseudomonas fluorescens during
establishment of ectomycorrhizal symbiosis between Laccaria
bicolor and Douglas fir. Appl Environ Microbiol 63: 139–144.
Gadd GM (1999) Fungal production of citric and oxalic acid:
importance in metal speciation, physiology and biochemical
processes. Adv Microbiol Physiol 41: 47–92.
Garbaye J (1994) Helper bacteria: a new dimension to the
mycorrhizal symbiosis. New Phytol 128: 197–210.
Gianinazzi S & Schuepp H (1994) Impact of Arbuscular
Mycorrhizas on Sustainable Agriculture and Natural Ecosystems.
Birkhauser Verlag, Basel.
Gittins R (1985) Canonical Analysis, a Review with Applications in
Ecology. Springer-Verlag, Berlin.
Grant WD & West AW (1986) Measurement of ergosterol,
diaminopimelic acid and glucosamine in soil: evaluation as
indicators of microbial biomass. J Microbiol 6: 47–53.
Hart MM, Reader RJ & Klironomos JN (2003) Plant coexistence
mediated by arbuscular mycorrhizal fungi. Trends Ecol Evol 18:
418–423.
Heinemeyer O, Insam H, Kaiser EA & Walenzik G (1989) Soil
microbial biomass and respiration measurements: an
automated technique based on infrared gas analysis. Plant Soil
116: 77–81.
Holt JA (1987) Carbon mineralization in Northeastern Australia:
the role of termites. J Trop Ecol 3: 255–263.
Holt JA & Lepage M (2000) Termites and soil properties.
Termites: Evolution, Sociality, Symbioses, Ecology, (Abe T,
Bignell DE & Higashi M, eds), pp. 389–407. Kluwer Academic
Publishers, Dordrecht.
Hooker JE & Black KE (1995) Arbuscular mycorrhizal fungi as
components of sustainable soil–plant systems. Crit Rev
Biotechnol 15: 201–212.
Hoskuldsson A (1988) PLS regression methods. J. Chemometr 2:
211–228.
Jouquet P (2002) De la structure biogenique au phenotype
etendu: le termite champignonniste comme ingenieur de
l’ecosysteme. PhD thesis, University of Paris 6, Paris.
Jouquet P, Boulain N, Gignoux J & Lepage M (2004) Association
between subterranean termites and grasses in a West African
savanna: spatial analysis shows a significant role for
Odontotermes n. pauperans. Appl Soil Ecol 27: 99–107.
King EO, Ward MK & Raney DE (1954) Two simple media for the
demonstration of pyocyanine and fluorescein. J Lab Clin Med
44: 301–307.
Kumar S, Tamura K, Jakobsen IB & Nei M (2001) MEGA2.2:
Molecular Evolutionary Genetics Analysis software.
Bioinformatics 17: 1244–1245.
Lavelle P (1996) Diversity of soil fauna and ecosystem function.
Biol Int 33: 3–16.
Lavelle P (1997) Faunal activities and soil processes: adaptative
strategies that determine ecosystem function. Adv Ecol Res 27:
93–132.
Lee KE & Wood TG (1971) Termites and Soils. Academic Press,
London.
Linderman RG (1988) Mycorrhizal interactions with the
rhizosphere microflora: the mycorrhizosphere effect.
Phytopathology 78: 366–371.
Lobry de Bruyn L & Conacher AJ (1990) The role of termites and
ants in soil modification: a review. Aust J Soil Res 28: 55–93.
Marx DH (1969) The influence of ectotropic mycorrhizal fungi
on the resistance of pine roots to pathogenic infections. I.
Antagonism of mycorrhizal fungi to root pathogenic fungi and
soil bacteria. Phytopathology 59: 153–163.
Meiklejohn J (1965) Microbiological studies on large termite
mounds. Rhodesia, Zambia and Malawi. J Agric Res 3: 67–79.
Mohindra P & Mukerji KG (1982) Fungal ecology of termite
mounds. Rev Ecol Biol Sol 19: 351–361.
Okello-Oloya T, Spain AV & John RD (1985) Selected chemical
characteristics of the mounds of two species of Amitermes
(Isoptera, Termitinae) and their adjacent surface soils from
northeastern Australia. Rev Ecol Biol Sol 22: 291–311.
FEMS Microbiol Ecol 56 (2006) 292–303c� 2006 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. No claim to original French government works
302 R. Duponnois et al.
Okello-Oloya T, Spain AV & John RD (1986) Comparative
growth of two pasture plants from northeastern Australia on
the mound materials of grass- and litter-feeding termites
(Isoptera: Termitidae) and on their associated surface soils.
Rev Ecol Biol Sol 23: 381–392.
Olsen SR, Cole CV, Watanabe FS & Dean LA (1954) Estimation of
available phosphorus in soils by extraction with sodium
bicarbonate. Circular, Vol. 939, p. 19. US Department of
Agriculture, Washington DC.
Pearson W & Lipman DJ (1988) Improved tools for biological
sequence comparison. Proc Natl Acad Sci USA 85: 2444–2448.
Phillips JM & Hayman DS (1970) Improved procedures for
clearing roots and staining parasitic and vesicular–arbuscular
mycorrhizal fungi for rapid assessment of infection. Trans Br
Mycol Soc 55: 158–161.
Rouland-Lefevre C (2000) Symbiosis with fungi. Termites:
Evoultion, Sociality, Symbioses, Ecology, (Abe T, Bignell DE &
Higashi M, eds), pp. 289–306. Kluwer Academic Publishers,
Dordrecht.
Sands WA (1969) The association of termites and fungi. Biology of
Termites, Vol. 1, (Krishna K & Weesner FM, eds), pp. 495–524.
Academic Press, London.
Sannasi A & Sundara-Rajulu G (1967) Occurrence of
antimicrobial substance in the exudates of physiogastric queen
termite, Termes redemanni Wasmann. Curr Sci India 16:
436–437.
Sanon K (1999) La symbiose mycorhizienne chez quelques
Cesalpiniacees et Euphorbiacees des forets du sud-ouest du
Burkina Faso. PhD thesis, University of Nancy 1, Nancy.
Smith SE & Read DJ (1997) Mycorrhizal Symbiosis. 2nd edn.
Academic Press, London.
Spain AV, Gordon V, Reddell P & Correll R (2004)
Ectomycorrhizal fungal spores in the mounds of tropical
Australian termites (isopteran). Eur J Soil Biol 40: 9–14.
Ter Braak CJF (1986) Canonical correspondence analysis: a new
eigenvector technique for multivariate direct gradient analysis.
Ecology 67: 1167–1179.
Thioulouse J, Chessel D, Doledec S & Olivier JM (1997) Ade-4: a
multivariate analysis and graphical display software. Stat
Comput 7: 75–83.
Thomas RJ (1987a) Distribution of Termitomyces and other fungi
in the nests and major workers of several Nigerian
Macrotermitinae. Soil Biol Biochem 19: 335–341.
Thomas RJ (1987b) Factors affecting the distribution and activity
of fungi in the nests of Macrotermitinae. Soil Biol Biochem 19:
343–349.
Thompson JD, Higgins DG & Gibson TJ (1994) CLUSTALW:
improving the sensitivity of progressive multiple sequence
alignment through sequence weighting, position-specific gap
penalties and weight matrix choice. Nucleic Acids Res 22:
4673–4680.
Van der Hejden MGA, Klironomos JN, Ursic M, Moutoglis P,
Streitwolf-Engel R, Boller T, Wiemken A & Sanders IR (1998)
Mycorrhizal fungal diversity determines plant biodiversity
ecosystem variability and productivity. Nature 396: 69–72.
West AW & Sparling GP (1986) Modifications to the substrate-
induced respiration method to permit measurements of
microbial biomass in soils of differing water contents. J
Microbiol Methods 5: 177–189.
Wood TG & Thomas RJ (1989) The mutualistic association
between Macrotermitinae and Termitomyces. Insect–Fungus
Interactions, (Wilding N, Collins NM, Hammond PM &
Webber JF, eds), pp. 69–92. Academic Press, London.
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
303Termite mounds enhance ectomycorrhizal symbiosis
ORIGINAL PAPER
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.
Keywords Ectomycorrhizas . Arbuscular mycorrhizas .
Fungal diversity .Microbial functionalities .
Uapaca bojeri . Madagascar
Introduction
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
Mycorrhiza (2007) 17:195–208DOI 10.1007/s00572-006-0095-0
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.
Gen
us r
elat
ive
freq
uenc
y (%
)
0
5
10
15
20
25
30
35
Russu
la
Amanita
Cantha
rellu
s
Boletu
s
Leccin
um
Tricho
loma
Sclero
derm
a
Afrobo
letus
Xeroco
mus
Rubino
bolet
us
Gyrop
orus
Suillus
0
5
10
15
20
25
Rus
sula
Am
anita
Can
thar
ellu
s
Bol
etus
Lecci
num
Tricho
lom
a
Scler
oder
ma
Afro
bole
tus
Xer
ocom
us
Rub
inob
oletus
Gyr
opor
us
Suillu
s
Num
ber
of
spec
ies
per
gen
us
a
b
Fig. 1 a Structure of theectomycorrhizal community(above-ground diversity)expressed as genus relativefrequency (b). Number ofspecies per genus
Mycorrhiza (2007) 17:195–208 199
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
20
40
60
80
100
0 1 2 3 4 5 6
AM
co
lon
izat
ion
(%
of
roo
t fr
agm
ents
) o
r
ecto
my
corr
hiz
al c
olo
niz
atio
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
Treatments Shoot biomass (mg per plant) Ectomycorrhizal colonization (%) RMD (%)a Rb Ec
Disinfected soilControl 135ad 0a – 28.7a 4.7aScleroderma sp. Sc1 192c 62.1c 29.1a 30.3a 6.9cNondisinfected soilControl 165b 18.2b – 29.7a 6.1bScleroderma sp. Sc1 240d 58.6c 30.4a 30.7a 7.7dSoil Treatment (ST) Se NS NS SFungal inoculation (FI) S NS S SFI × ST NSf NS NS NS
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
References
Allen EB, Allen MF, Helm DJ, Trappe JM, Molina R, Rincon E(1995) Patterns and regulation of mycorrhizal plant and fungaldiversity. Plant Soil 170:47–62
Andrade G, Mihara KL, Linderman RG, Bethlenfalvay GJ (1998) Soilaggregation status and rhizobacteria in the mycorrhizosphere.Plant Soil 202:89–96
Bâ AM, Thoen D (1990) First synthesis of ectomycorrhizas betweenAfzelia africana Sm. (Caesalpinioideae) and native fungi fromWest Africa. New Phytol 114:99–103
Bâ AM, Garbaye J, Dexheimer J (1991) Influence of fungalpropagules during the early stage of the time sequence ofectomycorrhizal colonization on Afzelia africana seedlings. CanJ Bot 69:2442–2447
Brundrett MC (2002) Coevolution of roots and mycorrhizas of landplants. New Phytol 154:275–304
Brundrett MC, Piche Y, Peterson RL (1985) A developmental study ofthe early stages in vesicular-arbuscular mycorrhizal formation.Can J Bot 63:184–194
Buscot F, Munch JC, Charcosset JY, Gardes M, Nehls U, Hampp R(2000) Recent advances in exploring physiology and biodiversityof ectomycorrhizas highlight the functioning of these symbiosesin ecosystems. FEMS Microbiol Rev 24:601–614
Buyck B, Thoen D, Walting R (1996) Ectomycorrhizal fungi of theGuinea–Congo region. Proc R Soc Edinb 104:313–333
Casper BB, Cahill JF (1998) Population-level responses to nutrientheterogeneity and density by Abutilon theophrasti (Malvaceae): anexperimental neighbourhood approach. Am J Bot 85:1680–1687
Cazares E, Trappe JM (1993) Vesicular endophytes in roots of thePinaceae. Mycorrhiza 2:153–156
Chilvers GA, Lapeyrie FF, Horan DP (1987) Ectomycorrhizal vsendomycorrhizal fungi within the same root system. New Phytol107:441–448
Culhane AC, Perriere G, Considine EC, Cotter TG, Higgins DG (2002)Between-group analysis of microarray data. Bioinformatics18:1600–1608
Deacon JW, Donaldson SJ, Last FT (1983) Sequences and interactionsof mycorrhizal fungi on birch. Plant Soil 71:257–262
Degens BP, Harris JA (1997) Development of a physiologicalapproach to measuring the metabolic diversity of soil microbialcommunities. Soil Biol Biochem 29:1309–1320
Degens BP, Vojvodic-Vukovic M (1999) A sampling strategy toassess the effects of land use on microbial functional diversity insoils. Aust J Soil Res 37:593–601
Degens BP, Schipper LA, Sparling GP, Duncan LC (2001) Is themicrobial community in a soil with reduced catabolic diversityless resistant to stress or disturbance? Soil Biol Biochem33:1143–1153
Dhillion SS (1994) Ectomycorrhizae, arbuscular mycorrhizae, andRhizoctonia sp. of alpine and boreal Salix spp. in Norway. ArctAlp Res 26:304–307
Dickie IA, Reich PB (2005) Ectomycorrhizal fungal communities atforest edges. J Ecol 93:244–255
Diédhiou AG, Guèye O, Diabaté M, Prin Y, Duponnois R, Dreyfus B, BâAM (2005) Contrasting responses to ectomycorrhizal inoculation inseedlings of six tropical African tree species. Mycorrhiza 16:11–17
Dolédec S, Chessel D (1987) Rythmes saisonniers et composantesstationnelles en milieu aquatique I-Description d’un plan d’observa-tions complet par projection de variables. Acta Oecol 8:403–426
Ducousso M, Béna G, Bourgeois C, Buyck B, Eyssartier G, VinceletteM, Rabevohitra R, Randrihasipara L, Dreyfus B, Prin Y (2004) Thelast common ancestor of Sarcolonaceae and Asian dipterocarp treeswas ectomycorrhizal before the India–Madagascar separation,about 88 million years ago. Mol Ecol 13:231–236
Duponnois R, Garbaye J (1991) Techniques for controlled synthesisof the Douglas fir—Laccaria laccata ectomycorrhizal symbiosis.Ann For Sci 48:239–251
Duponnois R, Plenchette C (2003) A mycorrhiza helper bacteriumenhances ectomycorrhizal and endomycorrhizal symbiosis ofAustralian Acacia species. Mycorrhiza 13:85–91
Duponnois R, Plenchette C, Thioulouse J, Cadet P (2001) Themycorrhizal soil infectivity and arbuscular mycorrhizal fungalspore communities in soils of different aged fallows in Senegal.Appl Soil Ecol 17:239–251
Duponnois R, Diédhiou S, Chotte JL, Sy MO (2003) Relativeimportance of the endomycorrhizal and/or ectomycorrhizalassociations in Allocasuarina and Casuarina genera. Can JMicrobiol 49(4):281–287
Duponnois R, Founoune H, Masse D, Pontanier R (2005) Inoculationof Acacia holosericea with ectomycorrhizal fungi in a semiaridsite in Senegal: growth response and influences on the mycor-rhizal soil infectivity after 2 years plantation. For Ecol Manag207:351–362
Dutton MV, Evans CS (1996) Oxalate production by fungi: its role inpathogenicity and ecology in soil environment. Can J Microbiol42:881–895
Egerton-Warburton L, Allen MF (2001) Endo- and ectomycorrhizas inQuercus agrifolia Nee. (Fagaceae): patterns of root colonizationand effects on seedling growth. Mycorrhiza 11:283–290
Elliot LF, Lynch JM (1994) Biodiversity and soil resilience. In:Greenland DJ, Szabolcs I (eds) Soil resilience and sustainableland use. CAB International, Wallingford, UK, pp 353–364
Estaun V, Save R, Biel C (1997) AM inoculation as a biological tool toimprove plant re-vegetation of a disturbed soil with Rosmarinusofficinalis under semi-arid conditions. Appl Soil Ecol 6:223–229
Farley RA, Fitter AH (1999) The responses of seven co-occurringwoodland herbaceous perennials to localized nutrient-richpatches. J Ecol 87:849–859
Founoune H, Duponnois R, Bâ AM (2002a) Influence of the dualarbuscular endomycorrhizal/ectomycorrhizal symbiosis on thegrowth of Acacia holosericea in glasshouse conditions (A. Cunn.ex G. Don). Ann For Sci 59:93–98
Founoune H, Duponnois R, Bâ AM, Sall S, Branger I, Lorquin J,Neyra M, Chotte JL (2002b) Mycorrhiza helper bacteriastimulate ectomycorrhizal symbiosis of Acacia holosericea withPisolithus albus. New Phytol 153:81–89
Franson RI, Bethlenfalvay GJ (1989) Infection unit method ofvesicular-arbuscular mycorrhizal propagule determination. SoilSci Soc Am J 53:754–756
Frey P, Frey-Klett P, Garbaye J, Berge O, Heulin T (1997) Metabolic andgenotypic fingerprinting of fluorescent pseudomonads associatedwith the Douglas fir Laccaria bicolor Mycorrhizosphere. ApplEnviron Microbiol 63:1852–1860
Frey-Klett P, Chavatte M, Clausse ML, Courrier S, Le Roux C,Raaijmakers J, Martinotti MG, Pierrat JP, Garbaye J (2005)Ectomycorrhizal symbiosis affects functional diversity of rhizo-sphere fluorescent pseudomonads. New Phytol 165:317–328
Green GM, Sussman RW (1990) Deforestation history of the easternrain forests of Madagascar from satellite images. Science248:212–215
Heim R (1970) Particularités remarquables des Russules tropicalesPelliculariae lilliputiennes: Les complexes annulata et radicans.Bull Soc Mycol France 86:59–77
Heinemeyer O, Insam H, Kaiser EA, Walenzik G (1989) Soilmicrobial biomass and respiration measurements: an automatedtechnique based on infrared gas analysis. Plant Soil 116:77–81
Herrera MA, Salamanca CP, Barea JM (1993) Inoculation of woodylegumes with selected arbuscular mycorrhizal fungi and rhizobiato recover desertified Mediterranean ecosystems. Appl EnvironMicrobiol 59:129–133
Mycorrhiza (2007) 17:195–208 207
Horton TR, Bruns TD (2001) The molecular revolution in ectomycor-rhizal ecology: peeking into the black-box. Mol Ecol 10:1855–1871
Jasper DA, Abbot LK, Robson AD (1991) The effect of soildisturbance on vesicular-arbuscular mycorrhizal fungi in soilsfrom different vegetation types. New Phytol 118:471–476
Landeweert R, Hoffland E, Finlay RD, Kuyper TW, van Breemen N(2001) Linking plants to rock: ectomycorrhizal fungi mobilizenutrients from minerals. Trends Ecol Evol 16:248–254
Lapeyrie FF, Chilvers GA (1985) An endomycorrhiza-ectomycorrhizasuccession associated with enhanced growth of Eucalyptus dumosaseedlings planted in a calcareous soil. New Phytol 100:93–104
Lebreton JD, Sabatier R, Banco G, Bacou AM (1991) Principalcomponent and correspondence analyses with respect to instrumen-tal variables: an overview of their role in studies of structure-activityand species-environment relationships. In: Devillers J, Karcher W(eds) Applied multivariate analysis in SAR and environmentalstudies. Kluwer, pp 85–114
Lee SS (1998) Root symbiosis and nutrition. In: Appanah S, TurnbullJMA (eds) Review of dipterocarps: taxonomy, ecology andsylviculture. CIFOR, Bogor, Indonesia, pp 99–114
Lee SS, Alexander IJ, Watling R (1997). Ectomycorrhizas andputative ectomycorrhizal fungi of Shorea leprosula Miq.(Dipterocarpaceae). Mycorrhiza 7:63–81
Linderman RG (1988) Mycorrhizal interactions with the rhizospheremicroflora: the mycorrhizosphere effect. Phytopathology78:366–371
Lodge DJ, Wentworth TR (1990) Negative associations among VA-mycorrhizal fungi and some ectomycorrhizal fungi inhabiting thesame root system. Oikos 57:347–356
Lowry PPII, Schatz GE, Phillipson PB (1997) The classification ofnatural and anthropogenic vegetation in Madagascar. In: Good-man SM, Patterson BD (eds) Natural change and human impactin Madagascar. Smithsonian Institute Press, Washington, DC, pp93–123
Magurran AE (1988) Ecological diversity and its measurement.Croom Helm, London
Mansfeld-Giese K, Larsen J, Bodker L (2002) Bacterial populationsassociated with mycelium of the arbuscular mycorrhizal fungusGlomus intraradices. FEMS Microbiol Ecol 41:133–140
Marx DH (1991) The practical significance of ectomycorrhizae inforest establishment. Ecophysiology of forest trees. MarcusWallenberg Found Symp Proc 7:54–90
McGee P (1989) Variation in propagule numbers of vesicular-arbuscular mycorrhizal fungi in a semi-arid soil. New Phytol92:28–33
Molina R, Massicotte H, Trappe JM (1992) Specificity phenomena inmycorrhizal symbioses: community-ecological consequences andpractical applications. In: Allen MF (ed) Mycorrhizal function-ing. Chapman and Hall, New York, NY, USA, pp 357–423
Molina R, Myrold D, Li CY (1994) Root symbiosis of red alder:technological opportunities for enhanced regeneration and soilimprovement. In: Hibbs DE, DeBell DS, Tarrant RF (eds) Thebiology and management of red alder. Oregon State UniversityPress, Corvallis, OR, pp 23–46
Moyersoen B, Fitter A (1999) Presence of arbuscular mycorrhizas intypically ectomycorrhizal host species from Cameroon and NewZealand. Mycorrhiza 8:247–253
Ochs M (1996) Influence of humidified and non-humidified naturalorganic compounds on mineral dissolution. Chem Geol 132:119–124
Olsen SR, Cole, CV, Watanabe FS, Dean LA (1954) Estimation ofavailable phosphorus in soils by extraction with sodiumbicarbonate. Circular, vol 939. US Department of Agriculture,Washington, DC, p 19
Perry DA, Amaranthus MP, Borchers JG, Borchers SL, Brainerd RE(1989) Bootstrapping in ecosystems. Bioscience 39:230–237
Phillips JM, Hayman DS (1970) Improved procedure for clearingroots and staining parasitic and vesicular-arbuscular fungi forrapid assessment of infections. Trans Br Mycol Soc 55:158–161
Pirozynski KA, Malloch DW (1975) The origin of land plants: amatter of mycotropism. Biosystems 6:153–164
Plenchette C, Fortin JA, Furlan V (1983) Growth responses of severalplant species to mycorrhizae in a soil of moderate P-fertility. I.Mycorrhizal dependency under field conditions. Plant Soil70:199–209
Plenchette C., Declerck S, Diop T, Strullu DG (1996) Infectivity ofmonoaxenic subcultures of the AM fungus Glomus versiformeassociated with Ri-TDNA transformed root. Appl MicrobiolBiotechnol 46:545–548
Rao CR (1964) The use and interpretation of principal componentanalysis in applied research. Sankhya A 26:329–359
Read DJ, Duckett JG, Francis R, Ligrone R, Russell A (2000)Symbiotic fungal associations in “lower” land plants. PhilosTrans R Soc Lond Ser B-Biol Sci 355:815–830
Requena N, Perez-Solis E, Azcon-Aguilar C, Jeffries P, Barea JM(2001) Management of indigenous plant-microbe symbioses aidsrestoration of desertified ecosystems. Appl Environ Microbiol67:495–498
Riviere T, Natarajan K, Dreyfus B (2006) Spatial distribution ofectomycorrhizal basidiomycete Russula subsect. Foetentinaepopulations in a primary dipterocarp rainforest. Mycorrhiza16:143–148
Sanon K, Bâ AM, Dexheimer J (1997) Mycorrhizal status of somefungi fruiting beneath indigenous trees in Burkina Faso. For EcolManag 98:61–69
Schreiner RP, Mihara KL, McDaniel KL, Bethlenfalvay GJ (2003)Mycorrhizal fungi influence plant and soil functions andinteractions. Plant Soil 188:199–209
Smith S, Read J (1997) Mycorrhizal symbiosis, 2nd edn. Clarendon,Oxford
Thioulouse J, Chessel D, Dolédec S, Olivier JM (1997) ADE-4: amultivariate analysis and graphical display software. Stat Comput7:75–83
Thoen D, Bâ AM (1989) Ectomycorrhizae and putative ectomycor-rhizal fungi of Afzelia africana and Uapaca guineensis inSouthern Senegal. New Phytol 113:549–559
Vagen TG, Andrianorofanomezana MAA, Andrianorofanomezana S(2006a) Deforestation and cultivation effects on characteristics ofoxisols in the highlands of Madagascar. Geoderma 131:190–200
Vagen TG, Walsh MG, Shepherd KD (2006b) Stable isotopes forcharacterisation of trends in soil carbon following deforestaionand land use change in the highlands of Madagascar. Geoderma135:133–139
Valentine LL, Fieldler TL, Hart AA, Petersen CA, Berninghausen HK,Southworth D (2004) Diversity of ectomycorrhizas associatedwith Quercus garryana in southern Oregon. Can J Bot 82:123–135
van der Hejden MGA, Klironomos JN, Ursic M, Moutoglis P,Streitwolf-Engel R, Boller T, Wiemken A, Sanders IR (1998)Mycorrhizal fungal diversity determines plant biodiversityecosystem variability and productivity. Nature 396:69–72
Wardle DA, Giller KE, Barker GM (1999) The regulation andfunctional significance of soil biodiversity in agro-ecosystems.In: Wood D, Lenné JM (eds) Agrobiodiversity: characterisation,utilisation and management. CABI, London, pp 87–121
Watling R, Lee SS (1998) Ectomycorrhizal fungi associated withmembers of the Dipterocarpaceae in Peninsular Malaysia-II. JTrop For Sci 10:421–430
West AW, Sparling GP (1986) Modifications of the substrate-inducedrespiration method to permit measurements of microbial biomassin soils of differing water contents. J Microbiol Methods 5:177–189
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
characteristics, (2) exotic-invaded soil significantly
reduces the early growth and ectomycorrhization of
U. bojeri seedlings and (3) L. bojeriana decreased
these negative effects of the exotic tree species by
facilitating ectomycorrhizal establishment and conse-
quently improved the U. bojeri early growth. This
study provides evidence that L. bojeriana can facilitate
the ectomycorrhizal infection of U. bojeri and miti-
gates the negative effects of the introduction of exotic
tree species on the early growth of the native tree
R. Baohanta � H. Ramanankierana �R. Rasolomampianina � N. Rakotoarimanga �H. Randriambanona
Laboratoire de Microbiologie de l’Environnement,
Centre National de Recherches sur l’Environnement,
BP 1739, Antananarivo, Madagascar
J. Thioulouse
Laboratoire de Biometrie et Biologie Evolutive,
CNRS, UMR 5558, Universite Lyon 1,
69622 Villeurbanne, France
Y. Prin � A. Galiana
CIRAD, Laboratoire des Symbioses Tropicales et
Mediterraneennes (LSTM), UMR 113 CIRAD/INRA/
IRD/SupAgro/UM2, Campus International de Baillarguet,
TA A-82/J, Montpellier, France
E. Baudoin � M. Lebrun � R. Duponnois (&)
IRD, Laboratoire des Symbioses Tropicales et
Mediterraneennes (LSTM), UMR 113 CIRAD/INRA/
IRD/SupAgro/UM2, Campus International de Baillarguet,
TA A-82/J, Montpellier, France
e-mail: [email protected]
Present Address:R. Duponnois
Laboratoire Ecologie & Environnement, Unite associee au
CNRST, URAC 32, Faculte des Sciences Semlalia,
Universite Cadi Ayyad, Marrakech, Morocco
123
Biol Invasions
DOI 10.1007/s10530-012-0238-5
species. From a practical point of view, the use of
ectotrophic early-successional shrub species should be
considered to improve forest resaturation after exotic
invasion.
Keywords Ectomycorrhizas � Uapaca bojeri �Exotic tree species � Degraded forest ecosystems �Nurse plant � Restoration ecology � Revegetation
strategies
Introduction
Numerous agricultural practices lead to soil degrada-
tion and losses of biodiversity in tropical areas. These
anthropogenic impacts do not only degrade natural
plant communities (population structure and species
diversity) but also physico-chemical and biological
soil properties such as nutrient availability, microbial
activity, and soil structure (Styger et al. 2007). In order
to reverse this loss of fertility and to limit soil erosion,
some revegetation programmes have been undertaken
in Madagascar using fast-growing exotic trees. Refor-
estation with eucalyptus (E. robusta, E. rostrata,
E. camaldulensis) and later pine (P. khesya, P. patula)
provided wood for the region (Gade 1996). By the
1930s, plantations have been set out by local commu-
nities, institutions, and individuals (Parrot 1925).
However, exotic trees can threaten ecosystems or
habitats by altering ecological interactions among
native plants (Rejmanek 2000; Callaway and Ridenour
2004) that could compromise their role in sustainable
development. Exotic plants can act directly on native
plant communities by allelopathic effects or by higher
performance in an introduction site that influence
vegetation dynamics, community structure, and com-
position (del Moral and Muller 1970; Thebaud and
Simberloff 2001). They also can alter biochemical
cycling compared with native plants (Ashton et al.
2005). As exotic and native plants have different
evolutionary histories and traits, it has been also
suggested that plant introduction could affect below-
ground soil microbial communities (Hawkes et al.
2005; Batten et al. 2006; Kisa et al. 2007; Kivlin and
Hawkes 2011). Among soil microbial communities,
mycorrhizal fungi are considered as key components
of the sustainable soil–plant system (Johansson et al.
2004; Dickie and Reich 2005). This symbiotic process
influences soil development as well as plant growth
(Schreiner et al. 2003; Duponnois et al. 2007).
Numerous studies have shown that ectomycorrhizal
(ECM) vegetation is highly dependent on ECM fungi
for their growth and survival (Smith and Read 2008).
Limitation of the presence, abundance, and commu-
nity composition of ectomycorrhizal fungi can result
from natural (Terwilliger and Pastor 1999) or anthro-
pogenic disturbance (Jones et al. 2003) and the lack of
established ectomycorrhizal fungi in soils may limit
the establishment or re-establishment of ECM tree
species seedlings (Marx 1991). It has been well
demonstrated that exotic plant species could disrupt
mutualistic associations involved in native ecological
associations (Callaway and Ridenour 2004; Kisa et al.
2007; Remigi et al. 2008; Faye et al. 2009) that could
limit the natural regeneration of native tree species.
However, these negative impacts on soil microbiota
may be counterbalanced by utilizing mycorrhizal
native species that enhance the abundance, diversity,
and function of mycorrhizal propagules in soil (Kisa
et al. 2007; Faye et al. 2009). Recent studies have
shown that some early-successional shrubs can
preserve and/or increase the abundance and diversity
of mycorrhizal propagules of AM fungi (Ouahmane
et al. 2006) or ectomycorrhizal fungi (Dickie et al.
2004) and subsequently facilitate forest woody species
growth. Improvement of seedling growth by pioneer
shrubs, also called the ‘‘nurse plant effect’’, is a
general facilitative process (Niering et al. 1963).
Nurse plants facilitate vegetation growing beneath
their canopies by ameliorating the physical environ-
ment and by increasing soil fertility (Franco and Nobel
1988; Callaway and Pennings 2000; Scarano 2002).
In Madagascar, the impacts of exotic tree species
such as pine and eucalypts on diversity and abundance
of mycorrhizal fungal communities as well as on the
early growth of endemic tree species remain unknown.
The aims of the present study were to determine in situ
and under glasshouse conditions the impact of Euca-
lyptus camaldulensis and Pinus patula (two exotic tree
species) on soil chemical characteristics, microbial
activities and on ECM community structures. We
hypothesized that soil microbial activities and mycor-
rhizal communities will differentiate under these exotic
species leading to a decrease of the early growth of a
native tree species, Uapaca bojeri. We further hypoth-
esized that an enhancement of ectomycorrhizal diversity
provided by an early-successional ectomycorrhizal
R. Baohanta et al.
123
shrub, Leptolena bojeriana, would minimize the neg-
ative effects of these exotic species and consequently
improve U. bojeri growth through a well-developed
ectomycorrhizal root colonization. Finally, we tested
the hypothesis that L. bojeriana could act as a natural
provider for ectomycorrhizal propagules and could
preserve the abundance and diversity of ectomycorrhi-
zal fungi in stressful environments.
Materials and methods
Study area
The field experiment was conducted within the central
part of Madagascarian highland sclerophyllous forest in
a forest located at 50 km to the west of Antananarivo
(Arivonimamo site). The average annual rainfall was
1,398 mm with a average monthly temperature of
26 �C. The vegetation is a mosaic of U. bojeri islands
and very scattered individuals of introduced tree species,
P. patula and E. camaldulensis. These trees dominate an
understorey mainly composed by early-successional
plant species such as Leptolaena bojeriana, Leptolaena
pauciflora, Erica sp., Helychrisum rusillonii, Aphloia
theaformis, Psiadia altissima, Rhus taratana, Vaccini-
um emirnensis, Rubus apelatus and Trema sp. L.
bojeriana was the most representative plant species in
this site with a cover contribution of about 43 %.
Analysis of the mycorrhizal status of trees
and early-successional plant species
Root samples were collected during the rainy season.
Root identity was ascertained by tracing from the
trunk to the fine root tips. Samples of 1–5 g (fresh
weight) of fine roots were washed under running water
and stored at 4 �C for further examination. Fine roots
were examined for ECM infection under a dissecting
microscope. Morphological parameters following
Agerer (1987–1996) such as mantle color and struc-
ture, branching pattern and characteristics of rhizo-
morphs were used to categorize ectomycorrhizas into
morphological type (morphotype) groups. For AM
infection, fine roots were stained following the method
of Phillips and Hayman (1970). The root pieces were
placed on a slide for microscopic observation under
250 magnification (Brundrett 1991). About fifty 1-cm
root pieces were randomly chosen from each root
sample collected from each plant species.
Bioassays of soils collected under exotic tree
species (E. camaldulensis and P. patula)
and the native tree species (U. bojeri)
Seven adult trees of each exotic species and of
U. bojeri were randomly chosen in an approximately
5 ha area in the Arivonimamo forest. In order to avoid
disruption of soil and more particularly changes in
mycorrhizal networks, seven intact blocks of soil were
collected near each adult tree (about 50 cm from the
trunk). Seven additional intact blocks were collected
at 10-15 m from any targeted tree species (E. camal-
dulensis, P. patula, and U. bojeri trees) or other known
ectomycorrhizal plants. Intact monoliths of soil were
cut with shovel and immediately transferred into
15 cm diameter, 16 cm height plastic pots.
In addition, soil samples were taken near each soil
block from the 0–10 cm layer and stored in sealed
plastic bags at field moisture content at 4 �C for further
measurements. For each soil sample, pH of a water soil
suspension was determined. The total organic carbon
(TOC) was measured according to the ANNE method
(Aubert 1978) and the total nitrogen by the Kjeldahl
method. The available and total phosphorus soil
contents were analyzed by colorimetry (Olsen et al.
1954). Acid and alkaline phosphatase activities were
measured using p-nitrophenol benzene as substrate
(Schinner et al. 1996), and production of the
p-nitrophenol product was determined colorimetri-
cally at 650 nm. Fluorescein diacetate (FDA) hydro-
lysis was assayed to provide a measurement of the
microbial global activity (Alef 1998).
Seeds of U. bojeri collected in the Arivonimamo
forest were surface sterilized in hydrogen peroxide for
10 min, rinsed and soaked in sterile distilled water for
12 h, and germinated on 1 % agar. The germinating
seeds were used when rootlets were 1–2 cm long. One
pre-germinated seed was planted per pot filled with
intact monolith of soil. The pots were randomized in
the greenhouse and seedlings grown under natural
light (daylight of approximately 12 h, average daily
temperature of 25 �C). They were watered regularly
with tap water without fertilizer.
After 5 months of culturing, U. bojeri seedlings
were gently uprooted from the pots in order to keep the
Restoring native forest ecosystems
123
root systems intact and to avoid root disruption. Then,
they were gently washed with running water. The
percentage of ectomycorrhizal short roots (number of
ectomycorrhizal short roots/total number of short
roots) was assessed under a dissecting microscope
by counting all single root tips. Ectomycorrhizal or
non-ectomycorrhizal short roots were detected accord-
ing to the presence or absence of fungal mantle and
mycelium and to the presence or lack of root hairs. In
each treatment, ECM root tips were classified by
morphotypes based on characteristics of their mantle
and extra-matrical mycelium (branching, surface
color, texture, emanating hyphae, and rhizomorphs
(Agerer 1995). All morphological types of ectomy-
corrhizas were stored at -20 �C in 700 ll CTAB lysis
buffer (2 % cetylammoniumbromide; 100 mM Tris–
HCl, 20 mM EDTA, 1.4 M NaCl) before molecular
analysis. Three ectomycorrhizas randomly selected
from each morphotype groups were screened by RFLP
analysis and one sample of each unique RFLP patterns
was sequenced.
DNA was extracted from root tips using Qiagen
DNeasy Plant Mini Kits (Qiagen SA, Courtaboeuf,
France) following the manufacturer’s recommenda-
tions. Fungal mitochondrial rDNA extracts were
amplified with ML5 and ML6 primers (White et al.
1990) and restriction digested HaeIII or HinfI
enzymes. Then, one sample of each individual RFLP
type was sequenced with the ABI Prism BigDye
Terminator Cycle sequence kit (Applied Biosystems,
Foster City, CA, USA) and analyzed on an applied
Biosystems model 310 DNA sequencer (Perkin-
Elmer). Sequences were aligned by using Clustal X
1.80 (Thompson et al. 1997) and alignment was
subsequently manually corrected using Genedoc
(Nicholas and Nicholas 1997). All sequences were
identified according to BLAST analysis at the NCBI
page http://www.ncbi.nlm.nih.gov/blast/Blast.cgi, using
default settings. Sequences were deposited in
GenBank.
For each U. bojeri seedlings, the oven dry weight
(1 week at 65 �C) of the aerial and root part was then
measured. After drying, plant tissues were ground,
ashed (500 �C), digested in 2 ml HCl 6N and 10 ml
HNO3 N for nitrogen and then analyzed by colorim-
etry for P (John 1970). For nitrogen (Kjeldahl)
determination, they were digested in 15 ml H2SO4
(36N) containing 50 g l-1 of salicylic acid.
Impact of early-successional ectomycorrhizal
shrub, Leptolena bojeriana on the characteristics
of soils collected under exotic tree species
(E. camaldulensis and P. patula) and the native tree
species (U. bojeri) and on U. bojeri early growth
Seeds of L. bojeriana were collected from the
Arivonimamo forest. They were surface sterilized
and were pre-germinated for 1 week in Petri dishes on
humid filter paper. A germinated seed was then
transplanted into each of plastic pots filled with soil
monoliths sampled as described above under exotic
and native tree species. One set of pots was unplanted.
There were 3 replicates for the unplanted pots and 6 for
the planted pots. The pots were randomized in a
greenhouse under natural light (daylight of approxi-
mately 12 h, average daily temperature of 25 �C) and
watered daily with deionized water. After 4 months of
growth, half of the L. bojeriana seedlings were cut and
their aerial parts discarded without any disruptions of
the cultural soil and L. bojeriana root systems.
Removal of aerial parts allowed to test the capacity
of L. bojeriana seedlings to act as a provider of
ectomycorrhizal propagules without any competitive
processes between each plant species for C acquisition
and consequently to reduce symbiosis costs. Then, one
pre-germinated seed of U. bojeri (treated as previously
described) was planted per pot randomized in the
greenhouse and seedlings were cultivated under
natural light (daylight of approximately 12 h, average
daily temperature of 25 �C). They were watered
regularly with tap water without fertilizer. There were
3 treatments: (1) control (without pre-cultivation with
L. bojeriana), (2) pre-cultivation and dual cultivation
with L. bojeriana (L. bojeriana treatment), and (3) pre-
cultivation dual cultivation with L. bojeriana without
aerial parts (L. bojeriana WA treatment). After
5 months of cultivation, measurements of chemical
and enzymatic soil characteristics as well as U. bojeri
ectomycorrhizal status, growth, and leaf mineral
contents (N, P) were determined as described before.
Statistical analysis
Plant growth measurements and soil characteristics
were treated with one-way analysis of variance and
means were compared with the Newman–Keul multi-
ple range test (p \ 0.05). The fungal colonization
R. Baohanta et al.
123
indexes were transformed by arcsin (ffiffiffixp
) before
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:
Bondarcevomyces taxi, UA2: Amanita sp., UA3: Telephoroid
mycorrhizal sp., UC3: Russula exalbicans, UA4: Uncultured
ECM homobasidiomycete Clone E2, UB6: Boletellus projec-tellus, UC2: Boletus rubropunctus, UB5: Coltricia perennis,
UB4: Xerocomus chrysenteron
Restoring native forest ecosystems
123
d = 1
EcaU EcaUL
EcaULc
PpaU
PpaUL PpaULc
BaSU
BaSUL BaSULc
UboU
UboUL UboULc
pH
P
N
OM
FDA
AcP
AlkP
SB
RB
ER
PN
PP PC1
PC2
H
A
B
Fig. 2 Results of the PCA
on the data table of soil,
plant, and microbial activity
parameters. a Correlation
circle of all the parameters.
The 12 variables are:
pH = pH, P = total
phosphorus (mg kg-1),
N = total nitrogen (%),
OM = total organic matter
(%), FDA = total
enzymatic activity,
AcP = acid phosphatase,
AlkP = alkaline
phosphatase, SB = shoot
biomass (g), RB = root
biomass (g),
ER = ectomycorrhizal rate
(%), PN = leaf nitrogen
(%), PP = leaf phosphorus
(mg.kg-1), H = Shannon
diversity index of
ectomycorrhizal fungi.
b Map of sample scores on
the first two principal
components. Samples are
coded as follows. The first
three characters correspond
to the soil origin: Eca = soil
collected under E.camaldulensis, Ppa = soil
collected under P. palida,
Ubo = soil collected under
U. bojeri, BaS = bare soil.
The treatment applied to the
U. bojeri seedlings is coded
as folows. U = Uapacaplant alone, UL = Uapacaplant ? L. bojeriana,
Ulc = Uapaca plant ?
L. bojeriana cut after
4 months cultivation. For
example, sample coded
‘‘EcaULc’’ is a U. bojeriseedling grown in soil
collected under
E. camaldulensis in which a
plant of L. bojeriana was
grown and cut after 4 month
cultivation
R. Baohanta et al.
123
Leptolena plant was cut and only the root system was
left before planting U. bojeri seedlings. It was also
interesting to notice that this effect was the same for
bare soil, for soils collected under exotic tree species
or for soil collected under a Uapaca adult tree. On the
same graphic (Fig. 2b), the PC2 showed the soil origin
effect (dotted arrows pointing upward), corresponding
to the negative influence of exotic tree species
(E. camaldulensis, P. patula) on root biomass. Root
biomass was higher in soils collected under U. bojeri
adult tree and lower in soils collected under exotic tree
species. Bare soils have an intermediate position.
Conversely, pH and total organic matter are higher in
soils collected under exotic tree species.
For each soil origins, the impact of L. bojeriana
(with or without aerial parts) on soil characteristics,
U. bojeri growth, and ectomycorrhizal communities
was indicated in Tables 5, 6, and 7. For the bulk soil
origin and compared with the control, the treatment
with L. bojeriana without aerial parts provided the
highest positive effects on pH, soluble P, soil N
content, organic matter content and on microbial
enzymatic activities (Table 5). The dual cultivation of
L. bojeriana with or without aerial parts significantly
improved shoot and root biomass and mineral nutri-
tion of U. bojeri seedlings (N, P) (Table 6). Ectomy-
corrhizal colonization was significantly improved
when the dual cultivation was performed with
L. bojeriana without aerial parts (Table 6). Strong
modifications in the composition of ectomycorrhizal
communities occurred in the treatments with
L. bojeriana (Table 7). RFLP types, UC3 and UC2
recorded in the control treatment, were not found in
the dual cultivation treatments and replaced by the
RFLP types UA1, UA2, and UB4. The RFLP type
UB6 was only recorded in the treatment with entire
L. bojeriana seedlings (Table 7).
For the U. bojeri soil origin, dual cultivation with
entire L. bojeriana seedlings increased all the mea-
sured soil parameters except for pH (Table 5). Elim-
inating the aerial parts of L. bojeriana seedlings led to
higher increases of N, organic matter soil contents and
FDA activity but to a lower enhancement of soil
soluble P content (Table 5). Dual cultivation had
significantly improved plant nutrient (N and P) uptake
with highest data for the treatment without aerial parts
(Table 6). No significant effect has been found on root
growth and root/shoot ratio but shoot growth of
U. bojeri seedlings was significantly improved with
L. bojeriana without aerial parts. Ectomycorrhizal
colonization was significantly increased when U.
bojeri seedlings were cultivated with L. bojeriana
without aerial parts (Table 6). This positive impact
was also recorded on the composition of ectomycor-
rhizal communities with the same RFLP types (except
for UA3) as those found in the control treatment (UA1,
UA2 and UA4) and two others only detected with the
presence of L. bojeriana seedlings (Table 7).
With E. camaldulensis soil, dual cultivation treat-
ments significantly improved soil pH, nitrogen con-
tent, and enzymatic activities with highest effects
found in L. bojeriana seedlings without aerial parts for
soil nitrogen content and FDA activity (Table 5).
Opposite effects have been found for soil P content
and soil organic matter (depressive effect provided by
L. bojeriana seedlings without aerial parts). Dual
cultivation treatments have enhanced the growth of U.
bojeri seedlings and ectomycorrhizal colonization but
no significant differences have been found between
both L. bojeriana treatments (with or without aerial
parts) and no effects have been recorded on the root/
shoot values (Table 6). The presence of L. bojeriana
seedlings allowed the development of some RFLP
types not detected in the control treatment (UA1, UA2,
UA3, UA4), increased the establishment of UB6 but
limited UB4 multiplication (Table 7).
For the P. patula soil origin, dual cultivation
treatments significantly improved soil P content and
enzymatic activities, whereas the presence of entire
L. bojeriana seedlings significantly decreased soil
nitrogen and organic matter contents (Table 5).
U. bojeri shoot growth and leaf foliar contents (N, P)
have been significantly promoted by L. bojeriana
seedlings (entire or not) (Table 6), and ectomycorrhi-
zal colonization was higher in the dual cultivation
treatment involving L. bojeriana seedlings without
aerial parts (Table 6). Only UB6 RFLP type was
detected in all the treatments, whereas UC3 recorded
in the control treatment was absent in the dual
cultivation treatments (Table 7). An opposite pattern
was found with UA1 and UA4 RFLP types (Table 7).
Discussion
This study clearly shows that (1) the introduction of
exotic tree species induces significant changes in the
soil chemical characteristics, microbial activities and
Restoring native forest ecosystems
123
Ta
ble
5E
ffec
to
fL
.bo
jeri
an
a/U
.bo
jeri
succ
essi
on
(pre
-cu
ltiv
atio
nw
ith
L.b
oje
ria
na
and
du
alcu
ltiv
atio
nw
ith
L.b
oje
ria
na
seed
lin
gs
wit
hae
rial
par
tso
rw
ith
ou
tae
rial
par
ts)
on
soil
chem
ical
char
acte
rist
ics
and
enzy
mat
icac
tiv
itie
s
Tre
atm
ents
pH
H2O
So
lP
4T
ota
lN
5T
ota
lO
M6
FD
A7
Ac
P8
Alk
P9
Bu
lkso
il
Co
ntr
ol1
5.7
10
(0.0
1)
a11
2.0
0(0
.06
)a
0.0
22
(0.0
01
)a
4.2
0(0
.06
)a
32
.0(6
.4)
a4
98
.8(3
1.9
)a
27
4.6
(6.2
)a
L.
bo
jeri
an
a2
5.9
(0.0
1)
b4
.47
(0.0
9)
b0
.02
4(0
.00
1)
a6
.33
(0.0
4)
b4
6.9
(1.4
)ab
1,0
46
.4(5
2.1
)b
35
9.5
(11
3.7
)ab
L.
bo
jeri
an
aW
A3
6.2
(0.0
2)
c5
.50
(0.1
1)
c0
.10
3(0
.00
1)
b9
.65
(0.0
3)
c5
7.5
(3.6
)b
1,3
34
.5(8
2.6
)c
38
3.7
(22
.1)
b
U.
bo
jeri
soil
Co
ntr
ol
5.4
(0.0
1)
b5
.35
(0.0
3)
a0
.30
1(0
.00
1)
a7
.32
(0.0
1)
a5
.2(0
.36
)a
71
5.6
(19
.5)
a4
04
.1(1
1.6
)a
L.
bo
jeri
an
a5
.4(0
.01
)b
6.8
0(0
.06
)c
0.4
12
(0.0
01
)b
8.3
2(0
.04
)b
49
.8(3
.4)
b9
80
.7(2
3.4
)b
51
2.2
(22
.3)
b
L.
bo
jeri
an
aW
A5
.3(0
.02
)a
6.3
2(0
.06
)b
0.4
23
(0.0
01
)c
8.7
2(0
.07
)c
63
.5(2
.2)
c1
,04
4.3
(24
.9)
b5
82
.5(1
7.7
)b
E.
cam
ald
ule
nsi
sso
il
Co
ntr
ol1
5.3
(0.0
07
)a
9.2
3(0
.03
)c
0.0
54
(0.0
01
)a
15
.76
(0.0
9)
b6
.1(1
.6)
a1
,21
3.5
(19
.9)
a2
14
.3(5
.6)
a
L.
bo
jeri
an
a2
6.3
(0.0
09
)c
4.4
3(0
.09
)b
0.0
64
(0.0
02
)b
15
.80
(0.0
6)
b2
1.4
(3.1
)b
1,4
47
.2(4
8.1
)b
41
7.1
(26
.3)
b
L.
bo
jeri
an
aW
A3
5.4
(0.0
06
)b
3.7
0(0
.06
)a
0.0
71
(0.0
01
)c
14
.25
(0.0
3)
a6
8.3
(5.3
)c
1,5
97
.6(8
.3)
c3
94
.4(4
3.6
)b
P.
pa
tula
soil
Co
ntr
ol
6.2
(0.0
1)
a3
.36
(0.0
9)
a0
.08
7(0
.00
1)
b1
4.4
7(0
.09
)b
22
.2(3
.8)
a5
58
.3(5
5.2
)a
28
8.5
(4.5
)a
L.
bo
jeri
an
a6
.3(0
.01
)a
7.6
0(0
.11
)c
0.0
73
(0.0
01
)a
14
.05
(0.0
3)
a1
00
.4(8
.6)
c1
,25
7.1
(37
.5)
b5
67
.9(1
8.3
)c
L.
bo
jeri
an
aW
A6
.2(0
.01
)a
4.4
0(0
.06
)b
0.0
90
(0.0
01
)b
14
.68
(0.0
6)
b6
6.4
(4.4
)b
1,5
94
.9(4
9.3
)c
33
1.4
(14
.5)
b
1U
.b
oje
riw
ith
ou
tp
re-
and
du
alcu
ltiv
atio
nw
ith
L.
bo
jeri
an
a.
2P
re-c
ult
ivat
ion
wit
hL
.b
oje
ria
na
and
du
alcu
ltiv
atio
nw
ith
L.
bo
jeri
an
ase
edli
ng
sw
ith
aeri
alp
arts
.3
Pre
-
cult
ivat
ion
wit
hL
.b
oje
ria
na
and
du
alcu
ltiv
atio
nw
ith
L.
bo
jeri
an
ase
edli
ng
sw
ith
ou
tae
rial
par
ts.
4S
olu
ble
ph
osp
ho
rus
(mg
kg
-1).
5T
ota
ln
itro
gen
(%).
6T
ota
lo
rgan
icm
atte
r
(%).
7T
ota
lm
icro
bia
lac
tiv
ity
(lg
of
hy
dro
lyze
dF
DA
h-
1g
-1
of
soil
).8
Aci
dp
ho
sph
atas
eac
tiv
ity
(lg
p-n
itro
ph
eno
lg
-1
of
soil
h-
1).
9A
lkal
ine
ph
osp
hat
ase
acti
vit
y(l
gp-
nit
rop
hen
ol
g-
1o
fso
ilh
-1).
10
Sta
nd
ard
erro
ro
fth
em
ean
.11
Dat
ain
the
sam
eco
lum
nan
dfo
rea
chso
ilo
rig
info
llo
wed
by
the
sam
ele
tter
are
no
tsi
gn
ifica
ntl
yd
iffe
ren
t
acco
rdin
gto
the
New
man
–K
euls
test
(p\
0.0
5)
R. Baohanta et al.
123
on ectomycorrhizal communities, (2) exotic-invaded
soil significantly reduces the early growth and ecto-
mycorrhization of U. bojeri seedlings, and (3) ecto-
trophic early-successional shrub species such as
L. bojeriana could lower these negative effects
provided by E. camaldulensis and P. patula by
facilitating ectomycorrhizal establishment and conse-
quently improved the U. bojeri early growth.
Numerous studies have reported that the introduc-
tion of exotic tree species has an environmental impact
on soil characteristics (i.e., soil nutrient contents,
water dynamics, etc.) (Smith et al. 2000; Sicardi et al.
2004) but with opposite results on soil biofunctioning
indicators. For instance, Sicardi et al. (2004) reported
that the conversion of pasture land to planted Euca-
lyptus grandis forest decreased FDA hydrolysis, acid
and alkaline phosphatase activities that are directly
involved in the transformation of soil organic matter.
On the opposite, other studies have shown higher
availability of nitrogen in exotic-invaded soils
(Kourtev et al. 1999; Ehrenfeld et al. 2001). Our
results are in accordance with these previous studies
for soil N contents. However, we report higher rates of
acid phosphatase activity under exotic plant species
(P. patula and E. camaldulensis) that probably result
from the more acid conditions encountered under
these two exotic species and in contrast suppress
alkaline phosphatase activities (Acosta-Martinez and
Tabatai 2000; Kramer and Green 2000). These results
are in accordance with those of Kourtev et al. (2002) as
the higher rates of acid phosphatase reflected the
organic-rich horizons with large amounts of recalci-
trant compounds which accumulate under E. camal-
dulensis and P. patula.
All these biological changes have resulted to a
lowest early growth of U. bojeri seedlings and in
particular to a decrease of ectomycorrhiza formation.
A previous study suggested that Pinus spp. was enable
to associate with native fungi in exotic habitats leading
to unsuccessful establishment when ECM fungi are
lacking (Mikola 1970). It agrees with our data where
this tree species selected a few ectomycorrhizal
Table 6 Effect of L. bojeriana/U. bojeri succession (pre-culti-
vation with L. bojeriana and dual cultivation with L. bojerianaseedlings with aerial parts or without aerial parts) on the growth
and ectomycorrhizal colonization of U. bojeri seedlings in soils
collected under Uapaca bojeri, Eucalyptus camaldulensis, Pinuspatula and from a bulk soil after 5 month culture in glasshouse
conditions
Treatments SB4 RB5 RB:SB6 N7 P8 ECM9
Bulk soil
Control1 131 (11)10 a11 113 (12) a 0.88 (0.13) b 0.89 (0.06) a 71.1 (7.3) a 36 (2.1) a
L. bojeriana2 277 (11) b 140 (10) ab 0.51 (0.04) a 3.02 (0.12) b 253.4 (10.9) b 42 (6) a
L. bojeriana WA3 309 (26) b 166 (3) b 0.55 (0.04) ab 3.08 (0.27) b 332.1 (29.1) b 90.3 (3.2) b
U. bojeri soil
Control 125 (15) a 295 (35) a 2.37 (0.16) b 0.85 (0.1) a 94.1 (9.9) a 73.7 (3.2) a
L. bojeriana 222 (38) ab 242 (38) a 1.21 (0.33) a 2.14 (0.32) b 197.7 (34.1) b 78 (2.1) a
L. bojeriana WA 332 (19) b 219 (39) a 0.67 (0.14) a 3.58 (0.19) c 303.9 (14.1) c 90.7 (2.4) b
E. camaldulensis soil
Control1 83 (0.9) a 27 (4) a 0.34 (0.08) a 0.65 (0.07) a 62.3 (7.3) a 16.3 (2.4) a
L. bojeriana2 233 (41) b 99 (6) b 0.45 (0.09) a 2.30 (0.41) b 194.6 (35.5) b 65.3 (3.3) b
L. bojeriana WA3 250 (42) b 129 (12) b 0.57 (0.17) a 3.17 (0.57) b 268.6 (44.9) b 79.3 (4.1) b
P. patula soil
Control 85 (12) a 119 (10) a 1.42 (0.12) b 0.65 (0.09) a 58.9 (8.7) a 29.3 (5.5) a
L. bojeriana 233 (9) b 146 (27) a 0.62 (0.11) a 2.28 (0.10) b 181.3 (5.7) b 30.3 (2.4) a
L. bojeriana WA 333 (66) b 127 (7) a 0.41 (0.08) a 3.90 (0.78) b 278.1 (53.9) b 65.3 (1.5) b
1 U. bojeri without pre- and dual cultivation with L. bojeriana. 2 Pre-cultivation with L. bojeriana and dual cultivation with
L. bojeriana seedlings with aerial parts. 3 Pre-cultivation with L. bojeriana and dual cultivation with L. bojeriana seedlings without
aerial parts. 4 Shoot biomass (mg dry weight). 5 Root biomass (mg dry weight). 6 Root:shoot ratio. 7 N leaf mineral content (mg per
plant). 8 P leaf mineral content (mg per plant). 9 Ectomycorrhizal colonization (%). 10 Standard error of the mean. 11 Data in the
same column and for each soil origin followed by the same letter are not significantly different according to the Newman–Keuls test
(p \ 0.05)
Restoring native forest ecosystems
123
symbionts such as Russula exalbicans. This ectomy-
corrhizal genus was largely distributed in tropical
areas (Ducousso et al. 2004; Riviere et al. 2006;
Diedhiou et al. 2010) and frequently recorded under
tropical tree species (Riviere et al. 2005, 2006). In
contrast to pine, it has been suggested that Eucalyptus
spp. (i.e., E. robusta) was able to contract ectomycor-
rhizal associations in their introduction area with most
of the native ectomycorrhizal symbionts (Tedersoo
et al. 2007). Our results partially corroborated these
data since ectomycorrhizal community associated
with U. bojeri seedlings grown in soil collected under
E. camaldulensis was more diverse than that found in
soil sampled under P. patula. However, E. camaldul-
ensis has negatively influenced the ectomycorrhizal
establishment and consequently U. bojeri seedling
growth largely than that which has been measured
with P. patula soil. It is well known that Eucalyptus
drastically alters the vegetation development where
Eucalyptus litter accumulates through the release of
allelochemicals (del Moral and Muller 1970). Hence,
this allelopathic effect could limit the U. bojeri growth
seedling and in particular root system development
leading to a lower ectomycorrhiza establishment.
Uapaca bojeri seedlings growing in soil collected
under U. bojeri adult tree showed much higher
ectomycorrhizal infection and growth than those
growing in the soil collected at a distance from
established ectomycorrhizal vegetation. These data
are consistent with results of previous works where it
has been demonstrated that a lack of ectomycorrhizal
infection distant from ectomycorrhizal vegetation or
from adult tree that provides ectomycorrhizal propa-
gules to the young seedlings could influence nutrient
uptake and growth of seedling (Baxter and Dighton
2001; Lilleskov et al. 2002; Dickie and Reich 2005;
Kisa et al. 2007). Moreover, it has been suggested that
a rapid and early integration of seedlings into
ectomycorrhizal mycelium radiating from mother
plants could significantly improve survival and growth
Table 7 Relative abundance of RFLP types harvested in
U. bojeri seedlings in the cultural patterns with L. bojeriana(pre-cultivation with L. bojeriana and dual cultivation with
L. bojeriana seedlings with aerial parts or without aerial parts)
in soils collected under Uapaca bojeri, Eucalyptus camaldul-ensis, Pinus patula and from a bulk soil after 5 month culture
in glasshouse conditions
Treatments Relative abundance of RFLP types (%)
UA1 UD1 UA2 UA3 UC3 UA4 UB6 UC2 UB5 UB4
Bulk soil
Control1 0.0 0.0 0.0 0.0 89.4 0.0 0.0 3.1 7.5 0.0
L. bojeriana2 26.5 0.0 27.9 0.0 0.0 0.0 7.4 0.0 0.0 38.2
L. bojeriana WA3 19.3 0.0 49.5 0.0 0.0 0.0 0.0 0.0 0.0 31.2
U. bojeri soil
Control 51.5 0.0 43.0 3.7 0.0 1.8 0.0 0.0 0.0 0.0
L. bojeriana 13.0 0.0 18.5 0.0 19.6 19.6 29.3 0.0 0.0 0.0
L. bojeriana WA 14.7 0.0 16.7 0.0 11.8 22.5 34.3 0.0 0.0 0.0
E. camaldulensis soil
Control1 0.0 11.9 0.0 0.0 58.7 0.0 11.9 0.0 0.0 17.5
L. bojeriana2 23.8 0.0 0.0 20.6 0.0 12.8 42.8 0.0 0.0 0.0
L. bojeriana WA3 20.2 0.0 12.1 19.2 0.0 25.3 23.2 0.0 0.0 0.0
P. patula soil
Control 0.0 63.2 0.0 0.0 20.8 0.0 16.0 0.0 0.0 0.0
L. bojeriana 22.6 0.0 0.0 0.0 0.0 28.3 49.1 0.0 0.0 0.0
L. bojeriana WA 17.8 0.0 0.0 0.0 0.0 35.6 46.6 0.0 0.0 0.0
1 U. bojeri without pre- and dual cultivation with L. bojeriana. 2 Pre-cultivation with L. bojeriana and dual cultivation with
L. bojeriana seedlings with aerial parts. 3 Pre-cultivation with L. bojeriana and dual cultivation with L. bojeriana seedlings
without aerial parts. UA1: Russula earlei, UD1: Bondarcevomyces taxi, UA2: Amanita sp., UA3: Telephoroid mycorrhizal sp.,
UC3: Russula exalbicans, UA4: Uncultured ECM homobasidiomycete Clone E2, UB6: Boletellus projectellus, UC2: Boletusrubropunctus, UB5: Coltricia perennis, UB4: Xerocomus chrysenteron
R. Baohanta et al.
123
of seedlings (Janos 1980, 1996; Onguene and Kuyper
2002). Our data support these observations with
U. bojeri in a Madagascarian highland forest and
showed that this tree species acts as a mother tree or
nurse tree by promoting ectomycorrhizal formation and
seedling growth. High root/shoot ratio has been iden-
tified as an important factor allowing plants to exploit
reduced resource availability due to patchiness in
distribution, both for water and nutrients (Reader et al.
1992). These high ratios would be of great importance
in the regeneration process of native tree species
especially during periods of drought or where nutrient
resources are heterogeneously distributed. Hence, the
exotic tree species (P. patula and E. camaldulensis)
could limit the growth of U. bojeri young regeneration,
whereas the presence of U. bojeri mother tree facilitated
the early development of U. bojeri seedlings.
In tropical forests, one of the main biological
processes that ensure recovery rates of tree species
depends on the amount and activity of mycorrhizal
inoculum. Ectomycorrhizal mycelia radiating from
mother tree roots function as a source of ectomycorrhi-
zal infection for neighboring host plants and more
particularly for young tree regeneration (Jonsson et al.
1999; Matsuda and Hijii 2004; Nara 2005). In addition
plants could become connected to a common mycor-
rhizal network that could be highly beneficial for growth
and fitness of seedlings (Nara 2005). When ectomycor-
rhizal potential (abundance and diversity of ectomy-
corrhizal propagules) is lowered following natural or
anthropogenic disturbance (Allen 1987; Jones et al.
2003), seedling establishment is limited and it is
necessary to reinforce ectomycorrhizal infection poten-
tial. It has been previously demonstrated that a
herbaceous ectomycorrhizal perennial of prairies, He-
lianthemum bicknellii, could permit the survival of
ectomycorrhizal propagules and create patches of high
ectomycorrhizal infection potential that facilitate the
establishment of Quercus, an ectomycorrhizal tree
species (Dickie et al. 2004). From the present study,
similar effects have been provided by the ectomycor-
rhizal shrub species, L. bojeriana. Among ectotrophic
early-successional plants recorded in the studied area,
Leptolaena genus was highly represented and facilitated
ectomycorrhizal infection and growth of U. bojeri
seedlings but also enhanced soil chemical characteris-
tics and enzymatic activities. Since L. bojeriana shared
ectomycorrhizal fungi with U. bojeri (i.e. Russula
earlei, Amanita sp., etc.), this shrub species has
significantly enhanced ectomycorrhizal colonization
of U. bojeri seedlings. This nursing effect was more
particularly recorded in the treatments with exotic-
invaded soils. In the P. patula and E. camaldulensis
soils, L. bojeriana stimulated U. bojeri total growth by
2.39 and 3.49, respectively, whereas this positive
effect was 1.39 with the U. bojeri soil. This result
supports the hypothesis that facilitation generally
increasing in importance with increasing abiotic stress
(Liancourt et al. 2005). In addition N, P nutrient uptake
of U. bojeri seedlings was significantly enhanced in the
dual cultivation treatments. Foliar N and P contents
were significantly correlated with ectomycorrhizal
colonization. Hence, by facilitating ectomycorrhizal
propagule multiplication, L. bojeriana enhanced ecto-
mycorrhizal infection of U. bojeri that is known to
improve plant nutrient uptake (Dickie et al. 2002). In
addition, U. bojeri nutrition may benefit from the
ectomycorrhizal network radiating from L. bojeriana
root systems that explores a larger volume of soil than
U. bojeri alone. These connections could lead to N and P
or carbon transfers between U. bojeri and L. bojeriana
seedlings via mycorrhizal linkages (Simard et al. 1997).
No significant effect has been recorded between treat-
ments with entire L. bojeriana seedlings and L. bojeri-
ana seedlings without aerial parts. It suggests that no
competitive interactions occur between each plant
species. The association in a common mycelial network
of each plant species has probably lowered the cost of
establishing mycorrhizal infection (Newman 1988).
This study provides evidence that L. bojeriana can
facilitate the ectomycorrhizal infection of U. bojeri
and mitigates the negative effects of the introduction
of exotic tree species on the early growth and
ectomycorrhizal formation of the native tree species.
However, the mechanisms involved in this nursing
effect have to be elucidated since multiple abiotic and
biotic factors are involved. From a practical point of
view, the use of ectotrophic early-successional shrub
species has to be considered in tropical areas to
improve the performances of reafforestation programs
with native tree species.
References
Acosta-Martinez V, Tabatai MA (2000) Enzyme activities in a
limed agricultural soil. Biol Fert Soil 31:85–91
Agerer R (1987–1996) Colour atlas of ectomycorrhizae. Ein-
horn-Verlag Eduard Dietenberger, Schwabisch Gmund
Restoring native forest ecosystems
123
Agerer R (1995) Anatomical characteristics of identified ecto-
mycorrhizas: an attempt towards a natural classification.
In: Varma A, Hock B (eds) Mycorrhiza: structure, function,
molecular biology and biotechnology. Springer, Berlin,
pp 687–734
Alef K (1998) Estimation of the hydrolysis of fluorescein
diacetate. In: Alef K, Nannipieri P (eds) Methods in applied
soil microbiology and biochemistry. Academic Press,
London, pp 232–233
Allen MF (1987) Re-establishment of mycorrhizas on Mount
St Helens: migration vectors. Trans Br Mycol Soc 88:
413–417
Ashton IW, Hyatt LA, Howe KM, Gurevitch J, Lerdau MT
(2005) Invasive species accelerate decomposition and litter
nitrogen loss in a mixed deciduous forest. Ecol Appl
15:1263–1272
Aubert G (1978) Methodes d’Analyse des sols. Edition CRDP,
Marseille, p 360
Batten K, Scow K, Davies K, Harrison S (2006) Two invasive
plants alter soil microbial community composition in ser-
pentine grasslands. Biol Invasions 8:217–230
Baxter JW, Dighton J (2001) Ectomycorrhizal diversity alters
growth and nutrient acquisition of grey birch (Betulapopulifolia) seedlings in host-symbiont culture conditions.
New Phytol 152:139–149
Brundrett MC (1991) Mycorrhizas in natural ecosystems. In:
Macfayden A, Begon M, Fitter AH (eds) Advances in
ecological research, vol 21. Academic Press, London,
pp 171–313
Callaway RM, Pennings SC (2000) Facilitation may buffer
competitive effects: indirect and diffuse interactions
among salt marsh plants. Am Nat 156:416–424
Callaway RM, Ridenour WM (2004) Novel weapons: invasive
success and the evolution of increased competitive ability.
Front Ecol Environ 2:436–443
del Moral R, Muller CH (1970) The allelopathic effects of
Eucalyptus camaldulensis. Am Midl Nat 83:254–282
Dickie IA, Reich PB (2005) Ectomycorrhizal fungal commu-
nities at forest edges. J Ecol 93:244–255
Dickie IA, Koide RT, Steiner KC (2002) Influence of estab-
lished trees on mycorrhizas, growth and nutrition of
Quercus rubra seedlings. Ecol Monogr 72:505–521
Dickie IA, Guza RC, Krasewski SE, Reich PB (2004) Shared
ectomycorrhizal fungi between a herbaceous perennial
(Helianthemum bicknellii) and oak (Quercus) seedlings.
New Phytol 164:375–382
Diedhiou AG, Selosse M-A, Galiana A, Diabate M, Dreyfus B,
Ba AM, de Faria SM, Bena G (2010) Multi-host ectomy-
corrhizal fungi are predominant in a Guinean tropical
rainforest and shared between canopy trees and seedlings.
Environ Microbiol 12:2219–2232
Dray S, Dufour AB (2007) The ade4 package: implementing the
duality diagram for ecologists. J Stat Soft 22:1–20
Ducousso M, Bourgeois C, Buyck B, Eyssartier G, Vincelette
M, Rabevohitra R, Bena G, Randrihasipara L, Dreyfus B,
Prin Y (2004) The last common ancestor of Sarcolaenaceae
and Asian dipterocarp trees was ectomycorrhizal before the
India-Madagascar separation, about 88 million years ago.
Mol Ecol 13:231–236
Duponnois R, Plenchette C, Prin Y, Ducousso M, Kisa M, BaAM, Galiana A (2007) Use of mycorrhizal inoculation to
improve reafforestation process with Australian Acacia in
Sahelian ecozones. Ecol Eng 29:105–112
Ehrenfeld JG, Kourtev PS, Huang WS (2001) Changes in soil
functions following invasions of exotic understory plants in
deciduous forests. Ecol Appl 11:1287–1300
Faye A, Krasova-Wade T, Thiao M, Thioulouse J, Neyra M,
Prin Y, Galiana A, Ndoye I, Dreyfus B, Duponnois R
(2009) Controlled ectomycorrhization of an exotic legume
tree species Acacia holosericea affects the structure of root
nodule bacteria community and their symbiotic effective-
ness on Faidherbia albida, a native Sahelian Acacia. Soil
Biol Biochem 41:1245–1252
Franco AC, Nobel PS (1988) Interactions between seedlings of
Agave deserti and the nurse plant Hilaria rigida. Ecology
69:1731–1740
Gade DW (1996) Deforestation and its effects in highland
Madagascar. Mt Res Dev 16:101–116
Hawkes CV, Wren IF, Herman DJ, Firestone MK (2005) Plant
invasion alters nitrogen cycling by modifying the soil
nitrifying community. Ecol Lett 8:976–985
Janos DP (1980) Vesicular-arbuscular mycorrhizae affect
lowland tropical rain forest plant growth. Ecology 61:
151–162
Janos DP (1996) Mycorrhizas, succession and rehabilitation of
deforested lands in the humid tropics. In: Frankland JC,
Magan N, Gadd GM (eds) Fungi and environment change.
Cambridge University Press, Cambridge, pp 129–161
Johansson JF, Paul LR, Finlay RD (2004) Microbial inter-
actions in the mycorrhizosphere and their significance
for sustainable agriculture. FEMS Microbiol Ecol 48:
1–13
John MK (1970) Colorimetric determination in soil and plant
material with ascorbic acid. Soil Sci 68:171–177
Jones MD, Durall DM, Cairney JWG (2003) Ectomycorrhizal
fungal communities in young forest stands regenerating
after clearcut logging. New Phytol 157:399–422
Jonsson L, Dahlberg A, Nilsson MC, Karen O, Zackrisson O
(1999) Continuity of ectomycorrhizal fungi in self-regen-
erating boreal Pinus sylvestris forests studied by comparing
mycobiont diversity on seedlings and mature trees. New
Phytol 142:151–162
Kisa M, Sanon A, Thioulouse J, Assigbetse K, Sylla S, Spichiger
R, Dieng L, Berthelin J, Prin Y, Galiana A, Lepage M,
Duponnois R (2007) Arbuscular mycorrhizal symbiosis
can counterbalance the negative influence of the exotic tree
species Eucalyptus camaldulensis on the structure and
functioning of soil microbial communities in a Sahelian
soil. FEMS Microbiol Ecol 62:32–44
Kivlin SN, Hawkes CV (2011) Differentiating between effects
of invasion and diversity: impacts of aboveground plant
communities on belowground fungal communities. New
Phytol 189:526–535
Kourtev PS, Huang WZ, Ehrenfeld JG (1999) Differences in
earthworm densities and nitrogen dynamics in soils under
exotic and native plant species. Biol Invasions 1:237–245
Kourtev PS, Ehrenfeld JG, Haggblom M (2002) Exotic plant
species alter the microbial community structure and func-
tion in the soil. Ecology 83:3152–3166
Kramer S, Green DM (2000) Acid and alkaline phosphatase
dynamics and their relationship to soil microclimate in a
semiarid woodland. Soil Biol Biochem 32:179–188
R. Baohanta et al.
123
Liancourt P, Callaway RM, Michalet R (2005) Stress tolerance
and competitive-response ability determine the outcome of
biotic interactions. Ecology 86:1611–1618
Lilleskov EA, Hobbie EA, Fahey TJ (2002) Ecto- mycorrhizal
fungal taxa differing in response to nitrogen deposition also
differ in pure culture organic nitrogen use and natural
abundance of nitrogen isotopes. New Phytol 154:219–231
Marx DH (1991) The practical significance of ectomycorrhizae
in forest establishment. Ecophysiology of ectomycorrhiz-
aeof forest trees, Marcus Wallenberg Foundation Sympo-
sia proceedings 7:54–90
Matsuda Y, Hijii N (2004) Ectomycorrhizal fungal communities
in an Abies firma forest, with special reference to ecto-
mycorrhizal associations between seedlings and mature
trees. Can J Bot 82:822–829
Mikola P (1970) Mycorrhizal inoculation in afforestation. Int
Rev For Res 3:123–196
Nara K (2005) Ectomycorrhizal networks and seedling estab-
lishment during early primary succession. New Phytol
169:169–178
Newman EI (1988) Mycorrhizal links between plants: their
functioning and ecological significance. Adv Ecol Res
18:243–270
Nicholas KB, Nicholas HB (1997) Genedoc: a toll for editing
and annotating multiple sequence alignments. Distributed
by the authors
Niering WA, Whittaker RH, Lowe CH (1963) The saguaro: a
population in relation to environment. Science 142:15–23
Olsen SR, Cole CV, Watanabe FS, Dean LA (1954) Estimation
of available phosphorus in soils by extraction with sodium
bicarbonate. Circular, vol 939. US Department of Agri-
culture, Washington, DC, p 19
Onguene NA, Kuyper TW (2002) Importance of the ectomy-
corrhizal network for seedling survival and ectomycorrhiza
formation in rain forests of south Cameroon. Mycorrhiza
12:13–17
Ouahmane L, Hafidi M, Kisa M, Boumezzough A, Thioulouse J,
Plenchette C, Duponnois R (2006) Some Mediterranean
plant species (Lavandula spp. and Thymus satureioides) act
as ‘‘plant nurses’’ for the early growth of Cupressus at-lantica. Plant Ecol 185:123–134
Parrot A (1925) Le reboisement de Madagascar par le moyen
des forets de ‘‘fokon olona’’. Bulletin Economique (An-
tananarivo) 1–2:55–57
Phillips JM, Hayman DS (1970) Improved procedures for
clearing and staining parasitic and vesicular-arbuscular
mycorrhizal fungi for rapid assessment of infection. Trans
Br Mycol Soc 55:158–160
R Development Core Team (2010) R: a language and environ-
ment for statistical computing. R Foundation for Statistical
Computing, Vienna, Austria. ISBN: 3-900051-07-0.
http://www.R-project.org/
Reader RJ, Jalili A, Grime JP, Spencer RE, Matthews N (1992)
A comparative study of plasticity in seedling rooting depth
in drying soil. J Ecol 81:543–550
Rejmanek M (2000) Invasive plants: approaches and predic-
tions. Aust Ecol 25:497–506
Remigi P, Faye A, Kane A, Deruaz M, Thioulouse J, Cissoko M,
Prin Y, Galiana A, Dreyfus B, Duponnois R (2008) The
exotic legume tree species Acacia holosericea alters
microbial soil functionalities and the structure of the ar-
buscular mycorrhizal community. Appl Environ Microb
74:1485–1493
Riviere T, Natarajan K, Dreyfus B (2005) Spatial distribution of
ectomycorrhizal Basidiomycete Russula subsect. Foeten-
tinae populations in a primary dipterocarp rainforest.
Mycorrhiza 16:143–148
Riviere T, Diedhiou AG, Diabate M, Senthilarasu G, Natarajan
K, Ducousso M, Verbeken A, Buyck B, Dreyfus B, Bena G,
Ba AM (2006) Diversity of ectomycorrhizal Basidiomy-
cetes in West African and Indian tropical rain forests.
Mycorrhiza 17:415–428
Scarano FR (2002) Structure, function and floristic relationships
of plant communities in stressful habitats marginal to the
Brazilian Atlantic Rainforest. Ann Bot 90:517–524
Schinner F, Ohlinger R, Kandeler E, Margesin R (1996)
Methods in soil biology. Springer, Berlin
Schreiner RP, Mihara KL, Mc Danield H, Benthlenfalvay GJ
(2003) Mycorrhizal fungi influence plant and soil functions
and interactions. Plant Soil 188:199–209
Sicardi M, Garcia-Prechac F, Frioni L (2004) Soil microbial
indicators sensitive to land use conversion from pastures to
commercial Eucalyptus grandis (Hill ex Maiden) planta-
tions in Uruguay. Appl Soil Ecol 27:125–133
Simard SW, Perry DA, Jones MD, Myrold DD, Durall DM,
Molina R (1997) Netb transfer of carbon between ecto-
mycorrhizal tree species in the field. Nature 388:579–582
Smith S, Read J (2008) Mycorrhizal symbiosis, 3rd edn. Aca-
demic Press, London, p 800
Smith OH, Petersen GW, Needelman BA (2000) Environmental
indicators of agroecosystems. Adv Agron 69:75–97
Styger E, Rakotondramasy HM, Pfeffer MJ, Fernandes ECM,
Bates DM (2007) Influence of slash-and-burn farming
practices on fallow succession and land degradation in the
rainforest region of Madagascar. Agric Ecosyst Environ
119:257–269
Tedersoo L, Suvi T, Beaver K, Koljalg U (2007) Ectomycor-
rhizal fungi of the Seychelles: diversity patterns and host
shifts from the native Vateriopsis seychellarum (Diptero-
carpaceae) and Intsia bijuga (Caesalpiniaceae) to the
introduced Eucalyptus robusta (Myrtaceae), but not Pinuscaribaea. New Phytol 175:321–333
Terwilliger J, Pastor J (1999) Small mammals, ectomycorrhizae,
and conifer succession in beaver meadows. Oikos
85:83–94
Thebaud C, Simberloff D (2001) Are plants really larger in their
introduced ranges? Am Nat 157:231–236
Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins
DG (1997) The ClustalX windows interface: flexible
strategies for multiple sequence alignment aided by quality
analysis tools. Nucl Acids Res 24:4876–4882
White TJ, Burns T, Lee S, Taylor J (1990) Amplification and
direct sequencing of fungal ribosomal RNA genes for
phylogenetics. In: Innis MA, Gelfand DH, Sninsky JJ,
White TJ (eds) PCR protocols: a guide to methods and
applications. Academic Press, San Diego, pp 315–322
Restoring native forest ecosystems
123
