Productions Scientifiques

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

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 1

- 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 2

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

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

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

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 6

(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

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.

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

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

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)

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

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.

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

SYNTHESE DES ENSEIGNEMENTS DISPENSES, DES PROJETS de recherche MENES ET PRODUCTIONS SCIENTIFIQUES

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.

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

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) 18

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.

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

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

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

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

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,

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.

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 termite mounds enhance the ectomycorrhizal symbiosis between Acacia holosericea A.Cunn. Ex G. Don and Scleroderma dictyosporum isolates Robin Duponnois1, Komi Assikbetse2, Heriniaina Ramanankierana3, Marija Kisa1, Jean Thioulouse4 & Michel Lepage5,6

1Institut de Recherche pour le Developpement,´ Laboratoire des Symbioses Tropicales et Mediterran´ eennes,´ 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 Abstract de Recherche pour le Developpement,´ UMR 113 CIRAD/INRA/IRD/AGRO-M/UM2, The hypothesis of the present study was that the termite mounds of Macrotermes Laboratoire des Symbioses Tropicales subhyalinus (MS) (a litter–forager termite) were inhabited by a specific microflora et Mediterran´ eennes´ (LSTM), 34398 that could enhance with the ectomycorrhizal fungal development. We tested the Montpellier, France. Tel.: (33) (0)4 67 59 38 effect of this feeding group mound material on (i) the ectomycorrhization 82; fax: (33) (0)4 67 59 38 02; symbiosis between Acacia holosericea (an Australian Acacia introduced in the e-mail: [email protected] sahelian areas) and two ectomycorrhizal fungal isolates of Scleroderma dictyosporum (IR408 and IR412) in greenhouse conditions, (ii) the functional diversity of Received 31 August 2005; revised 14 October soil microflora and (iii) the diversity of fluorescent pseudomonads. The results 2005; accepted 17 October 2005. First published online 8 February 2006. showed that the termite mound amendment significantly increased the ectomycorrhizal expansion. MS mound amendment and ectomycorrhizal inoculation doi:10.1111/j.1574-6941.2006.00089.x induced strong modifications of the soil functional microbial diversity by promoting the multiplication of carboxylic acid catabolizing microorganisms. Editor: Ralf Conrad The phylogenetic analysis showed that fluorescent pseudomonads mostly belong to the Pseudomonads monteillii species. One of these, P. monteillii isolate KR9, Keywords increased the ectomycorrhizal development between S. dictyosporum IR412 and termitaria; fluorescent pseudomonads; A. holosericea. The occurrence of MS termite mounds could be involved in the ectomycorrhizal symbiosis; Acacia holosericea. expansion of ectomycorrhizal symbiosis and could be implicated in nutrient flow and local diversity.

Introduction growth, the hyphae that grow outwards from the mycorrhizae into the surrounding soil interact with other soil microorgan- In recent decades, there has been increasing evidence that isms and constitute an important pathway for the transloca- soil microorganisms have an important effect on soil fertility tion of energy-rich plant compounds to the soil. The and plant health (Gianinazzi & Schuepp,¨ 1994). Amongst expanding mycorrhizal mycelium exploits a larger volume of the microbial populations living in the rhizosphere, myco- soil that would otherwise be inaccessible to plant roots. As rrhizal fungi have been found to be essential components of mycorrhizal symbiosis modifies the microbial communities of sustainable soil–plant systems (Amato & Ladd, 1988; Beth- its surrounding soil through changes in root exudation, this lenfalvay & Linderman, 1992; Hooker & Black, 1995; Van microbial compartment is usually named the ‘mycorrhizo- der Hejden et al., 1998; Hart et al., 2003; Dickie & Reich, sphere’ (Linderman, 1988), rather than the rhizosphere. The 2005). Over 80% of all land plants form some type of mycorrhizosphere includes the more specific term ‘hypho- symbiotic association with mycorrhizal fungi. By increasing sphere’, which refers only to the zone surrounding individual the absorptive surface area of their host plant, this fungal hyphae. Numerous studies have described the effect of the symbiosis influences plant growth and the uptake of nu- mycorrhizosphere on bacterial communities, such as fluores- trients, particularly phosphorus, a highly immobile element cent pseudomonads (Frey et al., 1997; Founoune et al., 2002a) in the soil, which thus frequently limits plant growth in or rhizobia (Duponnois & Plenchette, 2003). However, some tropical areas. In addition to this positive effect on plant bacteria belonging to the mycorrhizosphere compartment may

c 2006 Federation of European Microbiological Societies FEMS Microbiol Ecol 56 (2006) 292–303 Published by Blackwell Publishing Ltd. No claim to original French government works Termite mounds enhance ectomycorrhizal symbiosis 293 promote the development of mycorrhizal symbiosis (Garbaye, Table 1. Biological and chemical characteristics of Macrotermes sub- 1994). These bacterial strains have been named mycorrhiza hyalinus mound powder helper bacteria (MHB), and the MHB effect has been recorded Biological and chemical characteristics M. subhyalinus in different plant–fungus combinations (Dunstan et al., 1998; 1 1 NH4 (mgNg of dry mound powder) 9.4 1 Founoune et al., 2002b; Duponnois & Plenchette, 2003). NO3 (mgNg of dry mound powder) 3408.9 Mycorrhizal establishment usually depends on the plant Available P (mgg1 of dry mound powder) 3.5 species, soil type, soil phosphorus and mycorrhizal fungal Microbial biomass (mgCg1 of dry mound powder) 22.5 species (Smith & Read, 1997). The mycorrhizosphere effect Fluorescent pseudomonads 79.3 2 1 will therefore be influenced by soil disturbance (grazing or (10 CFU g of dry mound powder) Actinomycetes ( 102 CFU g1 of dry mound powder) 39.5 erosion) and by the impact of natural events in ecosystem Ergosterol (mgg1 of dry mound powder) 0.316 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, (about 5 kg each) were crushed and passed through a 2 mm modulate the availability of resources for other species, such sieve before use. as microorganisms and plants (Lavelle, 1997). For example, The chemical and microbiological analyses have been fruit bodies of the ectomycorrhizal fungus Scleroderma spp. described in a previous study (Table 1) (Duponnois et al., 1 are regularly observed around the termite mounds of 2005). The NH4 and NO3 contents were measured Macrotermes subhyalinus (a litter–forager termite) in the according to the method of Bremner, 1965, whereas avail- south of Burkina Faso (K. Sanon, pers. commun.) and able phosphorus was determined according to Olsen et al. Australia (Spain et al., 2004). In order to explain this (1954). The content of ergosterol was determined using the positive effect of the termite mound on fungal fructification, method of Grant & West (1986). The fumigation–extraction we hypothesized that the epigeal mound material was method was used to estimate the microbial biomass (Amato inhabited by a specific microflora that enhanced ectomyco- & Ladd, 1988). The enumeration of colony-forming units rrhizal fungal development. was carried out on King’s B agar medium for the fluorescent In order to verify this hypothesis, we tested the effect of pseudomonads (King et al., 1954) and on actinomycete the mound material of this feeding group on the ectomyco- isolation agar medium (Difco Laboratories, Detroit, MI) rrhizal symbiosis between Acacia holosericea (an Australian for the actinomycetes. The isolates of fluorescent pseudo- Acacia introduced in sahelian areas) and two ectomyco- monads were randomly selected (18 bacterial strains), rrhizal fungal isolates of Scleroderma dictyosporum (isolates purified, subcultured on King’s B medium and cryopre- IR408 and IR412), which are known to form ectomyco- served at 80 1C in glycerol 60%-TSB (tryptic soy broth, rrhizae with A. holosericea seedlings in pot experiments. The 3gL1) culture [1/1, volume in volume (v/v)]. influence of mound material amendment on the functional diversity of soil microflora was also assessed. As it has been Molecular characterization of fluorescent demonstrated previously that most MHB belong to the pseudomonad isolates fluorescent pseudomonad group (Frey-Klett et al., 1997), and that termite mounds of M. subhyalinus are inhabited by Fluorescent pseudomonads were grown overnight on TSB this bacterial genus (Duponnois et al., 2005), we investigated agar plates at 28 1C. For each strain, a single colony was their diversity and their effect on IR412 ectomycorrhizal picked up and suspended in 50 mL of lysis buffer [0.05 M establishment. 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/ Materials and methods v) with sterile distilled water. The primers rD1 (50-AAGCT- TAAGGAGGTGATCCAGCC-30) and fD1 (50-AGAGTTT- Chemical and microbiological analysis of the GATCCTGGCTCAG-30) were used to amplify the 16S sampled epigeal mounds rDNA gene (Frey-Klett et al., 1997). PCR was performed in Five termite mounds of Macrotermes subhyalinus were a GeneAmp PCR System 2400 thermal cycler (Perking- collected in a shrubby savanna, 50 km north of Ouagadou- Elmer, Foster City, CA) using PureTaq Ready-To-Go PCR gou, near the village of Yaktenga (Burkina Faso). The soil beads (Amersham Biosciences, Orsay, France), 1 mM of each was shallow and rich in gravel above the hardpan level. Large primer and 3 mL of bacterial cell suspension in 25 mL hydromorphic spots intertwined with the deepest soils reaction mixtures. The mixture was submitted to 5 min of characterized the landscape. Macrotermes mounds were initial denaturation, followed by 35 cycles at 94 1C for 1 min, predominantly localized on deeper soils. Termite mounds 55 1C for 45 s and 72 1C for 1.5 min. A final elongation step

FEMS Microbiol Ecol 56 (2006) 292–303 c 2006 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. No claim to original French government works 294 R. Duponnois et al. was performed at 72 1C for 5 min. PCR products (7 mL) were (120 1C, 20 min). This substrate was moistened to field digested in a total volume of 20 mLat371C for 2 h using capacity with 300 mL of liquid MMN medium. The jars 10 U of the endonucleases HaeIII and MspI (Promega, were sealed with a cotton float and autoclaved (120 1C, Charbonnie`res, France), as described by the manufacturer. 20 min). Finally, 10 fungal plugs were placed aseptically into Restriction fragments were separated by horizontal electro- each glass jar and incubated for 6 weeks at 28 1C in the dark. phoresis in a 2.5% (weight in volume, w/v) Metaphor gel One strain of fluorescent pseudomonad (Pseudomonas sp. (FMC, Rockland, ME). After 2 h of running at 80 V, the gel KR9) was randomly chosen from the selected bacterial was stained for 30 min with ethidium bromide (1 mg L1) isolates. It was grown in 0.3% TSB (Difco Laboratories) and integrated with the Image Analysis software BIOCAPT liquid medium for 3 days at 28 1C on a rotary shaker, (Vilbert Lourmat, Paris, France) under UV light. centrifuged (2400 g, 20 min) and suspended in 0.1 M

The amplified DNA fragments were purified using a MgSO4. The final concentration of the bacterial suspension Qiaquick PCR purification kit (Qiagen, Courtaboeuf, France), was about 108 CFU mL1, estimated by enumeration on a and then ligated into the pGEM-T vector and transformed into plate count agar medium (King’s B medium) (King et al., cells (Escherichia coli DH5a) according to the instructions of 1954). This suspension was used as inoculum. the manufacturer (pGEM-T easy vector system, Promega). PCR amplification with the primers T7 and Sp6 was per- Effect of the mound powder on IR408 and IR412 formed directly from the selected white colonies (presumed ectomycorrhizal development transformants) to confirm the presence of the insert of the correct size. The plasmid insert from a clone representing each Seeds of Acacia holosericea, originating in Ndioum/Podor isolate was sent for sequencing (Genome Express, Montreuil, (Senegal), were surface sterilized with concentrated 18 M France).Thesequencedatawerecomparedwithgenelibraries sulphuric acid for 60 min. The acid solution was decanted [GenBank and European Molecular Biology Laboratory off and the seeds were washed for 12 h in four rinses of (EMBL)] using BLAST (Heinemeyer et al., 1989) and FASTA sterile distilled water. The seeds were then transferred (Pearson & Lipman, 1988) programs. aseptically to Petri dishes filled with 1% (w/v) agar–water Twenty-eight different Pseudomonas species were re- medium. These plates were incubated at 25 1C in the dark. trieved from the Ribosome Database Project (RDP) (http:// The germinating seeds were used when the rootlets were www.cme.m-su.edu/RDP) for phylogenetic comparison 1–2 cm in length. with our Pseudomonas isolates. The phylogenetic analysis Acacia holosericea seedlings were grown in 1 L pots filled was performed using the MEGA (Molecular Evolutionary with soil collected from a millet field near Ouagadougou Genetics Analysis) version 2.1 package (Kumar et al., 2001). (Burkina Faso). Before use, the soil was crushed, passed Multiple sequence alignments were carried out using the through a 2 mm sieve and autoclaved for 40 min at 140 1C. CLUSTALW program (Thompson et al., 1994). Phylogenetic One week after autoclaving, its chemical and physical analysis was performed by the neighbour-joining method, characteristics were as follows: pH (H2O) 5.6; clay, 4.6%; and the relative support for groups was determined on the fine silt, 0.0%; coarse silt, 0.8%; fine sand, 25.5%; coarse basis of 1000 bootstrap trees. sand, 69.1%; carbon, 0.204%; nitrogen, 0.04%; carbon/ The nucleotide sequence obtained in this study has been nitrogen, 5.2; soluble phosphorus, 0.0043 mg g1; total deposited in the GenBank database and assigned Accession phosphorus, 0.116 mg g1. The soil was mixed with 10% number AY327816. (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 Glasshouse experiment treatments: control (C), fungal isolate inoculation (IR408 and IR412), termite mound amendment (MS) and fungal Fungal and bacterial inoculum inoculum and termite mound added together to the soil The ectomycorrhizal fungi, strains IR408 and IR412, have (IR4081MS and IR412 1 MS). The plants were placed in a been identified as Scleroderma dictyosporum on the basis of glasshouse (25 1C by day, 15 1C by night, 10 h photoperiod) rDNA internal transcribed spacer phylogeny (Sanon, 1999). and watered regularly with tap water without the addition of They were isolated from sporocarps under Uapaca guineen- fertilizer. They were arranged in a randomized complete sis in the province of Houet (Burkina Faso). The fungal block design with eight replicates per treatment. isolates were maintained on modified Melin–Norkrans After 4 months of culture, the plants were collected and (MMN) agar (Marx, 1969) at 25 1C. The ectomycorrhizal their root systems were gently washed under running tap fungal inoculum was prepared according to Duponnois & water. The oven dry weight (1 week at 65 1C) of the shoot Garbaye (1991). Glass jars were filled with 600 mL of a was measured. Some nodules were detected along the root vermiculite–peat moss mixture (4/1, v/v) and autoclaved systems despite disinfection of the soil and the seed surface.

The root nodules were counted. The root systems were cut Table 2. Organic compounds and their concentrations used to assess into 1 cm root pieces and mixed. The percentage of ectomy- patterns of ISCP of soil treatments corrhizal colonization [(number of ectomycorrhizal short Organic substrates Organic substrates roots/total number of short roots) 100] was determined Amino acids (15 mM) Carboxylic acids (100 mM) under a stereomicroscope at 40 magnification on a L-Glutamine 2-Keto-glutaric acid random sample of at least 100 short roots per root system. L-Arginine 3-Hydroxybutyric acid The arbuscular mycorrhizal fungal colonization was assessed L-Serine Ascorbic acid after clearing and staining the root pieces according to the L-Histidine D-quinic acid method of Phillips & Hayman (1970). The root pieces were Phenylalanine DL-malic acid L-Asparagine Formic acid placed on a slide for microscopic observations at 250 L-Tyrosine Fumaric acid magnification (Brundrett et al., 1985). About 50 1 cm root L-Glutamic acid Gallic acid pieces were observed per plant. Arbuscular mycorrhizal L-Lysine Gluconic acid colonization was expressed in terms of the fraction of the L-Cystein Ketobutyric acid root length with mycorrhizal internal structures (vesicles or Malonic acid hyphae): [(length of colonized root fragments/total length Carbohydrates (75 mM) Oxalic acid of root fragments) 100]. The dry weight (65 1C, 1 week) of D-Glucose Succinic acid D-Mannose Tartaric acid the roots was then determined. Sucrose Tri-sodium citrate 1 The soil from each pot was mixed and kept at 4 C for the Uric acid assessment of the catabolic diversity of microbial commu- Amides (15 mM) nities. D-Glucosamine Polymer (100 mM) N-methyl-D-Glucamine Cyclohexane Succinamide Assessment of the catabolic diversity of microbial ICSP, in situ catabolic potential. communities The microbial functional diversity in soil treatments was assessed by the determination of the in situ catabolic as used in the previous glasshouse experiment. The substrate potential patterns of microbial communities (Degens & was mixed with 10% (v/v) IR412 fungal inoculum or with a Harris, 1997). A range of amino acids, carbohydrates, 10% vermiculite–peat mixture (4/1, v/v) for treatments organic acids, amides and a polymer were screened for without fungus. Immediately after planting, the young differences in substrate-induced respiration (SIR) respon- seedlings from the experimental group were inoculated with 8 siveness between soil treatments (Table 2). The substrate 5 mL of fluorescent pseudomonad KR9 suspension (10 concentrations providing optimum SIR responses are in- bacterial cells), whereas those from the control group were dicated in Table 2 (Degens & Harris, 1997). Four replicates inoculated with 5 mL of 0.1 M MgSO4 solution. The plants (soil samples) were randomly chosen from each treatment. were placed in a glasshouse (25 1C by day, 15 1C by night, One gram equivalent of dry weight soil for each sample was 10-h photoperiod) and watered regularly with tap water suspended in 2 mL of sterile distilled water in 10 mL bottles without the addition of fertilizer. The pots were arranged in a randomized complete block design with eight replicates (West & Sparling, 1986). CO2 production from basal respiratory activity in the soil samples was also determined per treatment. by adding 2 mL of sterile distilled water to 1 g equivalent of After 4 months of culture, the shoot and root biomass, dry weight soil. The bottles were immediately closed and the number of nodules and the percentage of ectomycor- kept at 28 1C for 4 h after the addition of the substrate rhizal colonization were determined for each plant in each treatment, as described above. 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 Statistical analysis meter (Heinemeyer et al., 1989). Results were expressed as The data were treated with one-way analysis of variance. micrograms of CO2 per gram of soil per hour. Means were compared using Fisher’s protected least significant difference test (P o 0.05). The percentages of myco- Effect of the fluorescent pseudomonad isolate rrhizal colonization were transformed by arcsin (sqrt) before KR9 on IR412 ectomycorrhizal development statistical analysis. Co-inertia analysis was performed for Seeds of A. holosericea were prepared as described above; A. plant growth, mycorrhizal colonization indices and SIR holosericea seedlings were individually grown in 1 L pots responses. Co-inertia analysis (Chessel & Mercier, 1993; filled with the same autoclaved sandy soil (140 1C, 40 min) Doledec´ & Chessel, 1994) is a multivariate analysis

(a) M 1 2 3 4 5 6 7 8 9 10 11 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- 700 sion no. AY032725), P. mosselii (Accession no. AF072688), P. putida (Accession no. AB029257), P. plecoglossicida (Ac- cession no. AB09457) and P. monteillii (Accession no. 200 100 AF064458). Phylogenetic analysis with other selected Pseudomonas species from RDP was performed with the neighbour-join- (b) M1234567891011 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).

800 Effect of termite mound amendment on IR408 500 and IR412 ectomycorrhiza formation After 4 months of culture, the shoot growth of Acacia 100 holosericea seedlings was significantly stimulated by both ectomycorrhizal fungal strains in comparison with the Fig. 1. Gel electrophoresis of PCR-amplified 16S rDNA fragments of noninoculated treatment (control) (Table 3). The termite fluorescent pseudomonad isolates digested with HaeIII (a) and MspI (b). mound amendment also significantly improved the shoot Lanes 1–11: fluorescent pseudomonads isolated from termite mounds of biomass (Table 3). No significant differences were recorded Macrotermes subhyalinus. Lane M, 100 bp molecular size ladder. between the M. subhyalinus treatment and the termite mound amendment/ectomycorrhizal fungal inoculation (Table 3). Compared with the control, Scleroderma dictyos- technique that describes the relationships between two data porum IR408 significantly enhanced the root growth of tables. Numerous methods have been suggested for this [e.g. A. holosericea seedlings, whereas no significant effects were canonical analysis (Gittins, 1985), canonical correspondence recorded with S. dictyosporum IR412 (Table 3). In the soil analysis (Ter Braak, 1986) and partial least squares regres- amended with the termite mound, the root growth was sion Hoskuldsson,¨ 1988], but one of the simplest, from a significantly higher than that recorded in the control (Table theoretical point of view, is co-inertia analysis. Computa- 3). This termite mound effect was significantly enhanced tions and graphical displays were prepared with free ADE-4 when ectomycorrhizal fungi were inoculated (Table 3). No software (Thioulouse et al., 1997), available at http://pbil. significant differences were recorded between the treatments univ-lyon1.fr/ADE-4/. with regard to the total number of nodules per plant. The arbuscular mycorrhizal colonization indices were very low Results and not significantly different between the soil treatments (Table 3). No ectomycorrhizal short roots were detected in Genotypic fingerprinting of fluorescent the soil amended with the termite mound (Fig. 3). The pseudomonad strains isolated from termite ectomycorrhizal colonization indices of A. holosericea seed- mounds of Macrotermes subhyalinus lings inoculated with S. dictyosporum IR408 and IR412 were not significantly different (13.5%). The termite mound Eighteen randomly chosen fluorescent pseudomonad strains amendment significantly increased ectomycorrhizal forma- were subjected to PCR/restriction fragment length poly- tion, which reached around 25% (Fig. 3). morphism (RFLP) analysis. PCR conditions allowed the amplification of a single DNA fragment of the 16S rDNA Catabolic diversity of microbial communities in gene with the same size of 1000 bp for all 18 Pseudomonas soil treatments isolates studied. Digestion of the PCR products with two restriction enzymes (HaeIII and MspI) did not show any Co-inertia analysis of the relationship between plant growth, polymorphism in the patterns of the 16S rDNA fragments mycorrhizal formation and SIR responses is shown in Fig. 4. (Fig. 1). The four figures (Fig. 4a–d) can be superimposed to allow

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) Fig. 2. Dendrogram showing neighbour-joining P. resinovorans (AJ308314) analysis of 16S rDNA from some ﬂuorescent pseu- P. aeruginosa (AJ308297) domonads retrieved from the Ribosome Database E. coli (AJ01859) Project. The sequence obtained in this study is indicated in bold. Accession numbers are indicated in parentheses. 0.06 0.04 0.02 0.00

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 Shoot biomass Root biomass Arbuscular mycorrhizal Total number of Treatments (mg dry weight) (mg dry weight) colonization index (%) nodules per plant Control 261 aÃ 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 Ã Data in the same column followed by the same letter are not significantly different according to the one-way analysis of variance (P o 0.05). the analysis of the relationships between these variables. The ing to the inoculated treatments (IR408 and IR412) are Monte-Carlo test showed that there was a statistically shifted towards the right of the figures, which correspond to significant, although not extremely strong, relationship higher root and shoot biomass. The positive effect of (P = 0.025). Figure 4a and 4c shows the positive effect of Macrotermes subhyalinus mound powder amendment on fungal inoculation on plant growth: the points correspond- plant growth is also clearly visible: treatments MS, IR408 1

30 plant growth was significantly higher than that measured c c when IR412 was inoculated alone; ectomycorrhizal coloni- 25 zation was also significantly increased (from 28.3% to 48.5%) (Table 5). The total biomass of the plants correlated 20 significantly with the mycorrhizal rates (r2 = 0.78). Nodules b b 15 were observed in all treatments. Ectomycorrhizal inoculation significantly enhanced the number and total weight of 10 nodules per plant. This fungal positive effect was significantly increased when S. dictyosporum was co-inoculated 5 with KR9 (Table 5). The number and total biomass of a a Ectomycorrhizal colonization (%) nodules per plant were significantly linked with the myco- 0 rrhizal rates (r2 = 0.76 and r2 = 0.79, respectively). + MS IR408 IR412 Control

IR408+MS IR412+MS Discussion Fig. 3. Ectomycorrhizal formation of Scleroderma sp. IR408 and Scle- roderma dictyosporum IR412 on Acacia holosericea root systems in soil The main objectives of this study were to test the effect of a amended and not amended with Macrotermes subhyalinus mound Macrotermes subhyalinus mound structure amendment on powder after 4 months of culture in glasshouse conditions. Columns the formation of ectomycorrhizae between Acacia holo- indicated by the same letter are not significantly different according to sericea and two isolates of Scleroderma dictyosporum and to one-way analysis of variance (P o 0.05). MS, M. subhyalinus mound evaluate the role of fluorescent pseudomonads inhabiting powder amendment. the mound in these interactions. In a previous study, spores of ectomycorrhizal fungi were MS and IR412 1 MS are also shifted towards the right of the detected in the mounds of wood-, litter- and grass-feeding figure. This positive effect seems to be stronger for the IR412 termites (Spain et al., 2004). The authors showed that there strain than for the IR408 strain. With regard to SIR was a greater diversity and more concentrated populations responses, Figs 4b and 4d clearly show that the positive of ectomycorrhizal fungal spores in the mounds than in the effect of M. subhyalinus mound powder amendment corre- surrounding soil. They also detected basidiocarps of the sponds to a strong modification of the functional microbial common genera Pisolithus and Scleroderma species on the diversity for the three treatments. mound surfaces. This localization of fruit bodies indicated One-way analysis of variance confirmed these conclu- that the hyphae in the mounds originated from the nearest sions (Table 4). Ectomycorrhizal establishment was mainly putative host plants. In our study, no ectomycorrhizal short characterized by higher SIR responses with L-arginine, roots were detected in the M. subhyalinus treatment without whereas termite mound amendment was indicated by high- ectomycorrhizal fungal inoculation. This result seems to er SIR responses with sucrose, D-glucosamine, keto-glutaric, contradict the conclusions of Spain et al. (Spain et al., 2004). hydroxy-butyric, ascorbic, quinic, gluconic, keto-butyric, However, the termite mounds of M. subhyalinus were malonic, oxalic, succinic, tartaric and uric acids, trisodium collected in a shrubby savanna where all the plant species citrate and cyclohexane (Table 4). The SIR response with were associated with arbuscular mycorrhizal fungi (Dupon- gallic acid was significantly higher when termite mound and nois et al., 2001). As no potential ectomycorrhizal host tree ectomycorrhizal inoculum were both added to the soil species was present in these areas, termite mounds could be (Table 4). The highest catabolic richness was recorded in overspread by ectomycorrhizal short roots. In addition, in a the IR412 treatment, whereas the highest catabolic evenness previous study (Duponnois & Lesueur, 2005), the formation was recorded in the IR4081MS treatment (Table 4). of ectomycorrhizae was not observed after 4 months of culture when spores of ectomycorrhizal fungi were inoculated in the soil. Effect of a fluorescent pseudomonad strain In the present study, termite mound amendment signifi- (isolate KR9) on IR412 ectomycorrhizal cantly enhanced the ectomycorrhizal expansion of both development fungal isolates. This promoting effect could be attributed After 4 months of culture, S. dictyosporum IR412 had to: (1) the enhancement of plant growth (particularly root colonized A. holosericea seedlings and had significantly growth) induced by termite mound amendment; (2) inocu- increased shoot and root growth (Table 5). By contrast, no lation (via the termite mound) by a bacterial group (i.e. significant effect of the bacterial inoculant KR9 was recorded fluorescent pseudomonads) that could act as MHB (Du- on plant growth. When KR9 was co-inoculated with IR412, ponnois & Plenchette, 2003); and (3) the development of

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 Treatments

Organic substrates Control IR 408 IR 412 M. subhyalinus (MS) IR 4081MS IR 4121MS Ã 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

1 1 Data are expressed as mgCO2 g soil h . Ã Data in the same line followed by the same letter are not significantly different according to the one-way analysis of variance (P o 0.05). multitrophic interactions between the ectomycorrhizal sym- the present study, termite mound amendment stimulated biosis and the soil microflora. root growth, probably through an enhanced supply of Termite mounds (Isoptera) are a ubiquitous feature of nitrogen, which, in turn, increased the number of fungal tropical ecosystems, especially in savanna environments. infection sites. Through termite activities, large amounts of soil are trans- Recent studies have suggested that termite mounds could located from various depths of the soil profile (Holt & be sites of great bacterial and fungal diversity. Termite nests Lepage, 2000). In some areas, such termitaria represent a generally contain a diversity of fungi (Sannasi & Sundara- soil volume of more than 300 m3 above the ground. These Rajulu, 1967; Mohindra & Mukerji, 1982). In Macrotermes structures strongly influence their environment. In their bellicosus mound soil in Nigeria, Thomas (Thomas, 1987a) review, Lobry de Bruyn & Conacher (1990) reported a soil found 21 species of fungi. Other authors have found large quantity of up to 4.7 tonnes ha1 year1. This termite activity populations of active bacteria in termite mounds, different has a considerable influence on soil physical and chemical from those of the parent soil: eight functional bacterial properties (Lee & Wood, 1971; Lobry de Bruyn & Conacher, groups were found in a Macrotermes mound in Rhodesia 1990; Black & Okwakol, 1997; Holt & Lepage, 2000), and (Meiklejohn, 1965). The higher microbial diversity in ter- largely explains the termite role as ecosystem engineers. In mite mounds was attributed to higher organic matter levels

1 (a) −1 2 (b) 1.1 AMI −2 −0.8 1.1 −0.8 SB

9 RB 29 14 28 26 8 1 27 31 4 33 NN 23 2530 13 17 18 20 ECI 10 6 3 24 32 16 12 19 7 21 2 22 5 11

3 6 (c) −3 3 (d) −5 5 −3 −4

MS MS

IR 408 IR 412 + MS IR 412 + MS C

IR 412 IR 408 + MS C IR 412 IR 408 IR 408 + MS

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 Shoot biomass Root biomass Number of nodules Total nodule weight Ectomycorrhizal Treatments (mg dry weight) (mg dry weight) per plant per plant (mg) colonization (%) Control 532 aÃ 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 Ã Data in the same column followed by the same letter are not signiﬁcantly different according to the one-way analysis of variance (P o 0.05). and a better supply of nitrogen (Meiklejohn, 1965; Mohin- The population and composition of microbial groups dra & Mukerji, 1982), and to higher moisture levels and appear to vary according to the mound compartment higher substrate availability (Holt, 1987; Abbadie & Lepage, considered (Brauman, 2000). Increasing evidence demon- 1989). strates that termites are able to control the number of

c 2006 Federation of European Microbiological Societies FEMS Microbiol Ecol 56 (2006) 292–303 Published by Blackwell Publishing Ltd. No claim to original French government works Termite mounds enhance ectomycorrhizal symbiosis 301 microorganisms, and probably their diversity, in selected litter-feeding termites form fertile ‘islands’ in the savanna, parts of their mounds (Sannasi & Sundara-Rajulu, 1967; maintaining fertility in these, mostly highly weathered, soils Holt & Lepage, 2000). Previous microbiological studies of (Okello-Oloya et al., 1985, 1986; Lobry de Bruyn & Con- termite mounds have been carried out to compare the acher, 1990). This positive effect is generally attributed to cultures of microbial communities in grass-, litter- and the activity of termites, which translocate nutrient elements soil-feeding termite mounds (Duponnois et al., 2005). in food into their mounds. However, another translocation Fluorescent pseudomonads have been detected only in M. could be proposed, from the termite mound to the host subhyalinus mound powder. The phylogenetic analysis per- plant, mediated by ectomycorrhizal roots. formed in this study showed that these fluorescent pseudomonads mostly belonged to Pseudomonas monteillii species. It has been demonstrated that one isolate of P. monteillii (isolate HR13) can stimulate the establishment of ectomy- References corrhizal symbiosis in tropical conditions (Founoune et al., Abbadie L & Lepage M (1989) The role of subterranean fungus 2002b) and is considered as an MHB. This MHB effect has comb chambers (Isoptera, Macrotermitinae) in soil nitrogen been recorded with different fungal isolates, such as S. cycling in a preforest savanna (Coˆte d’Ivoire). Soil Biol Biochem dictyosporum, S. verrucosum, Pisolithus albus and P. tinctor- 8: 1067–1071. ius,onA. holosericea and other Australian Acacia species Amato M & Ladd JM (1988) Assay for microbial biomass based (Duponnois & Plenchette, 2003). As P. monteillii isolate KR9 on ninhydrin-reactive nitrogen in extracts of fumigated soils. stimulated ectomycorrhizal formation between S. dictyos- Soil Biol Biochem 20: 107–114. porum IR412 and A. holosericea, these bacterial strains Bethlenfalvay GJ & Linderman RG (1992) Mycorrhizae in present in M. subhyalinus mounds could also be involved in Sustainable Agriculture, ASA Special Publication No. 54. the enhancement of ectomycorrhizal formation recorded in Agronomy Society of America, Madison, WI. the present study. Black HIJ & Okwakol MJN (1997) Agricultural intensification, Macrotermes subhyalinus mound amendment and ecto- soil biodiversity and agrosystem function in the tropics: the mycorrhizal inoculation induced strong modifications of role of termites. Appl Soil Ecol 6: 37–53. functional microbial diversity. In particular, important soil Brauman A (2000) Effect of gut transit and mound deposit on microflora, able to use carboxylic acids, were detected soil organic matter transformations in the soil feeding termite: through high SIR responsiveness with these compounds. a review. Eur J Soil Biol 36: 117–125. Biological and biochemical weathering is mediated by Bremner JM (1965) Inorganic forms of nitrogen. Methods of Soil microorganisms that excrete organic acids, phenolic com- Analysis, Part 2. Agronomy Monographs, Vol. 9, (Black CA, ed.), pounds, protons and siderophores (Drever & Vance, 1994). pp. 1179–1237. Agronomy Society of America and Soil Science For instance, it is well known that many different fungal Society of America, Madison, WI. species produce these organic acids as the strongest chelators Brundrett MC, Piche Y & Peterson RL (1985) A developmental of trivalent metals (oxalate, malate and citrate) (Dutton & study of the early stages in vesicular–arbuscular mycorrhizal Evans, 1996; Gadd, 1999). In addition, amongst termites, formation. Can J Bot 63: 184–194. the Macrotermitinae subfamily (also called ‘fungus-growing Chessel D & Mercier P (1993) Couplage de triplets statistiques et liaison espe`ce-environnement. Biometrie´ et Environnement, termites’) plays a major role in African ecosystem function- (Lebreton JD & Asselain D, eds), pp. 15–44. Masson, Paris. ing, mainly in arid and semi-arid areas. 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FEMS Microbiol Ecol 56 (2006) 292–303 c 2006 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. No claim to original French government works

Mycorrhiza (2007) 17:195–208 DOI 10.1007/s00572-006-0095-0

ORIGINAL PAPER

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

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) to sites. They were identified as belonging to the ectomycor- describe the diversity of mycorrhizal fungal communities rhizal genera Afroboletus, Amanita, Boletus, Cantharellus, associated with Uapaca bojeri, an endemic Euphorbiaceae of Lactarius, Leccinum, Rubinoboletus, Scleroderma, Tricho- Madagascar, and (2) to determine the potential benefits of loma, and Xerocomus. Russula was the most frequent inoculation with mycorrhizal fungi [ectomycorrhizal and/or ectomycorrhizal genus recorded under U. bojeri.AM arbuscular mycorrhizal (AM) fungi] on the growth of this structures (vesicles and hyphae) were detected from the tree species and on the functional diversity of soil microflora. roots in all surveyed sites. In addition, this study showed that Ninety-four sporophores were collected from three survey this tree species is highly dependent on both types of : : : mycorrhiza, and controlled ectomycorrhization of this N. Ramanankierana N. Rakotoarimanga E. Randrianjohany Uapaca species strongly influences soil microbial catabolic L. Ramaroson diversity. These results showed that the complex symbiotic Laboratoire de Microbiologie de l’Environnement, Centre National de Recherches sur l’Environnement, status of U. bojeri could be managed to optimize its P.O. Box 1739, Antananarivo, Madagascar development in degraded areas. The use of selected : : mycorrhizal fungi such the Scleroderma Sc1 isolate in M. Ducousso Y. Prin A. Galiana nursery conditions could be of great interest as (1) this CIRAD, UMR 113 CIRAD/INRA/IRD/AGRO-M/UM2, Laboratoire des Symbioses Tropicales et Méditerranéennes fungal strain is very competitive against native symbiotic (LSTM), TA10/J, Campus International de Baillarguet, microflora, and (2) the fungal inoculation improves the 34398 Montpellier Cedex 5, France catabolic potentialities of the soil microflora.

J. Thioulouse . . CNRS, Laboratoire de Biométrie et Biologie Evolutive, Keywords Ectomycorrhizas Arbuscular mycorrhizas UMR 5558, Université Lyon 1, Fungal diversity. Microbial functionalities . 69622 Villeurbanne Cedex, France Uapaca bojeri . Madagascar

M. Kisa : R. Duponnois IRD, UMR 113 CIRAD/INRA/IRD/AGRO-M/UM2, Laboratoire des Symbioses Tropicales et Méditerranéennes (LSTM), TA10/J, Introduction Campus International de Baillarguet, 34398 Montpellier Cedex 5, France A high botanical diversity and a high degree of endemism R. Duponnois (*) characterize Madagascarian forests (Lowry et al. 1997), but IRD, Laboratoire Commun de Microbiologie IRD/ISRA/UCAD, they are often deforested for their conversion to agriculture. Centre de Recherche de Bel Air, −1 P.O. Box 1386, Dakar, Senegal Deforestation rates were estimated to be 0.11 Mha year e-mail: [email protected] between 1950 (7.6 Mha) and 1985 (3.8 Mha; Green and 196 Mycorrhiza (2007) 17:195–208

Sussman 1990). Disturbances of the vegetation cover are ing a range of simple organic substrates directly to the soil often accompanied by rapid erosion of surface soil that and measuring the short-term catabolic responses (Degens induces a loss or reduction of major physicochemical and and Harris 1997). Catabolic evenness, a component of biological soil properties (Vagen et al. 2006a,b). In microbial functional diversity is defined as the uniformity particular, it has been shown that mycorrhizal soil potential of substrate use and can be calculated from the CRPs was drastically reduced (Marx 1991; Jasper et al. 1991; (Degens and Harris 1997). Herrera et al. 1993; Dickie and Reich 2005). Hence, an Mycorrhizal fungi are ubiquitous components of most increase of this fungal inoculum potential is needed in both ecosystems throughout the world and are considered key natural and artificial revegetation processes (McGee 1989). ecological factors in governing the cycles of major plant However, the mycorrhizal status of the Madagascarian flora nutrients and in sustaining the vegetation cover (van der is poorly known. Typical ectomycorrhizal fungi were Hejden et al. 1998; Requena et al. 2001; Schreiner et al. reported more than 60 years ago (Heim 1970). More 2003). Two major forms of mycorrhizas are usually recently, mycological surveys show the large diversity of recognised: the arbuscular mycorrhizas (AM) and the the associated ectomycorrhizal fungi (Buyck et al. 1996; ectomycorrhizas (ECMs). AM symbiosis is the most Ducousso et al. 2004). The mycorrhizal inoculation of widespread mycorrhizal association type with plants that plants is very efficient in establishing plants on disturbed have true roots, i.e. pteridophytes, gymnosperms and soils (Estaun et al. 1997; Duponnois et al. 2001, 2005). The angiosperms (Read et al. 2000). They affect about 80– management of mycorrhizal symbiosis needs to investigate 90% land plants in natural, agricultural, and forest the presence, abundance, and community composition of ecosystems (Brundrett 2002). ECMs affect trees and mycorrhizal fungi associated with plants. Furthermore, shrubs, gymnosperms (Pinaceae) and angiosperms, and efficient fungal strains have to be selected to help tree are usually the result of the association of Homobasidio- establishment and also to improve soil quality (Franson mycetes with about 20 families of mainly woody plants and Bethlenfalvay 1989; Duponnois and Plenchette 2003; (Smith and Read 1997). These woody species are associ- Diédhiou et al. 2005; Duponnois et al. 2005). ated with a larger (compared to the AM symbiosis) The benefits of mycorrhizal symbiosis to the host plant diversity of fungi, comprising 4,000 to 6,000 species, have usually been considered a result from the close mainly Basidiomycetes and Ascomycetes (Allen et al. relationship between fungal symbionts and plant species. 1995; Valentine et al. 2004). However, it has been demonstrated that mycorrhizal The main objectives of this study were (1) to describe symbiosis has a great influence on the soil bacterial and the diversity of mycorrhizal fungal communities associated fungal communities in natural conditions (Frey et al. 1997; with Uapaca bojeri, an endemic Euphorbiaceae of Mada- Founoune et al. 2002a,b; Mansfeld-Giese et al. 2002; Frey- gascar and (2) to determine the potential benefits of Klett et al. 2005). This microbial compartment is common- inoculation with mycorrhizal fungi (ectomycorrhizal and/ ly named “mycorrhizosphere” (Linderman 1988) and is or AM fungi) on the growth of this tree species and on the usually divided in two different zones: one is subjected to functional diversity of soil microflora. the dual influence of the root and the mycorrhizal symbionts (the mycorrhizosphere) and, the other, under the influence of mycorrhizal hyphae (the hyphosphere). Materials and methods The microbial activities that occur in the hyphosphere are different from those recorded in the mycorrhizosphere Site description and sporophore sampling (Andrade et al. 1998). Hyphosphere microorganisms may influence mycorrhizal functions such as nutrient and water Three forests in Madagascar were visited at 2- to 3-week uptake carried out by the external hyphae of the mycorrhi- intervals during the sampling seasons, mid-November to zal fungi (Duponnois, unpublished data). Hence, the early February 1993, July–August 1994, and July to mid- association between the fungus and the host plant has been September 1995, to collect ectomycorrhizal fungi fruiting enlarged to the soil microflora to form a multitrophic under U. bojeri. The forests were located 50 km to the west mycorrhizal complex (Frey-Klett et al. 2005). The micro- of Antananarivo (Arivonimamo site as site A), 20 km to the bial functional diversity of each soil compartment includes south of Antsirabe (Ambositra site as site B), and 100 km a vast range of activities (nutrient transformations, decom- to the east of Toliara (Isalo site as site C). The mean annual position, etc.) and can be characterized by the measurement rainfall varied from 912.4 mm (site C), 1,428.8 mm (site of catabolic response profiles (CRPs; Degens and Harris A), to 1,554.4 mm (site B). The vegetation sampled 1997; Degens et al. 2001). The measurement of CRPs included savannas (sites A and B) and deciduous forests directly assesses the catabolic diversity of microbial (site C). The main chemical characteristics of the upper soil communities involved in decomposition activities by add- layer (0–20 cm) of these sites are shown in Table 1. Mycorrhiza (2007) 17:195–208 197

Table 1 Main-chemical characteristics of the upper soil layer (0– inoculum was prepared according to Duponnois and 20 cm) Garbaye (1991). Site Site A Site B Site C The AM fungus Glomus intraradices (Schenk and Smith, DAOM 181602, Ottawa Agricultural Herbarium) was pH (H2O) 4.96 5.37 4.54 multiplied on leek (Allium porrum L.) on Terragreen (Oil pH (KCl) 4.75 5.23 4.45 Dri UK) in glasshouse conditions. The culture substrate was Total C (%) 1.12 3.09 1.33 an attapulgite (calcined clay; average particle size, 5 mm) Total N (%) 0.07 0.15 0.91 Total organic matter (%) 1.92 5.31 2.28 from Georgia used for the propagation of AM fungi C/N 16.0 21.0 14.6 (Plenchette et al. 1996). After 12 weeks of culturing, the Total P (mg kg−1) 15.2 15.2 17.3 leek plants were uprooted and gently washed, and the roots Available P (mg g−1, Olsen et al. 1954) 3.42 7.01 5.24 were cut into 0.5-cm pieces bearing around 250 vesicles per centimeter. Non-mycorrhizal leek roots prepared as above Sporophores of putative epigeous ectomycorrhizal fungi were used for the control treatment without AM inoculation. were collected under U. bojeri, photographed, described as The seeds of the U. bojeri were surface sterilized as fresh material, preserved by oven-drying, and deposited at described above. The germinated seeds were individually the herbarium at Laboratoire de Microbiologie de l’Envi- grown in 1-l polythene bags filled with sterilized sandy soil ronnement (LME, Madagascar). In addition, roots of U. (140°C, 40 min) in which G. intraradices and/or Scleroder- bojeri were collected in each site, and fine roots were ma Sc1 were already inoculated. A control treatment without stained for AM according to the procedure of Phillips and fungi was included. After autoclaving, the soil chemical

Hayman (1970) and examined with light microscopy. characteristics were as follows: pH 5.01 (H2O); total carbon, 9.3%; total nitrogen, 0.06%; total phosphorus, 120.6 mg Time sequence of mycorrhizal colonization on U. bojeri kg−1. For ectomycorrhizal inoculation, the soil was mixed in glasshouse conditions with fungal inoculum (10/1; v/v). The treatments without fungus received an autoclaved mixture of moistened (MMN Surface forest soil (0- to 20-cm depth) was collected from medium) vermiculite/peat moss at the same rate. For the native stand of U. bojeri in site A, crushed, passed endomycorrhizal inoculation, one hole (1×5 cm) was made through a 2-mm sieve, carefully mixed, and distributed in in each pot and filled with 1-g fresh leek root (mycorrhizal 1-l pots. The seeds of U. bojeri collected in site A were for the experimental treatment or non-mycorrhizal for the surface sterilized in hydrogen peroxide for 10 min, rinsed control treatment without fungus). The holes were then and soaked in sterile distilled water for 12 h, and ger- covered with the same autoclaved soil. They were watered minated on 1% agar. After 1 week of incubation at 30°C in regularly with tap water without fertilizer. The pots were the dark, one pre-germinated seed was planted per pot. The arranged in a randomized complete block design with 25 seedlings were screened from the rain and grown under replicates per treatment. The seedlings were screened from natural light (daylight of approximately 12 h, average daily the rain and grown under natural light (daylight of temperature of 25°C). They were watered regularly with approximately 12 h, average daily temperature of 25°C). tap water without fertilizer. After 5 months of culture, the Uapaca plants were During 5 months, four plants per month were randomly uprooted, and the oven dry weight (1 week at 65°C) of the sampled, uprooted, and their root systems gently washed with shoot was measured. The root systems were gently washed, tap water. About 30 lateral roots were randomly chosen along cut into 1-cm root pieces, mixed, and the percentage of the tap root of each plant, cut into short pieces, and observed ectomycorrhizal short roots (number of ectomycorrhizal under a stereomicroscope (magnification ×40). All ECMs short roots/total number of short roots) was determined on were counted on each root fragment. Other root samples were a random sample of at least 100 short roots under a collected from each plant to detect AM structures using the stereomicroscope (magnification ×40). Then these root same procedure as before (Phillips and Hayman 1970). pieces were cleared and stained according to the method of Phillips and Hayman (1970). The root pieces were placed on Assessment of U. bojeri mycorrhizal dependency a slide for microscopic observation at 250× magnification (Brundrett et al. 1985). About 100 1-cm root pieces were A strain of Scleroderma sp. (strain Sc1) was isolated from a observed per plant. The extent of mycorrhizal colonization sporocarp collected in site A. This fungal isolate was was expressed in terms of the fraction of root length with the previously tested for its compatibility with U. bojeri in internal fungal structures (vesicles and arbuscules). The axenic conditions (data not shown). The fungal strain was relative mycorrhizal dependency was determined by express- maintained in Petri dishes on modified Melin–Norkrans ing the difference between the shoot dry weight of the (MMN) agar medium at 25°C (Marx 1991). The fungal mycorrhizal plant and the shoot dry weight of the non- 198 Mycorrhiza (2007) 17:195–208 mycorrhizal plant as a percentage of the shoot dry weight of and amino acids were added at 10 mM, whereas the the mycorrhizal plant (Plenchette et al. 1983). carbohydrates were added at 75 mM and the carboxylic acids at 100 mM (Degens and Vojvodic-Vukovic 1999). Influence of ectomycorrhizal inoculation on soil microbial The catabolic richness and catabolic evenness were catabolic diversity calculated to evaluate the catabolic diversity of both soil treatments. The catabolic richness, R, expressed the The Uapaca seedlings were grown in 1-l pots filled with number of substrates used by the microorganisms in each natural soil collected in site A. One part of the soil was soil treatment. The catabolic evenness, E,representingthe autoclaved (140°C, 40 min) and the other part was not variability of used substrates amongst the range of the disinfected before use. After autoclaving, its chemical substrates tested was calculated using the Simpson–Yule 2 characteristics were as follows: pH 5.2 (H2O); total C, index E ¼ 1 pi with pi=respiration as the response to 1.01%; total N, 0.08%; organic matter, 1.55%; C/N, 13.2; individual substrates/total respiration activity induced by total P, 11.9 mg kg−1. The native chemical characteristics of all substrates for a soil treatment (Magurran 1988). this soil are indicated in Table 1. The ectomycorrhizal inoculation with the Scleroderma isolate Sc1 was per- Statistical analysis formed as described above, and the same treatment was performed for the control treatment. They were watered The data were treated with one-way analysis of variance. The regularly with tap water without fertilizer. The pots were means were compared using the Newman and Keuls test (p< arranged in a randomized complete block design with ten 0.05). The percentages of the mycorrhizal colonization were replicates per treatment. The seedlings were screened from transformed by arcsin(sqrt) before the statistical analysis. the rain and grown under natural light (daylight of The between-group analysis (BGA, Dolédec and Chessel approximately 12 h, average daily temperature of 25°C). 1987; Culhane et al. 2002) was used to analyse the surface After 5 months of culture, Uapaca plants were uprooted, insulation resistance (SIR) responses in soil samples the shoot biomass and the ectomycorrhizal colonization inoculated with Scleroderma Sc1 and samples without were measured as described before. Most of the soil from 3 inoculation. BGA is a multivariate analysis technique randomly chosen pots in each treatment was carefully derived from principal components analysis (PCA). The mixed and kept at 4°C for further analysis. aim of PCA is to summarize a data table by searching The microbial catabolic diversity was measured by adding orthogonal axes on which the projection of the sampling a range of simple organic compounds to the soil and points (rows of the table) has the highest possible variance. determining the short-term respiration responses (Degens From a theoretical point of view, BGA is the particular and Harris 1997;Degensetal.2001). Each of the 31 case of PCA with respect to instrumental variables (principal substrates suspended in 2-ml sterile distilled water was component analysis with instrumental variables, Rao 1964; added to 1 g of moist soil in 10-ml bottles (West and Lebreton et al. 1991) where the instrumental variables table

Sparling 1986). The CO2 production from the basal is reduced to just one qualitative variable. This variable respiratory activity in the soil samples was measured by defines groups of rows in the data table, and BGA consists adding 2-ml sterile distilled water to 1 g of the equivalent of the PCA of the table of the means by groups. This table dry weight of soil. After the addition of the substrate has a number of rows equal to the number of groups, and solutions to the soil samples, the bottles were immediately the same number of columns as the original table. The aim sealed with a vacutainer stopper and incubated at 28°C for of this analysis is to separate the groups. This is also the

4 h in darkness. After 4 h, respired CO2 in the headspace of aim of discriminant analysis (also called canonical variates each bottle was determined by taking a 1-ml syringe sample analysis), but whilst discriminant analysis is limited to and analysing the CO2 concentration using an infrared gas tables that have a high number of samples compared to the analyser (Polytron IR CO2,Dräger™) in combination with a number of variables, BGA can be used even when the thermal flow meter (Heinemeyer et al. 1989). The results number of rows is less than the number of variables. BGA −1 −1 were expressed as μgCO2 g soil h . There were 10 can, thus, be considered as a robust alternative to amino acids (L-glutamine, L-serine, L-arginine, L-asparagine, discriminant analysis when the number of samples is low. L-cystein, L-histidine, L-lysine, L-glutamic acid, L-phenylala- A Monte Carlo test (permutation test) can be used to check nine, L-tyrosine), 3 carbohydrates (D-glucose, D-mannose, the significance of the differences between groups. This sucrose), 2 amides (D-glucosamine and succinamide), and 16 method consists, in performing many times, a random carboxylic acids (ascorbic acid, citric acid, fumaric acid, glu- permutation of the rows of the table (but not of the qualitative conic acid, quinic acid, malonic acid, α-ketoglutaric acid, variable defining the groups) followed by the recomputation α-ketobutyric acid, succinic acid, tartaric acid, uric acid, of the between-class inertia. By comparing the between-class oxalic acid, malic acid, hydroxybutyric acid). The amines inertia obtained in the normal analysis with the between-class Mycorrhiza (2007) 17:195–208 199 inertia obtained after randomization, we get an estimation of genera Afroboletus, Amanita, Boletus, Cantharellus, Lecci- the probability of meeting a situation similar to the observed num, Gyroporus, Rubinoboletus, Russula, Scleroderma, situation without differences between groups (i.e. a signifi- Suillus, Tricholoma, and Xerocomus (S 1). The highest cance test of the differences between groups). fungal diversity of the above-ground sporophores was The computations and graphical displays were made with recorded in site A (40 species), whereas only 27 and 29 the free ADE-4 software (Thioulouse et al. 1997)availablein fungal species were detected in sites B and C, respectively the Internet at http://www.pbil.univ-lyon1.fr/ADE-4/. (S 1). Russula was the most frequent ectomycorrhizal genus recorded under U. bojeri (32.9% of the above- ground sporophore diversity) followed by the genera Results Amanita (17.1%) and Cantharellus (Fig. 1a). Twenty-one different species were recorded for Russula followed by Sporophore survey Amanita (14 species) and the genera Cantharellus and Boletus (10 species; Fig. 1b). AM structures (vesicles and We collected 94 sporophores in three survey sites (S 1). hyphae) were detected from the roots in all surveyed sites. They were identified as belonging to the ectomycorrhizal Fig. 1 a Structure of the ectomycorrhizal community a 35 (above-ground diversity) expressed as genus relative 30 frequency (b). Number of species per genus 25

Genus relative frequency ( % ) 5

s s s s tu u u lu e il omus bolet opor Su Russula Amanita Boletus roderma obol o r Leccinum r Tricholomale bin Gy Cantharellus Sc Af Xeroc u R b 25

5 Number of species per genus

s s s s us tu u u lu e il omus ccinum bolet opor Su Russula Amanita Bolet obol o r Le r Tricholomaleroderma bin Gy Cantharellus Sc Af Xeroc u R 200 Mycorrhiza (2007) 17:195–208

Time sequence of mycorrhizal colonization on U. bojeri Table 2 Shoot growth, mycorrhizal development, and relative mycorrhizal dependency of U. bojeri seedlings 5 months after G. intraradices and/or Scleroderma sp. Sc1 inoculation in pot culture First, ECMs were recorded after 2 months (Fig. 2). Native ectomycorrhizal fungi colonized approximately 50% of the Treatments Shoot Ectomycorrhizal AM RMD lateral roots sampled after 5 months of culture (Fig. 2). AM biomass (mg colonization colonization (%)a structures were also observed after 2 months of culturing per plant) (%) (%) (Fig. 2). Control 91.1ab 0a 0a – Scleroderma 181.2b 8.7b 0a 47.6a Mycorrhizal dependency of U. bojeri seedlings sp. Sc1 G. intraradices 160.1b 0a 77.5b 42.7a The shoot dry weight of the plants inoculated with G. Scleroderma 360.3c 11.5b 82.5b 70.7b intraradices or Scleroderma sp. Sc1 was significantly sp. Sc1 + G. higher than in the control treatment (Tables 2 and 3). intraradices Compared to the control treatment, the shoot growth of a RMD Relative mycorrhizal dependency ectomycorrhized plants was stimulated 1.9 times, whereas it b Data in the same column followed by the same letter are not was 1.7 times for plants inoculated with G. intraradices significantly different according to the one-way analysis of variance (Table 2). When both fungal symbionts were co-inoculated, (p<0.05). the shoot dry weight significantly increased over the single inoculation treatments (Table 2). The shoot dry weight increased 2.1 times compared to the mean shoot dry weight Catabolic richness did not differ between the treatments of the single fungus treatments (G. intraradices alone or (Table 3). However, catabolic evenness was significantly Scleroderma sp. Sc1 alone). The dual fungal inoculation influenced by the soil treatments (autoclaved or not) and by did not significantly modify the establishment of ectomy- the fungal inoculation (Table 4). corrhizal and AM symbioses compared to the ectomycor- The BGA of the SIR responses for the four soil rhizal or AM colonization rates measured in the single treatments are presented in Fig. 3. The map of the soil inoculation treatments (Table 2). samples (Fig. 3b) shows that the four treatments (NDNI, NDI, DNI, and DI) were clearly separated. This result Influence of ectomycorrhizal inoculation on soil microbial indicates that the microbial communities were different (in catabolic diversity composition or at least in activity), according to the soil treatment. The map of the substrates (Fig. 3a) shows that, The growth of U. bojeri seedlings was significantly higher on the first axis, the use of four organic acids was highest in in the native soil than in the autoclaved soil (Table 3). non-autoclaved soil samples and in inoculated samples (left Ectomycorrhizal fungal inoculation significantly increased part of the figure: ketobutyric, ketoglutaric, oxalic, and shoot biomass of U. bojeri seedlings. There were no citric acids). The Monte Carlo test is significant (p=0.025). significant interactions between the autoclaving and the The soil autoclaving involved a lower rate of use of these inoculum treatments (Table 3). four organic acids, whereas fungal inoculation led to a higher rate. Moreover, the effect of inoculation seemed stronger in non-disinfected soil samples.

100 Discussion 80 The main results of this study show that (1) a large 60 diversity of sporophores was recorded under U. bojeri, (2) 40 U. bojeri formed AMs and ECMs in natural soils, (3) this

20 tree species is highly dependent on both types of mycorrhiza, and (4) controlled ectomycorrhization of U. bojeri 0 strongly influences soil microbial catabolic diversity. 0123456Our investigations show that forests dominated by U. AM colonization (% of root fragments) or Time (months) bojeri contain a wide range of sporophores belonging to at ectomycorrhizal colonization (% of short roots) (%) least four different fungal families: Russulaceae, Canthar- Fig. 2 Sequence of mycorrhizal colonization on U. bojeri seedlings in experiment 1 (square, AM colonization; diamond, total ectomycor- ellaceae, Boletaceae, and Amanitaceae. In tropical forests, rhizal colonization) these families of putative ectomycorrhizal fungi have been Mycorrhiza (2007) 17:195–208 201

Table 3 Shoot growth, mycorrhizal development, and relative mycorrhizal dependency of U. bojeri seedlings 5 months after Scleroderma sp. Sc1 inoculation in disinfected or nondisinfected soil

Treatments Shoot biomass (mg per plant) Ectomycorrhizal colonization (%) RMD (%)a Rb Ec

Disinfected soil Control 135ad 0a – 28.7a 4.7a Scleroderma sp. Sc1 192c 62.1c 29.1a 30.3a 6.9c Nondisinfected soil Control 165b 18.2b – 29.7a 6.1b Scleroderma sp. Sc1 240d 58.6c 30.4a 30.7a 7.7d Soil Treatment (ST) Se NS NS S Fungal inoculation (FI) S NS SS FI × ST NSf NS NS NS a RMD Relative mycorrhizal dependency b Catabolic richness c Catabolic evenness d 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) described under Afzelia africana, Monotes kerstingii, it has also been reported that roots of U. guineensis seedlings Uapaca guineensis, and U. somon in Africa (Thoen and growing in a forest soil were only colonized by AM fungi Bâ 1989; Sanon et al. 1997) and in Asia under dipterocarps (Moyersoen and Fitter 1999). The results of the present (Lee 1998). It is also well known that Russulaceae are often study confirmed the high occupancy of AM fungi recorded dominant in tropical rainforests of Africa, Asia, and on young seedlings (3-month-old root systems) and that AM Madagascar (Buyck et al. 1996; Lee et al. 1997; Watling structures appeared for the first time on the plant culture and Lee 1998; Riviere et al. 2006). The identification of followed by ECM colonization (Chilvers et al. 1987). this group in the tropics remains problematic as many A synergistic effect of dual AM/ECM inoculation was species are new and undescribed. A high diversity of described for Acacia holosericea inoculated with G. ectomycorrhizal fungi was associated with U. bojeri. With fasciculatum and Pisolithus albus (Founoune et al. 2002a, other tropical ectomycorrhizal tree species, Lee et al. b), but the involved mechanisms remained unknown. In (1997) recorded only 28 fungal species under Shorea contrast, in 1-year-old field seedlings of Quercus agrifolia leprosula, and Sanon et al. (1997) had identified 14 fungal with a high glomalean and ectomycorrhizal fungal load, species under U. guineensis and 11 species under U. somon coexistent mycorrhizal types constituted a cost during the in Burkina Faso. However, numerous studies in temperate establishment of young oaks and potentially limited their areas indicate little correlation between above-ground development (Egerton-Warburton and Allen 2001). These (sporophores) and below-ground (ECMs) fungal diversity authors suggested that the progressive shift to predomi- (Buscot et al. 2000; Horton and Bruns 2001). Further nantly ectomycorrhizal colonization with increasing plant molecular-based studies are needed to determine the fungal age become beneficial over time as it has been recorded diversity of ECMs associated with U. bojeri in natural with U. bojeri after AM/ECM inoculation in the present conditions. study. Most mycorrhizal species are generally associated with Pirozynski and Malloch (1975) hypothesised that the only one type of mycorrhiza, usually either AMs or ECMs AM habitat was a prerequisite for the early development of (Moyersoen and Fitter 1999). It has also been reported that land flora. Soil nutrient distribution in natural environments some plant species formed both AM and ECM (Molina et al. is typically heterogenous (Farley and Fitter 1999), and 1992). The dual symbiotic association is well documented mycorrhizas may allow plants growing in low nutrient for Populus (Lodge and Wentworth 1990), Salix (Dhillion patches to access resources in adjacent rich nutrient patches 1994), Eucalyptus (Lapeyrie and Chilvers 1985), Alnus (Casper and Cahill 1998). In addition, ectomycorrhizal (Molina et al. 1994), Acacia (Founoune et al. 2002a,b), fungi are not uniformly distributed in terms of their Pinaceae (Cazares and Trappe 1993), Quercus (Egerton- presence, abundance, or community composition (Dickie Warburton and Allen 2001), and Casuarinaceae (Duponnois and Reich 2005), and a lack of ectomycorrhizal fungi may et al. 2003), but it was unknown for U. bojeri,althoughit slow the invasion of disturbed sites by ectomycorrhizal was usually stated that this tree species was only colonized plants. Young seedlings of U. bojeri that form AM could by ectomycorrhizal fungi (Moyersoen and Fitter 1999). But survive in sites with low availability of ectomycorrhizal 202 Table 4 Description of putative ectomycorrhizal fungi collected from the three studied sites beneath U. bojeri

Species Prominent features Habitat Sites

Site Site Site A B C

Amanitaceae Amanita rubescens Gray White pinkish cap (8-cm diameter) covered with white powdered and flat scales, remnant veil Solitary, scarce x visible at the margin, white stem reddening by wound, often eaten by insect larvae Amanita virosa (Fr.) Bertillon White yellowish fruiting body (7- to 12-cm diameter), white and chinated stem (1.2-cm Patch of 5 to 6 individuals x x x diameter) with ring and cup at the base Amanita phalloides var. verna Bull White fruiting body (5.5- to 11-cm diameter), stem (0.6 diameter by 9.5 cm high) with a large Patch of 5 to 7 individuals x x X pendant ring and a bulbous cup at the base Amanita strobiliformis Bertillon White and big fruiting body (10 to 12 cm diameter), fleecy remnant veil on the cap, Solitary, scarce x club-shaped stem (2.2-cm diameter) with a ring Amanita cf. Baccata (Fr.) Gillet Big white fruiting body similar features than previous species but with no ring, stem (2-cm diameter Solitary, scarce x by 7 cm high) Amanita sp1 White finely scaled fruiting body (4- to 6- cm diameter) turning yellowish when ageing or by wound, Solitary, scarce x concoloured gills and flesh Amanita cf. Strobiloceovolvata White fruiting body (8.5- to 11-cm diameter), stem (1.2-cm diameter by 10.5 cm high) without ring, Patch of 3 to 4 individuals x x x Beeli well-developed bulbous cup at the base Amanita sp2 White and big species with a convex scaly cap (10- to 13-cm diameter by 9 to 10 cm high), strong Solitary, scarce x bulbous stem (3- to 4-cm diameter) with a pendant ring Amanita sp3 Pale grey cap (4.5-cm diameter) with few veil remanences on surface, bulbous stem (0.7 to 6 cm) Solitary, scarce x with grey chinates Amanita sp4 Yellow conical and mucronated cap (2.5- to 3-cm diameter), paler to whitish gills and stem (0.5-cm Solitary, scarce x diameter by 12 cm high), white scaly basal cup Amanita cf. cecilia (Berk. Yellow grey cap (4- to 5-cm diameter) with rised scales, white gills and concoloured stem (0.7-cm Solitary, scarce x et Broome) Bas diameter to 6 cm high), bulbous base covered by grey chinates and veil remanences Amanita sp5 Convex and grey purplish-blue cap (4 to 4.5 cm diameter) with grey flat scales at the centre and hairy Solitary, scarce x ones at the margin, white flesh and gills, white bulbous stem (0.9-cm diameter by 6 cm high) turning to grey by touch with a pendant ring Amanita sp6 Small white species (2- to 3-cm diameter) with yellowish scales, bulbous based stem with pendant Solitary, scarce x ring

Amanita sp7 Big white flat cap species (9- to 13-cm diameter) with veil remanences at the margin, strong bulbous Patch of 2 to 3 individuals x x x 17:195 (2007) Mycorrhiza stem (3- to 4-cm diameter) with a ring Boletaceae Rubinoboletus griseus Big red-pink and grey-brownish dry and smooth cap (10- to 12-cm diameter by 8 to 9 cm high), Patch of 5 to 6 individuals x x x white flesh (1.8 cm thick) partially burnishing after sectioning, pale reticulated hairy scaled stem, burnishing like pores by touch Gyroporus cf. cyanescens Big white yellowish smooth cap (10- to 12-diameter by 8–9 cm high), concoloured tubes and stem Patch of 3 to 4 individuals x x x (Bulliard Fr.) Quélet turning to blue by wound

Boletus sp1 Brownish to brown cap, with large darker flat scales, cylindrical and dark stem, red reticulated, Patch of 3 to 4 individuals x – becoming yellow at the base like rhizomorph, flesh and pores turning blue by wound 208 yoria(07 17:195 (2007) Mycorrhiza

Leccinum sp1 Small grey boletus (1.8- to 3-cm diameter by 3 to 4 cm high), yellow pores, red hairy scales on the Patch of 3 to 4 individuals x x x stem, base of the stem yellow like the rhizomophs Boletus sp2 Big brownish-brown wet cap (7- to 8-cm diameter to 12 to 15 cm high), white and smooth flesh Patch of 5 to 6 individuals x Xerocomus sp1 Brown scaly cap (8.5-cm diameter) showing white flesh between scales, white stem (1.4-cm Solitary, scarce x diameter by 5 to 6 cm high) with some red zone Leccinum sp2 Yellow grey scaled boletus (4.5- to 6-cm diameter by 6 to 7 cm high), stem yellow at the base and Solitary, scarce x –

red in its upper part, yellow blueishing pores 208 Boletus sp3 Brown cap (7.5-cm diameter) with red-pink pigments, yellow and red pores, greenishing and Solitary, scarce x blueishing tubes, yellowish stem with some red pigments Leccinum sp3 Red purplish-blue wet cap (7-cm diameter), yellow burnishing stem (0.8-cm diameter by 6 cm Solitary, scarce x high), concoloured yellow flesh and pores, blueishing after air exposure Boletus sp4 Big smooth and shiny red boletus (8- to 12-cm diameter by 7 to 8 cm high), yellowish stem with Patch of 2 to 3 individuals x x x some pink pigments, concoloured flesh (1.6 cm thick) Xerocomus sp2 Pale to dark brown scaly dry cap (5-cm diameter), white dirty stem (0.8-cm diameter by 4 cm high) Solitary, scarce x with a white-yellowish flesh, yellow greenish and pink pores Boletus sp5 Yellowish brown cap (8-cm diameter) with flat partially pink scales, yellow pores and stem (1.2-cm Solitary, scarce x diameter by 6 cm high), white flesh (1.6 cm thick) Boletus sp6 Dark brown scaly cap showing yellow flesh, pale concoloured pores and stem Solitary, scarce x Boletus sp7 Brown boletus with dry and silky cap (4.5-cm diameter), concoloured dark stem (2.2-cm diameter Solitary, scarce x by 5.2 cm high), white flesh (1.6 cm thick) rapidly turning to red, then black after air exposure Boletus sp8 Pale brown boletus with silky cap (5-cm diameter), white stem (1.5-cm diameter by 5.2 cm high) Solitary, scarce x and flesh (1.3 cm thick) turning purplish-blue after air exposure Leccinum sp4 Yellow and wet cap (3.5 cm) with hairy grey scales, yellow pores, yellow and red stem (0.5-cm Solitary, scarce x diameter) with dark scales and a narrow base Leccinum sp5 Yellowish-brown dry cap (3.5-cm diameter), red pores and lighter stem (0.6-cm diameter by 4 cm Solitary, scarce x high) turning to dark-brownish in section, white flesh turning burnish after air exposure Suillus sp2 Yellow and grey scaly cap (5-cm diameter), yellow pores covered by a yellow partial veil when Patch of 2 to 3 individuals x young, yellow stem (1.4-cm diameter by 4.5 cm high) with greenish grey scales, becoming very slimy Boletus sp9 Yellow brownish boletus (7- to 8-cm diameter) with a sticky surface, yellow pores and stem, Solitary, scarce x yellowish flesh (1.7 cm thick) Leccinum sp6 Pale brown cap (5- to 4-cm diameter) with red brownish scales at the centre, white pores and white Solitary, scarce x flesh turning rapidly to red, then black by wound Boletus sp10 Yellow brown boletus (4.5- to 5.7-cm diameter) with wet and smooth surface, yellow pores, yellow Solitary, scarce x stem (1.2 diameter by 3 cm high), white flesh (1 cm thick) Cantharellaceae Cantharellus sp1 Tall thick and lobed fasciculate bright yellow caps (4- to 6-cm diameter) forming patches of 4 to 5 Patch of 8 to 10 xxx individuals (12 cm), grained gills, pale yellow stem (1.8 cm), white flesh individuals Cantharellus sp2 Small orange-brownish cap (2- to 2.2-cm diameter), white pinkish gills, pink stem and white flesh Solitary, scarce x Cantharellus sp3 Yellowish to pale brown cap (3.5- to 3.2-cm diameter), yellow grained gills, pale yellow stem (0.6 Solitary, scarce x x x to 2.5 cm) 203 204 Table 4 (continued)

Species Prominent features Habitat Sites

Site Site Site A B C

Cantharellus sp4 Red orange cap (3.2- to 3.5-cm diameter), largely spaced yellowish grained gills, pale yellow to Patch of 8 to 10 xxx reddish stem (0.9 cm) individuals Cantharellus sp5 Pale brown cap (3.2- to 3.5-cm diameter), pale pink grained gills, white stem and flesh, turning to Solitary, scarce x x x yellow by touch or sectioning Cantharellus sp6 Red pinkish fasciculate caps (2.5-cm diameter) forming small patch (3.5 to 4 cm), yellowish grained Patchy x gills, pink orange stem and white fibrous flesh Cantharellus sp7 Small and fragile bright yellow cap (2- to 3.2-cm diameter), pale yellow gills, concoloured short Solitary, scarce x stem (0.3 cm) Cantharellus cf decolorans Eyss. Small pink orange cap (0.7- to 1.5-cm diameter, 2.5 to 3.5 cm high), concoloured gills and short Patch of 5 to 6 individuals x et Buyck stem (0.2 cm) Cantharellus cf. Cyanoxanthus Yellow and purple cap (4-cm diameter), pale pink grained gills, pale yellow stem (1.8 cm), fibrous Patch of 2 to 3 individuals x R. Heim flesh Cantharellus rubber R. Heim Pale pink cap (3.5- to 4-cm diameter), concoloured stem and gills Patch of 2 to 3 individuals x Russulaceae Russula subfistulosa Buyck White-greyish (darker at the centre) umbilicated cap (3- to 12-cm diameter) Solitary to patch of 3 xxx individuals Russula ochraceorivulosa Greyish to purplish-blue grey cap (7- to 8-cm diameter), convex cap with an undulating margin Solitary x x x Russula patouiillardi Pale yellow and purple (darker at the centre) dry scaly cap, white and purple stem Solitary to patch of 5 xxx individuals Russula liberiensis Buyck White-greyish fibrillose cap (3- to 12-cm diameter) turning brown when ageing, closely spaced Solitary to patch of 3 xxx decurrent gills individuals Russula cf. Cyanoxantha Pink to purple-red cap (5- to 15-cm diameter), white stem Patch of 2 to 3 individuals x Russula cellulata Buyck Brown scaly cap (3- to 9-cm diameter), closely spaced decurrent gills Patch of 2 to 3 individuals x Russula cf. archae R. Heim White smooth and flat cap (4.5- to 6-cm diameter) Solitary x Russula cf. nigricans White-greyish cap turning to brown when ageing, white flesh turn rapidly pink to red by air Solitary x exposure Russula cf. subfistulosa White-greyish convex cap (3- to 8-cm diameter) Solitary to patch of 4 x individuals

Russula sp3 White to pale yellow gluey and convex cap (3- to 13-cm diameter) Solitary to patch of 3 x 17:195 (2007) Mycorrhiza individuals Russula sp5 Yellow smooth umbilicated cap (6- to 12-cm diameter), with a very regular margin Patch of 2 to 3 individuals x x x Russula sp6 White-yellowish flat or slightly umbilicated cap (4- to 10-cm diameter), white flesh turning reddish Patch of 3 to 5 individuals x after air exposure Russula sp7 Dark grey to brown convex cap (3.5- to 8-cm diameter), involucrated margin, wet surface covered Solitary, rarely patchy x by orange to yellow layers, white-yellowish flesh Russula sp8 White convex to slightly umbilicate cap (4- to 13-cm diameter) turning brown when ageing, smooth Solitary, scarce x

surface with involucrated margin, white flesh turning reddish after air exposure – 208 yoria(07 17:195 (2007) Mycorrhiza

Russula sp10 Dark grey to brown when fully mature convex to flat cap (4- to 9-cm diameter), white flesh Patch of 2 to 4 individuals x Russula sp11 Small purple to purple-reddish umbilicate when young to flat when ageing cap (2- to 7-cm Solitary to patch of 3 x diameter), sticky surface, regular margin, adnate white to yellowish closely spaced gills, white flesh individuals Russula sp13 Brown-reddish convex and smooth glutinous cap (6- to 15-cm diameter), decurrent gills, white flesh Solitary x turning greyish by air exposure Russula sp14 Dark yellow to brown convex to flat sticky cap (4- to 10-cm diameter), adnate closely spaced gills Patch of 3 to 5 individuals x –

Russula sp15 Yellow to orange-yellow flat slightly umbilicated with an involucrated yellow margin cap (2- to Patch of 2 to 5 individuals x 208 8-cm diameter) with a smooth surface with small strias Russula sp16 Pink to reddish (darker at the centre) fragile convex glutinous cap, (2- to 6-cm diameter) with a Patch of 2 to 4 individuals x smooth or dusty surface, white flesh Russula sp17 Slightly umbilicated convex and glutinous cap (4- to 10-cm diameter), dark yellow tending to Solitary to patch of 4 x brown, yellow to pale orange closely spaced gills individuals Strobilomycetacea Afroboletus sp1 Brown-purple scaly cap (3- to12-cm diameter), fibrous stem, pale yellow flesh turning purplish by Patch of 3 to 5 individuals x air exposure Afroboletus sp2 Flat-convex dusty cap (3 to 10 cm diameter) with dark-brown to black scales, fibrous stem inflated Patch of 2 to 3 individuals x at the base, greyish-yellow flesh Sclerodermataceae Scleroderma sp1 Whitish to yellowish small pyriformic fruit bodies, size below 3 cm in diameter, dark grey gleba Solitary to patch of 5 x individuals Scleroderma sp2 Whitish to yellowish 3- to 7-cm diameter fruit bodies with grey spots at the top, dark grey gleba Solitary, rarely patchy x Tricholoma sp2 Yellow cap (3- to 9-cm diameter), dry surface, involucrated margin, thick widely spaced gills, Solitary to patch of 4 x yellow flesh keeping yellow even after exposure to air individuals Tricholoma sp3 Yellow-greyish cap (3- to 12-cm diameter), dry surface, white yellowish stalk, white flesh Solitary to patch of 4 x individuals Tricholoma sp4 Dark-grey cap (3- to 15-cm diameter), smooth dry surface, thick gills, white flesh Solitary x 205 206 Mycorrhiza (2007) 17:195–208

a 14 ectomycorrhizal fungus communities and facilitate the -26 2 establishment or re-establishment of the seedlings of -14 ectomycorrhizal tree species after the disturbance (Perry et al. 1989), thus, influencing plant succession from prairie or old field to savanna or woodland. Scleroderma species are considered “early-stage” symbionts (Deacon et al. 1983;Bâetal.1991) and can form 1 mycorrhizas with a wide range of tropical tree species such 5 4 8 17 as Afzelia africana (Bâ and Thoen 1990), A. quanzensis, 1011 9 16 291314 28 2725 2215 Isoberlinia doka, I. dalziellii, and Brachystegia speciformis 3 12 1917 2126 232018 24 (Sanon et al. 1997). In the present study, Scleroderma 2 6 7 isolate Sc1 increased Uapaca growth in disinfected and in non-disinfected soil, suggesting that this fungal strain was highly competitive against the native ectomycorrhizal mycota at least under the conditions of this pot-based experiment. In addition, ectomycorrhizal inoculation induced strong modification of the soil microflora functionalities and increased its catabolic microbial diversity. Elliott and Lynch (1994) hypothesised that microbial communities with reduced catabolic evenness are less resistant to stress b 60 -90 60 and disturbance. Microbial functional diversity is involved -90 in a large range of activities such as nutrient transformation, decomposition, etc. (Wardle et al. 1999). In particular, ectomycorrhizal fungi mobilize P and other essential plant nutrients directly from minerals through the excre- DNI tion of organic acids (Landeweert et al. 2001). Amongst NDI DI the total organic acids in the soil solution, low molecular weight organic acids are considered to be the most important biological weathering agents (Ochs 1996).

NDNI Oxalate, citrate, and malate produced by plant roots and soil microorganisms are the strongest chelators of trivalent metals (Landeweert et al. 2001). Oxalic acid, commonly produced by many different fungal species, has the highest acid strength (Dutton and Evans 1996). In the present study, SIR responses with all oxalic and citric acids increased in the fungal inoculated soil, suggesting that Scleroderma Sc1 and its associated microflora excreted higher amounts of such organic acids and induced a multiplication of microorganisms that utilize these avail- Fig. 3 BGA of the SIR responses with respect to the fungal able organic resources than noninoculated soil. treatments and soil treatments (DNI disinfected soil without fungal In conclusion, this study showed that U. bojeri has a inoculation, DI disinfected soil with fungal inoculation, NDNI nondisinfected soil without fungal inoculation, NDI nondisinfected complex symbiotic status that can be managed to optimize soil with fungal inoculation, NIND: 1 Ketobutyric acid, 2 ketoglutaric its development in degraded areas. In addition, the use of acid, 3 oxalic acid, 4 citric acid, 5 phenylalanine, 6 gluconic acid, 7 selected mycorrhizal fungi such the Scleroderma Sc1 glucose, 8 uric acid, 9 malic acid, 10 asparagine, 11 tartaric acid, 12 isolate in nursery conditions could be of great interest, as malonic acid, 13 gallic acid, 14 formic acid, 15 cystein, 16 histidine, 17 sucrose, 18 tyrosine, 19 glutamic acid, 20 succinic acid, 21 (1) this fungal strain appears competitive against native glucosamine, 22 succinamide, 23 mannose, 24 glutamine, 25 quinic symbiotic microflora and (2) the fungal inoculation acid, 26 lysine, 27 ascorbic acid, 28 serine, 29 arginine, 30 fumaric improves the catabolic potentialities of the soil microflora. acid, 31 hyroxybutyric acid However, further studies are needed to describe the belowground diversity of ectomycorrhizal fungi and to demon- fungi and develop ectomycorrhizas later as roots contact strate the potential interest of controlled mycorrhization in residual ECM communities. This mycorrhiza successional natural conditions in afforestation programs with U. bojeri process would promote the development of subsequent in Madagascar. Mycorrhiza (2007) 17:195–208 207

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Biol Invasions DOI 10.1007/s10530-012-0238-5

ORIGINAL PAPER

Restoring native forest ecosystems after exotic tree plantation in Madagascar: combination of the local ectotrophic species Leptolena bojeriana and Uapaca bojeri mitigates the negative inﬂuence of the exotic species Eucalyptus 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 Ó Springer Science+Business Media B.V. 2012

Abstract The objectives of this study were to two exotic tree species (Eucalyptus camaldulensis and determine the impact of two exotic tree species (pine Pinus patula) and in the same soils but previously and eucalypts) on the early growth of Uapaca bojeri cultured by L. bojeriana seedlings. This study clearly (an endemic tree species from Madagascar) via their shows that (1) the introduction of exotic tree species influence on soil chemical, microbial characteristics, induces significant changes in soil biotic and abiotic on ectomycorrhizal fungal community structures in a characteristics, (2) exotic-invaded soil significantly Madagascarian highland forest and to test the ability of reduces the early growth and ectomycorrhization of an early-successional ectomycorrhizal shrub, Lepto- U. bojeri seedlings and (3) L. bojeriana decreased lena bojeriana, to mitigate the impacts of these exotic these negative effects of the exotic tree species by species. Finally, we hypothesized that L. bojeriana facilitating ectomycorrhizal establishment and conse- could act as a natural provider for ectomycorrhizal quently improved the U. bojeri early growth. This propagules. Soil bioassays were conducted with study provides evidence that L. bojeriana can facilitate U. bojeri seedlings grown in soils collected under the ectomycorrhizal infection of U. bojeri and miti- the native tree species (U. bojeri and L. bojeriana) and gates the negative effects of the introduction of exotic tree species on the early growth of the native tree

R. Baohanta H. Ramanankierana E. Baudoin M. Lebrun R. Duponnois (&) R. Rasolomampianina N. Rakotoarimanga IRD, Laboratoire des Symbioses Tropicales et H. Randriambanona Me´diterrane´ennes (LSTM), UMR 113 CIRAD/INRA/ Laboratoire de Microbiologie de l’Environnement, IRD/SupAgro/UM2, Campus International de Baillarguet, Centre National de Recherches sur l’Environnement, TA A-82/J, Montpellier, France BP 1739, Antananarivo, Madagascar e-mail: [email protected]

J. Thioulouse Present Address: Laboratoire de Biome´trie et Biologie Evolutive, R. Duponnois CNRS, UMR 5558, Universite´ Lyon 1, Laboratoire Ecologie & Environnement, Unite´ associeéau 69622 Villeurbanne, France CNRST, URAC 32, Faculte´ des Sciences Semlalia, Universite´ Cadi Ayyad, Marrakech, Morocco Y. Prin A. Galiana CIRAD, Laboratoire des Symbioses Tropicales et Me´diterraneénnes (LSTM), UMR 113 CIRAD/INRA/ IRD/SupAgro/UM2, Campus International de Baillarguet, TA A-82/J, Montpellier, France 123 R. Baohanta et al. species. From a practical point of view, the use of influences soil development as well as plant growth ectotrophic early-successional shrub species should be (Schreiner et al. 2003; Duponnois et al. 2007). considered to improve forest resaturation after exotic Numerous studies have shown that ectomycorrhizal invasion. (ECM) vegetation is highly dependent on ECM fungi for their growth and survival (Smith and Read 2008). Keywords Ectomycorrhizas Uapaca bojeri Limitation of the presence, abundance, and commu- Exotic tree species Degraded forest ecosystems nity composition of ectomycorrhizal fungi can result Nurse plant Restoration ecology Revegetation from natural (Terwilliger and Pastor 1999) or anthro- strategies 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 Introduction demonstrated that exotic plant species could disrupt mutualistic associations involved in native ecological Numerous agricultural practices lead to soil degrada- associations (Callaway and Ridenour 2004; Kisa et al. tion and losses of biodiversity in tropical areas. These 2007; Remigi et al. 2008; Faye et al. 2009) that could anthropogenic impacts do not only degrade natural limit the natural regeneration of native tree species. plant communities (population structure and species However, these negative impacts on soil microbiota diversity) but also physico-chemical and biological may be counterbalanced by utilizing mycorrhizal soil properties such as nutrient availability, microbial native species that enhance the abundance, diversity, activity, and soil structure (Styger et al. 2007). In order and function of mycorrhizal propagules in soil (Kisa to reverse this loss of fertility and to limit soil erosion, et al. 2007; Faye et al. 2009). Recent studies have some revegetation programmes have been undertaken shown that some early-successional shrubs can in Madagascar using fast-growing exotic trees. Refor- preserve and/or increase the abundance and diversity estation with eucalyptus (E. robusta, E. rostrata, of mycorrhizal propagules of AM fungi (Ouahmane E. camaldulensis) and later pine (P. khesya, P. patula) et al. 2006) or ectomycorrhizal fungi (Dickie et al. provided wood for the region (Gade 1996). By the 2004) and subsequently facilitate forest woody species 1930s, plantations have been set out by local commu- growth. Improvement of seedling growth by pioneer nities, institutions, and individuals (Parrot 1925). shrubs, also called the ‘‘nurse plant effect’’, is a However, exotic trees can threaten ecosystems or general facilitative process (Niering et al. 1963). habitats by altering ecological interactions among Nurse plants facilitate vegetation growing beneath native plants (Rejmanek 2000; Callaway and Ridenour their canopies by ameliorating the physical environ- 2004) that could compromise their role in sustainable ment and by increasing soil fertility (Franco and Nobel development. Exotic plants can act directly on native 1988; Callaway and Pennings 2000; Scarano 2002). plant communities by allelopathic effects or by higher In Madagascar, the impacts of exotic tree species performance in an introduction site that influence such as pine and eucalypts on diversity and abundance vegetation dynamics, community structure, and com- of mycorrhizal fungal communities as well as on the position (del Moral and Muller 1970;The´baud and early growth of endemic tree species remain unknown. Simberloff 2001). They also can alter biochemical The aims of the present study were to determine in situ cycling compared with native plants (Ashton et al. and under glasshouse conditions the impact of Euca- 2005). As exotic and native plants have different lyptus camaldulensis and Pinus patula (two exotic tree evolutionary histories and traits, it has been also species) on soil chemical characteristics, microbial suggested that plant introduction could affect below- activities and on ECM community structures. We ground soil microbial communities (Hawkes et al. hypothesized that soil microbial activities and mycor- 2005; Batten et al. 2006; Kisa et al. 2007; Kivlin and rhizal communities will differentiate under these exotic Hawkes 2011). Among soil microbial communities, species leading to a decrease of the early growth of a mycorrhizal fungi are considered as key components native tree species, Uapaca bojeri. We further hypoth- of the sustainable soil–plant system (Johansson et al. esized that an enhancement of ectomycorrhizal diversity 2004; Dickie and Reich 2005). This symbiotic process provided by an early-successional ectomycorrhizal 123 Restoring native forest ecosystems shrub, Leptolena bojeriana, would minimize the neg- root pieces were randomly chosen from each root ative effects of these exotic species and consequently sample collected from each plant species. improve U. bojeri growth through a well-developed ectomycorrhizal root colonization. Finally, we tested the hypothesis that L. bojeriana could act as a natural Bioassays of soils collected under exotic tree provider for ectomycorrhizal propagules and could species (E. camaldulensis and P. patula) preserve the abundance and diversity of ectomycorrhi- and the native tree species (U. bojeri) zal fungi in stressful environments. Seven adult trees of each exotic species and of U. bojeri were randomly chosen in an approximately Materials and methods 5 ha area in the Arivonimamo forest. In order to avoid disruption of soil and more particularly changes in Study area mycorrhizal networks, seven intact blocks of soil were collected near each adult tree (about 50 cm from the The field experiment was conducted within the central trunk). Seven additional intact blocks were collected part of Madagascarian highland sclerophyllous forest in at 10-15 m from any targeted tree species (E. camal- a forest located at 50 km to the west of Antananarivo dulensis, P. patula, and U. bojeri trees) or other known (Arivonimamo site). The average annual rainfall was ectomycorrhizal plants. Intact monoliths of soil were 1,398 mm with a average monthly temperature of cut with shovel and immediately transferred into 26 °C. The vegetation is a mosaic of U. bojeri islands 15 cm diameter, 16 cm height plastic pots. and very scattered individuals ofintroduced tree species, In addition, soil samples were taken near each soil P. patula and E. camaldulensis. These trees dominate an block from the 0–10 cm layer and stored in sealed understorey mainly composed by early-successional plastic bags at field moisture content at 4 °C for further plant species such as Leptolaena bojeriana, Leptolaena measurements. For each soil sample, pH of a water soil pauciflora, Erica sp., Helychrisum rusillonii, Aphloia suspension was determined. The total organic carbon theaformis, Psiadia altissima, Rhus taratana, Vaccini- (TOC) was measured according to the ANNE method um emirnensis, Rubus apelatus and Trema sp. L. (Aubert 1978) and the total nitrogen by the Kjeldahl bojeriana was the most representative plant species in method. The available and total phosphorus soil this site with a cover contribution of about 43 %. contents were analyzed by colorimetry (Olsen et al. 1954). Acid and alkaline phosphatase activities were measured using p-nitrophenol benzene as substrate Analysis of the mycorrhizal status of trees (Schinner et al. 1996), and production of the and early-successional plant species p-nitrophenol product was determined colorimetri- cally at 650 nm. Fluorescein diacetate (FDA) hydro- Root samples were collected during the rainy season. lysis was assayed to provide a measurement of the Root identity was ascertained by tracing from the microbial global activity (Alef 1998). trunk to the fine root tips. Samples of 1–5 g (fresh Seeds of U. bojeri collected in the Arivonimamo weight) of fine roots were washed under running water forest were surface sterilized in hydrogen peroxide for and stored at 4 °C for further examination. Fine roots 10 min, rinsed and soaked in sterile distilled water for were examined for ECM infection under a dissecting 12 h, and germinated on 1 % agar. The germinating microscope. Morphological parameters following seeds were used when rootlets were 1–2 cm long. One Agerer (1987–1996) such as mantle color and struc- pre-germinated seed was planted per pot filled with ture, branching pattern and characteristics of rhizo- intact monolith of soil. The pots were randomized in morphs were used to categorize ectomycorrhizas into the greenhouse and seedlings grown under natural morphological type (morphotype) groups. For AM light (daylight of approximately 12 h, average daily infection, fine roots were stained following the method temperature of 25 °C). They were watered regularly of Phillips and Hayman (1970). The root pieces were with tap water without fertilizer. placed on a slide for microscopic observation under After 5 months of culturing, U. bojeri seedlings 250 magnification (Brundrett 1991). About fifty 1-cm were gently uprooted from the pots in order to keep the 123 R. Baohanta et al. root systems intact and to avoid root disruption. Then, Impact of early-successional ectomycorrhizal they were gently washed with running water. The shrub, Leptolena bojeriana on the characteristics percentage of ectomycorrhizal short roots (number of of soils collected under exotic tree species ectomycorrhizal short roots/total number of short (E. camaldulensis and P. patula) and the native tree roots) was assessed under a dissecting microscope species (U. bojeri) and on U. bojeri early growth by counting all single root tips. Ectomycorrhizal or non-ectomycorrhizal short roots were detected accord- Seeds of L. bojeriana were collected from the ing to the presence or absence of fungal mantle and Arivonimamo forest. They were surface sterilized mycelium and to the presence or lack of root hairs. In and were pre-germinated for 1 week in Petri dishes on each treatment, ECM root tips were classified by humid filter paper. A germinated seed was then morphotypes based on characteristics of their mantle transplanted into each of plastic pots filled with soil and extra-matrical mycelium (branching, surface monoliths sampled as described above under exotic color, texture, emanating hyphae, and rhizomorphs and native tree species. One set of pots was unplanted. (Agerer 1995). All morphological types of ectomy- There were 3 replicates for the unplanted pots and 6 for corrhizas were stored at -20 °C in 700 ll CTAB lysis the planted pots. The pots were randomized in a buffer (2 % cetylammoniumbromide; 100 mM Tris– greenhouse under natural light (daylight of approxi- HCl, 20 mM EDTA, 1.4 M NaCl) before molecular mately 12 h, average daily temperature of 25 °C) and analysis. Three ectomycorrhizas randomly selected watered daily with deionized water. After 4 months of from each morphotype groups were screened by RFLP growth, half of the L. bojeriana seedlings were cut and analysis and one sample of each unique RFLP patterns their aerial parts discarded without any disruptions of was sequenced. the cultural soil and L. bojeriana root systems. DNA was extracted from root tips using Qiagen Removal of aerial parts allowed to test the capacity DNeasy Plant Mini Kits (Qiagen SA, Courtaboeuf, of L. bojeriana seedlings to act as a provider of France) following the manufacturer’s recommenda- ectomycorrhizal propagules without any competitive tions. Fungal mitochondrial rDNA extracts were processes between each plant species for C acquisition amplified with ML5 and ML6 primers (White et al. and consequently to reduce symbiosis costs. Then, one 1990) and restriction digested HaeIII or HinfI pre-germinated seed of U. bojeri (treated as previously enzymes. Then, one sample of each individual RFLP described) was planted per pot randomized in the type was sequenced with the ABI Prism BigDye greenhouse and seedlings were cultivated under Terminator Cycle sequence kit (Applied Biosystems, natural light (daylight of approximately 12 h, average Foster City, CA, USA) and analyzed on an applied daily temperature of 25 °C). They were watered Biosystems model 310 DNA sequencer (Perkin- regularly with tap water without fertilizer. There were Elmer). Sequences were aligned by using Clustal X 3 treatments: (1) control (without pre-cultivation with 1.80 (Thompson et al. 1997) and alignment was L. bojeriana), (2) pre-cultivation and dual cultivation subsequently manually corrected using Genedoc with L. bojeriana (L. bojeriana treatment), and (3) pre- (Nicholas and Nicholas 1997). All sequences were cultivation dual cultivation with L. bojeriana without identified according to BLAST analysis at the NCBI aerial parts (L. bojeriana WA treatment). After page http://www.ncbi.nlm.nih.gov/blast/Blast.cgi,using 5 months of cultivation, measurements of chemical default settings. Sequences were deposited in and enzymatic soil characteristics as well as U. bojeri GenBank. ectomycorrhizal status, growth, and leaf mineral For each U. bojeri seedlings, the oven dry weight contents (N, P) were determined as described before. (1 week at 65 °C) of the aerial and root part was then measured. After drying, plant tissues were ground, Statistical analysis ashed (500 °C), digested in 2 ml HCl 6N and 10 ml

HNO3 N for nitrogen and then analyzed by colorim- Plant growth measurements and soil characteristics etry for P (John 1970). For nitrogen (Kjeldahl) were treated with one-way analysis of variance and determination, they were digested in 15 ml H2SO4 means were compared with the Newman–Keul multi- (36N) containing 50 g l-1 of salicylic acid. ple range test (p \ 0.05). The fungal colonization

123 Restoring native forest ecosystems

pffiffiffi indexes were transformed by arcsin ( x) before E. camaldulensis soils had intermediate TOC contents statistical analysis. A principal component analysis (Table 2). (PCA) was applied to the soil, plant, and microbial The acid phosphatase and FDA activities were parameters. The software used was the ade4 package significantly higher in the soils collected under the (Dray and Dufour 2007) for the R software for targeted tree species compared to the bulk soil but statistical computing (R Development Core Team these activities were higher in the soils sampled under 2010). 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 Results U. bojeri soil followed by the P. patula soil and finally by the bulk and E. camaldulensis soils (Table 2). Mycorrhizal status of trees and early-successional After 5 months of culturing, shoot and root bio- plant species in the Arivonimamo forest mass, total biomass of U. bojeri seedlings were significantly lower in the soil collected under All tree and shrub species recorded in the Arivonim- E. camaldulensis than in the other soil origins, amo forest formed mycorrhizas. Among these, 8 whereas the highest root and total growth were found presented AM infections and 5 were found with both in the U. bojeri soil (Table 3). Compared with the AM and ECM (Table 1). 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 Impact of targeted tree species on soil chemical the soil origins, root/shoot ratios ranged as follows: characteristics, ectomycorrhizal colonization, U. bojeri [ P. patula [ bulk soil (control) [ E. cam- and growth of U. bojeri seedlings aldulensis (Table 3). Nitrogen leaf contents were not significantly different among soil origins, whereas The highest soil acidity was recorded with the phosphorus foliar content of U. bojeri seedlings was E. camaldulensis origin followed by P. patula, significantly higher in the soil originating from around U. bojeri, and the bulk soil (Table 2). For N and P U. bojeri compared with P. patula soil (Table 3). soil contents, the opposite ranking was found with the Compared with the bulk soil, the extent of highest values recorded with E. camaldulensis soil ectomycorrhizal colonization was significantly higher (Table 2). The total organic matter in soil was in the soil collected under U. bojeri (73.7 %) and significantly higher in U. bojeri and the lowest value significantly lower in the E. camaldulensis soil was found in the bare soil whereas P. patula and (16.3 %) (Table 3). Structures of ectomycorrhizal

Table 1 Mycorrhizal Shrub and tree species Family Mycorrhizal status status of trees and early- successional plant species Leptolaena pauciﬂora Baker. Sarcolaenaceae ECM & AM in the Arivonimamo forest 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 ECM ectomycorrhizas, AM arbuscular mycorrhizas, Eucalyptus camaldulensis Dehn. Myrtacea ECM & AM ECM & AM co-existence of Pinus patula Schiede ex Schtdl. & Cham. Pinaceae ECM & AM arbuscular mycorrhizas and Uapaca bojeri L. Euphorbiaceae ECM & AM ectomycorrhizas 123 R. Baohanta et al.

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

1 2 pH (H2O) 5.26 (0.03) d 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 5.61 (0.05) a 6.69 (0.25) b 11.54 (0.65) c 15.33 (2.05) c (lg of hydrolyzed FDA h-1 g-1 of soil) Acid phosphatase activity 130.56 (31.8) a 314.01 (11.7) b 867.06 (50.7) c 586.51 (104.9) c (lg p-nitrophenol g-1 of soil h-1) Alkaline phosphatase activity 166.51 (6.91) a 302.54 (7.44) c 170.95 (8.47) b 82.54 (5.59) a (lg p-nitrophenol g-1 of soil h-1) 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) communities associated with U. bojeri root systems Responses of soil characteristics and U. bojeri in the different soil origins were signiﬁcantly different growth to the L. bojeriana cultivation (Table 4; Fig. 1). The RFLP types UA1 (Russula earlei), UA2 (Amanita sp.), UA3 (Thelephoroid A data table with 36 rows and 12 columns was symbiont), and UA4 (uncultured ECM fungus) constructed with the soil, plant, and microbial activity were only recorded on U. bojeri seedlings grown in parameters. The 12 variables were: pH, soluble U. bojeri soil, whereas in the soils collected under phosphorus, total nitrogen and total organic matter, exotic tree species, UD1 (Bondarcevomyces), UC3 total microbial activity, acid and alkaline phosphatase (Russula exalbicans), and UB6 (Boletellus projectel- activities, shoot and root biomass of U. bojeri lus) were found. In the bare soil, the RFLP type seedlings, ectomycorrhizal rate, leaf nitrogen and UC3 was mainly detected and two other types, phosphorus contents, and the Shannon diversity index UC2 (Boletus rubropunctus) and UB5 (Coltricia of the ectomycorrhizal fungal morphotypes. The 36 perennis) at lower abundances (Fig. 1). The RFLP rows corresponded to three samples of the four soil type UB4 (Xerocomus chrysenteron) was only origins: soil collected under E. camaldulensis, recorded in the E. camaldulensis soil treatment P. patula, U. bojeri, or bare soil. For each soil origin, (Fig. 1). three treatments were considered: U. bojeri seedling 123 Restoring native forest ecosystems

Table 4 Identification by ITS sequence of RFLP types for root system. The resulting data table was submitted to ectomycorrhizas collected on U. bojeri seedling after 5 month a principal component analysis (PCA) to describe the culturing in glasshouse conditions on soils collected under a main structures of this data set. native tree species (Uapaca bojeri), two exotic tree species (Pinus patula and Eucalyptus camaldulensis) and from the bare The Fig. 2 showed the results of this PCA. The soil (control) in the Arivonimamo forest upper part (Fig. 2a) graphic was the correlation circle RFLP GenBank Closest GenBank BLAST of all the parameters, and the lower part graphic types accession species expected (Fig. 2b) was the map of sample scores on the first two number value principal components. The correlation circle (Fig. 2a) showed that the first principal component (PC1) was UA1 AF518722 Russula earlei 2e-144 well correlated to plant growth, with better growth UD1 DQ534583 Bondarcevomyces taxi 3e-138 toward the right of the graphic (shoot biomass, leaf UA2 AM117659 Amanita sp. 0.0 phosphorus and leaf nitrogen contents) and also to the UA3 AJ509798 Telephoroid 1e-154 mycorrhizal sp. microbial activities (total microbial activity, acid and UC3 AY293269 Russula exalbicans 2e-170 alkaline phosphatase activity), to the ectomycorrhizal rate, and to the Shannon diversity index of ectomy- UA4 AY157720 Uncultured ECM 0.0 homobasidiomycete corrhizal fungi. The second principal component Clone E2 (PC2) was negatively correlated to root biomass UB6 DQ534582 Boletellus projectellus 0.0 increase and soil total nitrogen (downward arrows) UC2 FJ480421 Boletus rubropunctus 2e-171 and positively to organic matter and pH (upward UB5 None Coltricia perennis 2e-141 arrows). UB4 AD001659 Xerocomus 4e-173 The map of sample scores (Fig. 2b) showed on the chrysenteron PC1 the very strong effect of the L. bojeriana plant (solid arrows pointing right). This effect was positive, was planted alone, with a L. bojeriana seedling, or as it corresponded to an increase of U. bojeri seedling with a L. bojeriana seedling that aerial part was cut growth, of microbial activities, and of ectomycorrhizal after 4 months of cultivation, but keeping intact its fungal diversity. This effect was highest when the

Fig. 1 Similarities in ectomycorrhizal communities between Bondarcevomyces taxi, UA2: Amanita sp., UA3: Telephoroid U. bojeri seedlings growing in soils collected under Uapaca mycorrhizal sp., UC3: Russula exalbicans, UA4: Uncultured bojeri, Eucalyptus camaldulensis, Pinus patula and from a bulk ECM homobasidiomycete Clone E2, UB6: Boletellus projec- soil (d). Values are expressed by RFLP type percentages with tellus, UC2: Boletus rubropunctus, UB5: Coltricia perennis, regards to the soil treatments. UA1: Russula earlei, UD1: UB4: Xerocomus chrysenteron 123 R. Baohanta et al.

Fig. 2 Results of the PCA A on the data table of soil, PC2 plant, and microbial activity parameters. a Correlation circle of all the parameters. OM The 12 variables are: pH = pH, P = total pH AcP phosphorus (mg kg-1), N = total nitrogen (%), OM = total organic matter (%), FDA = total PN enzymatic activity, AcP = acid phosphatase, FDA AlkP = alkaline SB P PP phosphatase, SB = shoot H PC1 biomass (g), RB = root biomass (g), ER = ectomycorrhizal rate (%), PN = leaf nitrogen (%), PP = leaf phosphorus AlkP -1 (mg.kg ), H = Shannon ER diversity index of ectomycorrhizal fungi. N b Map of sample scores on the ﬁrst two principal components. Samples are RB coded as follows. The ﬁrst three characters correspond to the soil origin: Eca = soil collected under E. camaldulensis, Ppa = soil d = 1 collected under P. palida, B Ubo = soil collected under U. bojeri, BaS = bare soil. The treatment applied to the EcaU U. bojeri seedlings is coded EcaUL as folows. U = Uapaca PpaUL PpaULc plant alone, UL = Uapaca EcaULc plant ? L. bojeriana, Ulc = Uapaca plant ? PpaU L. bojeriana cut after BaSUL BaSULc 4 months cultivation. For example, sample coded ‘‘EcaULc’’ is a U. bojeri seedling grown in soil collected under BaSU E. camaldulensis in which a plant of L. bojeriana was grown and cut after 4 month cultivation UboULc UboUL

UboU

123 Restoring native forest ecosystems

Leptolena plant was cut and only the root system was L. bojeriana without aerial parts. Ectomycorrhizal left before planting U. bojeri seedlings. It was also colonization was significantly increased when U. interesting to notice that this effect was the same for bojeri seedlings were cultivated with L. bojeriana bare soil, for soils collected under exotic tree species without aerial parts (Table 6). This positive impact or for soil collected under a Uapaca adult tree. On the was also recorded on the composition of ectomycor- same graphic (Fig. 2b), the PC2 showed the soil origin rhizal communities with the same RFLP types (except effect (dotted arrows pointing upward), corresponding for UA3) as those found in the control treatment (UA1, to the negative influence of exotic tree species UA2 and UA4) and two others only detected with the (E. camaldulensis, P. patula) on root biomass. Root presence of L. bojeriana seedlings (Table 7). biomass was higher in soils collected under U. bojeri With E. camaldulensis soil, dual cultivation treat- adult tree and lower in soils collected under exotic tree ments significantly improved soil pH, nitrogen con- species. Bare soils have an intermediate position. tent, and enzymatic activities with highest effects Conversely, pH and total organic matter are higher in found in L. bojeriana seedlings without aerial parts for soils collected under exotic tree species. soil nitrogen content and FDA activity (Table 5). For each soil origins, the impact of L. bojeriana Opposite effects have been found for soil P content (with or without aerial parts) on soil characteristics, and soil organic matter (depressive effect provided by U. bojeri growth, and ectomycorrhizal communities L. bojeriana seedlings without aerial parts). Dual was indicated in Tables 5, 6, and 7. For the bulk soil cultivation treatments have enhanced the growth of U. origin and compared with the control, the treatment bojeri seedlings and ectomycorrhizal colonization but with L. bojeriana without aerial parts provided the no significant differences have been found between highest positive effects on pH, soluble P, soil N both L. bojeriana treatments (with or without aerial content, organic matter content and on microbial parts) and no effects have been recorded on the root/ enzymatic activities (Table 5). The dual cultivation of shoot values (Table 6). The presence of L. bojeriana L. bojeriana with or without aerial parts significantly seedlings allowed the development of some RFLP improved shoot and root biomass and mineral nutri- types not detected in the control treatment (UA1, UA2, tion of U. bojeri seedlings (N, P) (Table 6). Ectomy- UA3, UA4), increased the establishment of UB6 but corrhizal colonization was significantly improved limited UB4 multiplication (Table 7). when the dual cultivation was performed with For the P. patula soil origin, dual cultivation L. bojeriana without aerial parts (Table 6). Strong treatments significantly improved soil P content and modifications in the composition of ectomycorrhizal enzymatic activities, whereas the presence of entire communities occurred in the treatments with L. bojeriana seedlings significantly decreased soil L. bojeriana (Table 7). RFLP types, UC3 and UC2 nitrogen and organic matter contents (Table 5). recorded in the control treatment, were not found in U. bojeri shoot growth and leaf foliar contents (N, P) the dual cultivation treatments and replaced by the have been significantly promoted by L. bojeriana RFLP types UA1, UA2, and UB4. The RFLP type seedlings (entire or not) (Table 6), and ectomycorrhi- UB6 was only recorded in the treatment with entire zal colonization was higher in the dual cultivation L. bojeriana seedlings (Table 7). treatment involving L. bojeriana seedlings without For the U. bojeri soil origin, dual cultivation with aerial parts (Table 6). Only UB6 RFLP type was entire L. bojeriana seedlings increased all the mea- detected in all the treatments, whereas UC3 recorded sured soil parameters except for pH (Table 5). Elim- in the control treatment was absent in the dual inating the aerial parts of L. bojeriana seedlings led to cultivation treatments (Table 7). An opposite pattern higher increases of N, organic matter soil contents and was found with UA1 and UA4 RFLP types (Table 7). 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 Discussion with highest data for the treatment without aerial parts (Table 6). No significant effect has been found on root This study clearly shows that (1) the introduction of growth and root/shoot ratio but shoot growth of exotic tree species induces significant changes in the U. bojeri seedlings was significantly improved with soil chemical characteristics, microbial activities and 123 123

Table 5 Effect of L. bojeriana/U. bojeri succession (pre-cultivation with L. bojeriana and dual cultivation with L. bojeriana seedlings with aerial parts or without aerial parts) on soil chemical characteristics and enzymatic activities 4 5 6 7 8 9 Treatments pH H2O Sol P Total N Total OM FDA Ac P Alk P

Bulk soil Control1 5.710 (0.01) a11 2.00 (0.06) a 0.022 (0.001) a 4.20 (0.06) a 32.0 (6.4) a 498.8 (31.9) a 274.6 (6.2) a L. bojeriana2 5.9 (0.01) b 4.47 (0.09) b 0.024 (0.001) a 6.33 (0.04) b 46.9 (1.4) ab 1,046.4 (52.1) b 359.5 (113.7) ab L. bojeriana WA3 6.2 (0.02) c 5.50 (0.11) c 0.103 (0.001) b 9.65 (0.03) c 57.5 (3.6) b 1,334.5 (82.6) c 383.7 (22.1) b U. bojeri soil Control 5.4 (0.01) b 5.35 (0.03) a 0.301 (0.001) a 7.32 (0.01) a 5.2 (0.36) a 715.6 (19.5) a 404.1 (11.6) a L. bojeriana 5.4 (0.01) b 6.80 (0.06) c 0.412 (0.001) b 8.32 (0.04) b 49.8 (3.4) b 980.7 (23.4) b 512.2 (22.3) b L. bojeriana WA 5.3 (0.02) a 6.32 (0.06) b 0.423 (0.001) c 8.72 (0.07) c 63.5 (2.2) c 1,044.3 (24.9) b 582.5 (17.7) b E. camaldulensis soil Control1 5.3 (0.007) a 9.23 (0.03) c 0.054 (0.001) a 15.76 (0.09) b 6.1 (1.6) a 1,213.5 (19.9) a 214.3 (5.6) a L. bojeriana2 6.3 (0.009) c 4.43 (0.09) b 0.064 (0.002) b 15.80 (0.06) b 21.4 (3.1) b 1,447.2 (48.1) b 417.1 (26.3) b L. bojeriana WA3 5.4 (0.006) b 3.70 (0.06) a 0.071 (0.001) c 14.25 (0.03) a 68.3 (5.3) c 1,597.6 (8.3) c 394.4 (43.6) b P. patula soil Control 6.2 (0.01) a 3.36 (0.09) a 0.087 (0.001) b 14.47 (0.09) b 22.2 (3.8) a 558.3 (55.2) a 288.5 (4.5) a L. bojeriana 6.3 (0.01) a 7.60 (0.11) c 0.073 (0.001) a 14.05 (0.03) a 100.4 (8.6) c 1,257.1 (37.5) b 567.9 (18.3) c L. bojeriana WA 6.2 (0.01) a 4.40 (0.06) b 0.090 (0.001) b 14.68 (0.06) b 66.4 (4.4) b 1,594.9 (49.3) c 331.4 (14.5) 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 Soluble phosphorus (mg kg-1). 5 Total nitrogen (%). 6 Total organic matter (%). 7 Total microbial activity (lg of hydrolyzed FDA h-1 g-1 of soil). 8 Acid phosphatase activity (lg p-nitrophenol g-1 of soil h-1). 9 Alkaline phosphatase activity (lg p- nitrophenol g-1 of soil h-1). 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) .Boat tal. et Baohanta R. Restoring native forest ecosystems

Table 6 Effect of L. bojeriana/U. bojeri succession (pre-culti- collected under Uapaca bojeri, Eucalyptus camaldulensis, Pinus vation with L. bojeriana and dual cultivation with L. bojeriana patula and from a bulk soil after 5 month culture in glasshouse seedlings with aerial parts or without aerial parts) on the growth conditions and ectomycorrhizal colonization of U. bojeri seedlings in soils 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) on ectomycorrhizal communities, (2) exotic-invaded results are in accordance with these previous studies soil significantly reduces the early growth and ecto- for soil N contents. However, we report higher rates of mycorrhization of U. bojeri seedlings, and (3) ecto- acid phosphatase activity under exotic plant species trophic early-successional shrub species such as (P. patula and E. camaldulensis) that probably result L. bojeriana could lower these negative effects from the more acid conditions encountered under provided by E. camaldulensis and P. patula by these two exotic species and in contrast suppress facilitating ectomycorrhizal establishment and conse- alkaline phosphatase activities (Acosta-Martinez and quently improved the U. bojeri early growth. Tabatai 2000;Kra¨mer and Green 2000). These results Numerous studies have reported that the introduc- are in accordance with those of Kourtev et al. (2002)as tion of exotic tree species has an environmental impact the higher rates of acid phosphatase reflected the on soil characteristics (i.e., soil nutrient contents, organic-rich horizons with large amounts of recalci- water dynamics, etc.) (Smith et al. 2000; Sicardi et al. trant compounds which accumulate under E. camal- 2004) but with opposite results on soil biofunctioning dulensis and P. patula. indicators. For instance, Sicardi et al. (2004) reported All these biological changes have resulted to a that the conversion of pasture land to planted Euca- lowest early growth of U. bojeri seedlings and in lyptus grandis forest decreased FDA hydrolysis, acid particular to a decrease of ectomycorrhiza formation. and alkaline phosphatase activities that are directly A previous study suggested that Pinus spp. was enable involved in the transformation of soil organic matter. to associate with native fungi in exotic habitats leading On the opposite, other studies have shown higher to unsuccessful establishment when ECM fungi are availability of nitrogen in exotic-invaded soils lacking (Mikola 1970). It agrees with our data where (Kourtev et al. 1999; Ehrenfeld et al. 2001). Our this tree species selected a few ectomycorrhizal 123 R. Baohanta et al.

Table 7 Relative abundance of RFLP types harvested in in soils collected under Uapaca bojeri, Eucalyptus camaldul- U. bojeri seedlings in the cultural patterns with L. bojeriana ensis, Pinus patula and from a bulk soil after 5 month culture (pre-cultivation with L. bojeriana and dual cultivation with in glasshouse conditions L. bojeriana seedlings with aerial parts or without aerial parts) 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: Boletus rubropunctus, UB5: Coltricia perennis, UB4: Xerocomus chrysenteron symbionts such as Russula exalbicans. This ectomy- allelochemicals (del Moral and Muller 1970). Hence, corrhizal genus was largely distributed in tropical this allelopathic effect could limit the U. bojeri growth areas (Ducousso et al. 2004; Rivie`re et al. 2006; seedling and in particular root system development Die´dhiou et al. 2010) and frequently recorded under leading to a lower ectomycorrhiza establishment. tropical tree species (Rivie`re et al. 2005, 2006). In Uapaca bojeri seedlings growing in soil collected contrast to pine, it has been suggested that Eucalyptus under U. bojeri adult tree showed much higher spp. (i.e., E. robusta) was able to contract ectomycor- ectomycorrhizal infection and growth than those rhizal associations in their introduction area with most growing in the soil collected at a distance from of the native ectomycorrhizal symbionts (Tedersoo established ectomycorrhizal vegetation. These data et al. 2007). Our results partially corroborated these are consistent with results of previous works where it data since ectomycorrhizal community associated has been demonstrated that a lack of ectomycorrhizal with U. bojeri seedlings grown in soil collected under infection distant from ectomycorrhizal vegetation or E. camaldulensis was more diverse than that found in from adult tree that provides ectomycorrhizal propa- soil sampled under P. patula. However, E. camaldul- gules to the young seedlings could influence nutrient ensis has negatively influenced the ectomycorrhizal uptake and growth of seedling (Baxter and Dighton establishment and consequently U. bojeri seedling 2001; Lilleskov et al. 2002; Dickie and Reich 2005; growth largely than that which has been measured Kisa et al. 2007). Moreover, it has been suggested that with P. patula soil. It is well known that Eucalyptus a rapid and early integration of seedlings into drastically alters the vegetation development where ectomycorrhizal mycelium radiating from mother Eucalyptus litter accumulates through the release of plants could significantly improve survival and growth

123 Restoring native forest ecosystems of seedlings (Janos 1980, 1996; Onguene and Kuyper significantly enhanced ectomycorrhizal colonization 2002). Our data support these observations with of U. bojeri seedlings. This nursing effect was more U. bojeri in a Madagascarian highland forest and particularly recorded in the treatments with exotic- showed that this tree species acts as a mother tree or invaded soils. In the P. patula and E. camaldulensis nurse tree by promoting ectomycorrhizal formation and soils, L. bojeriana stimulated U. bojeri total growth by seedling growth. High root/shoot ratio has been iden- 2.39 and 3.49, respectively, whereas this positive tified as an important factor allowing plants to exploit effect was 1.39 with the U. bojeri soil. This result reduced resource availability due to patchiness in supports the hypothesis that facilitation generally distribution, both for water and nutrients (Reader et al. increasing in importance with increasing abiotic stress 1992). These high ratios would be of great importance (Liancourt et al. 2005). In addition N, P nutrient uptake in the regeneration process of native tree species of U. bojeri seedlings was significantly enhanced in the especially during periods of drought or where nutrient dual cultivation treatments. Foliar N and P contents resources are heterogeneously distributed. Hence, the were significantly correlated with ectomycorrhizal exotic tree species (P. patula and E. camaldulensis) colonization. Hence, by facilitating ectomycorrhizal could limit the growth of U. bojeri young regeneration, propagule multiplication, L. bojeriana enhanced ecto- whereas the presence of U. bojeri mother tree facilitated mycorrhizal infection of U. bojeri that is known to the early development of U. bojeri seedlings. improve plant nutrient uptake (Dickie et al. 2002). In In tropical forests, one of the main biological addition, U. bojeri nutrition may benefit from the processes that ensure recovery rates of tree species ectomycorrhizal network radiating from L. bojeriana depends on the amount and activity of mycorrhizal root systems that explores a larger volume of soil than inoculum. Ectomycorrhizal mycelia radiating from U. bojeri alone. These connections could lead to N and P mother tree roots function as a source of ectomycorrhi- or carbon transfers between U. bojeri and L. bojeriana zal infection for neighboring host plants and more seedlings via mycorrhizal linkages (Simard et al. 1997). particularly for young tree regeneration (Jonsson et al. No significant effect has been recorded between treat- 1999; Matsuda and Hijii 2004;Nara2005). In addition ments with entire L. bojeriana seedlings and L. bojeri- plants could become connected to a common mycor- ana seedlings without aerial parts. It suggests that no rhizal network that could be highly beneficial for growth competitive interactions occur between each plant and fitness of seedlings (Nara 2005). When ectomycor- species. The association in a common mycelial network rhizal potential (abundance and diversity of ectomy- of each plant species has probably lowered the cost of corrhizal propagules) is lowered following natural or establishing mycorrhizal infection (Newman 1988). anthropogenic disturbance (Allen 1987; Jones et al. This study provides evidence that L. bojeriana can 2003), seedling establishment is limited and it is facilitate the ectomycorrhizal infection of U. bojeri necessary to reinforce ectomycorrhizal infection poten- and mitigates the negative effects of the introduction tial. It has been previously demonstrated that a of exotic tree species on the early growth and herbaceous ectomycorrhizal perennial of prairies, He- ectomycorrhizal formation of the native tree species. lianthemum bicknellii, could permit the survival of However, the mechanisms involved in this nursing ectomycorrhizal propagules and create patches of high effect have to be elucidated since multiple abiotic and ectomycorrhizal infection potential that facilitate the biotic factors are involved. From a practical point of establishment of Quercus, an ectomycorrhizal tree view, the use of ectotrophic early-successional shrub species (Dickie et al. 2004). From the present study, species has to be considered in tropical areas to similar effects have been provided by the ectomycor- improve the performances of reafforestation programs rhizal shrub species, L. bojeriana. Among ectotrophic with native tree species. early-successional plants recorded in the studied area, Leptolaena genus was highly represented and facilitated ectomycorrhizal infection and growth of U. bojeri References seedlings but also enhanced soil chemical characteristics and enzymatic activities. Since L. bojeriana shared Acosta-Martinez V, Tabatai MA (2000) Enzyme activities in a limed agricultural soil. Biol Fert Soil 31:85–91 ectomycorrhizal fungi with U. bojeri (i.e. Russula Agerer R (1987–1996) Colour atlas of ectomycorrhizae. Ein- earlei, Amanita sp., etc.), this shrub species has horn-Verlag Eduard Dietenberger, Schwa¨bisch Gmu¨nd 123 R. Baohanta et al.

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