Dissertations / Theses on the topic 'Bacteria mycorrhiza'

Kyoto University (京都大学)
0048
新制・課程博士
博士(農学)
甲第14058号
農博第1728号
新制||農||961(附属図書館)
学位論文||H20||N4396(農学部図書室)
UT51-2008-F450
京都大学大学院農学研究科地域環境科学専攻
(主査)教授 二井 一禎, 教授 武田 博清, 教授 縄田 栄治
学位規則第4条第1項該当

La première étape de la thèse a consisté à comparer la diversité et la structure génétique des populations de champignons mycorhiziens à arbuscules (MA) associées à quatre espèces de médiques annuelles. Les résultats obtenus montrent que l’abondance des champignons MA différait, indiquant que la structure génétique de la communauté fongique a été influencée par l’espèce végétale. La deuxième étape de ce travail visait à tester l’hypothèse selon laquelle la longue histoire évolutive entre champignons MA et plantes ne se serait pas faite de façon indépendante des bactéries. La structure génétique des populations bactériennes associées aux racines mycorhizées et non de M. Truncatula a été comparée. Les communautés bactériennes différaient de façon significative, ces différences étant expliquées par des marqueurs moléculaires associés aux familles des Oxalobacteraceae et Comamonadaceae. Six isolats représentatifs des populations associées aux racines mycorhizées et deux souches de référence (Collimonas fungivorans Ter331 et Pseudomonas fluorescens C7R12) ont été testées afin de déterminer leur effet sur la mycorhization. Une souche appartenant aux Oxalobacteraceae (Collimonas sp. J5B4) et P. Fluorescens C7R12 ont amélioré la croissance du champignon AM et sa colonisation racinaire, confirmant partiellement l’hypothèse que les bactéries associées aux racines mycorhizées auraient un effet favorable sur la mycorhization. Enfin, la caractérisation de l’effet promoteur de P. Fluorescens C7R12 sur la mycorhization a montré que cet effet était spécifique du champignon MA et que la colonisation des cellules bactériennes différait sur les racines mycorhizées et non-mycorhizées
In the first part of the thesis, possible effect of the plant genotype on the genetic diversity and structure of the arbuscular mycorrhizal (AM) fungal community was assessed. Results indicated a similar diversity of AM fungi in the four Medicago species used. However, the abundance of AM fungi differed significantly upon the plant species, indicating preferential associations between AM fungal and plant genotypes. The second part of the thesis was based on the hypothesis that the long joint evolution of AM fungi and plants did not occur independently of the associated bacteria. To test this hypothesis, the genetic structure of bacterial communities associated with mycorrhizal and non mycorrhizal roots of M. Truncatula was compared. The bacterial communities differed significantly between mycorrhizal and non mycorrhizal roots, these differences being explained by sequences belonging to Oxalobacteraceae and Comamonadaceae families. Six bacterial isolates representative of the populations belonging to Oxalobacteraceae and Comamonadaceae were tested together with two reference strains (Collimonas fungivorans Ter331 and Pseudomonas fluorescens C7R12) for their effect on mycorrhization. One strain belonging to Oxalobacteraceae (Collimonas sp. J5B4) and P. Fluorescens C7R12 promoted both AM fungal growth and mycorrhization, partially confirming our hypothesis that bacteria preferentially associated with mycorrhizal roots would be beneficial to the symbiosis. Finally, the characterization of the promoting effect of P. Fluorescens C7R12 showed that it was fungal specific and that the pattern of colonization of bacterial cells on mycorrhizal and non mycorrhizal roots differed

Arbuscular mycorrhizal (AM) fungi are naturally occurring in roots of terrestrial plants. AM fungi are capable of benefiting the host plant through various mechanisms such as enhanced nutrient supply, alleviation of environmental stress and inhibition of plant fungal pathogens. AM fungal spore-associated bacteria have been previously isolated and shown to have plant growthpromoting (PGP) abilities by several authors. Some bacterial isolates are able to promote AM fungal colonisation of host plants and are known to be mycorrhizal helper bacteria (MHB). This study focused on the isolation of AM fungal spore-associated bacteria, characterization of the isolates according to plant growth promoting abilities and evaluation of their potential to enhance plant growth and mycorrhizal colonisation. AM fungi were extracted from soils sampled from natural indigenous forest sources, raspberry (Rubus idaeus cv. Heritage) and strawberry (Fragaria ananassa) farms in South Africa and from a raspberry (Rubus idaeus cv. Autumn Bliss) plantation in Argentina. A total of 52 sporeassociated bacteria were isolated from the external and internal surfaces of AM fungal spore morphotypes from the two countries. The bacterial isolates were evaluated for their PGP abilities such as phosphate solubilisation, indole-3-acetic acid production, ammonia production and inhibition of the fungal pathogens Fusarium oxysporum and Phythophthora nicotianae through mechanisms such as siderophore and/ or hydrolytic enzyme production. A total of 23 bacterial isolates from both South Africa and Argentina showing the most potential to be PGP, were identified molecularly as belonging to the genera Acinetobacter, Alcaligenes, Bacillus, Microbacterium, Micrococcus, Serratia and Staphylococcus. The ability of ten selected bacterial isolates showing multiple PGP capacity were evaluated for their plant growth promotion and mycorrhizal colonisation enhancement ability on raspberry (Rubus idaeus cv. Meeker). Significant differences in increased shoot and root dry weights were shown by the treatments compared to the uninoculated control. The highest increase in shoot and root dry weights were shown by South African (Bacillus mycoides) and Argentinean (Alcaligenes faecalis) isolates. AM fungal colonisation was significantly enhanced by the South African (Bacillus mycoides) and Argentinean (Micrococcus luteus) isolates compared to the AM fungal singly inoculated control.

South Africa is the world's second largest exporter of fresh citrus and is ranked 14th in citrus production. Fungal pathogens such as Phytophthora and Pythium cause economic losses as a result of root rot and brown rot. Mycorrhizal fungi are specialized members of the fungal community forming a mutualistic relationship with plant roots. Mycorrhizal fungal structures are known to associate with other soil microorganisms and these may contribute to improved plant growth. A diverse group of bacteria that interact with the mycorrhizal fungi are known as Mycorrhizal Helper Bacteria (MHB). The aim of this study was to investigate the role of arbuscular mycorrhiza and associated bacteria isolated from spores and determine whether they had any plant growth promoting potential. A total of 19 bacteria were isolated from arbuscular mycorrhizal spores and were molecularly identified as belonging to several Bacillus, Micrococcus, Onchrobactrum and Staphylococcus sp. All bacterial isolates were tested for plant growth promotion abilities. One Bacillus isolate was able to solubilise phosphate. Four isolates Micrococcus sp, Micrococcus leteus, Ochrobacterum sp and Ochrobacterum antropi were able to produce Indole Acetic Acid and three isolates showed potential to reduce growth of Phytophthora nicotianae, P. citrocola and P. citrophthora in in vitro plate cultures. Further tests using culture supernatants of the Bacillus sp, Micrococcus sp and Bacillus cereus confirmed their ability to inhibit or reduce growth of the three Phytophthora species in a 96 well bioassay. Bacillus sp and Bacillus cereus were able to inhibit Phytophthora spp by 95 to 100 % and Micrococcus spp was able to decrease pathogen growth by 60 to 94 %. These bacterial isolates were further evaluated for plant growth promoting abilities on citrus rough lemon seedlings alone or in combination with arbuscular mycorrhizal inoculum. Bacterial and mycorrhizal inoculants influence the increase in shoot and root biomass. Bacillus cereus in combination with mycorrhizal inoculum significantly increased seedling shoot to root ratio while root biomass was significantly increased with mycorrhizal inoculation. Due to the short duration of the trial mycorrhizal colonisation could not be assessed. It is evident that selected combinations of bacteria and mycorrhizal fungi could promote citrus seedling growth and potentially improve seedling health. Further studies under nursery conditions are recommended.

[Truncated abstract] Genus Burkholderia encompasses a diverse collection of bacteria that inhabit rhizospheres throughout the world. Species can provide beneficial returns for eukaryotes, such as nitrogen fixation and nodule formation in plants and biocontrol of cropping systems. Burkholderia members can also cause disease in various animals, fungi and plants. These seemingly conflicting characteristics point to the capacity of Burkholderia spp. to interact with diverse eukaryotes. Within terrestrial ecosystems, Burkholderia spp. must negotiate favourable outcomes with both the primary producers and the primary decomposers, namely plants and fungi. It is these ongoing negotiations which govern many rhizosphere processes and lead to niche differentiation for Burkholderia spp. This research set out to design an in vitro model for investigating Burkholderiaeukaryote interactions. Surface and cellular interactions between Burkholderia spp. and both plants and fungi were then investigated. Specifically, mechanisms of adherence and invasion of plant and fungal cells were studied. The Burkholderia spp. B. vietnamiensis and B. pseudomallei were applied to mycorrhizal fungus spores as well as to several plant species. Bacterial inoculation had varying effects on germination of plant and fungal dormant forms. B. vietnamiensis-inoculation consistently increased Gigaspora decipiens spore germination, while B. pseudomallei produced no significant change. The effect of B. vietnamiensis on Acacia colei seed germination was density dependant, resulting in either increases or decreases in seed germination rates. ... Detection of B. pseudomallei in surface waters and soils was improved by the use of a rapid on-site molecular method. The related species B. thailandensis and B. ubonensis were also cultured from northern Western Australia. Mycorrhizal spores were isolated from soils of melioidosis-endemic regions. Burkholderia spp., including B. pseudomallei and B. vietnamiensis were detected in extracts of these mycorrhizal spores. Therefore, associations of Burkholderia spp. with mycorrhizal spores extend beyond the in vitro setting. These studies have increased our understanding of several specific interactions between Burkholderia spp. and eukaryotes of the rhizosphere. Common themes in adherence and invasion have emerged. Burkholderia spp. are able to closely associate with eukaryotes and to gain access to protected niches. Such access helps to explain the persistence of these bacteria in the environment during periods of desiccation and nutrient limitation.

L'objectif de cette thèse est de contribuer à faire progresser les connaissances sur les interactions bénéfiques entre les plantes et les microorganismes en évaluant la contribution des systèmes de sécrétion de type III (SST3). Une synthèse des connaissances disponibles relatives aux SST3 chez les Pseudomonas non pathogènes, saprotrophes ou mutualistes, présentée chapitre I, montre que les SST3 ne sont pas cantonnés aux interactions parasites ou pathogènes avec les plantes. Dans l'étude expérimentale présentée chapitre II, nous avons utilisé différents génotypes de Medicago truncatula Gaertn. cv. Jemalong capables (Myc+) ou non (Myc-) d'établir une symbiose mycorhizienne. Ce travail nous a permis de montrer que les Pseudomonas spp. fluorescents possédant un SST3 (SST3+) sont préférentiellement associés aux racines mycorhizées des génotypes Myc+ de M. truncatula (J5 et TRV48) plutôt qu'aux racines du mutant Myc- (TRV25) et au sol nu. Ainsi, la plante seule n'est pas à l'origine de la présence accrue des Pseudomonas SST3+. La colonisation de la racine par les champignons mycorhizogènes à arbuscules (CMA), le développement du mycélium intraradiculaire et/ou la formation associée d'arbuscules, sont également déterminants. Dans l'étude présentée chapitre III, nous avons comparé les effets de la souche modèle promotrice de mycorhization (MHB) P. fluorescens C7R12 (SST3+) et de son mutant C7SM7 (SST3-), sur la mycorhization et la croissance de M. truncatula dans un sol non stérile. Ce travail a permis de montrer que le SST3 de C7R12 contribue à l'effet MHB de la bactérie. La promotion de la colonisation de la racine de M. truncatula par les CMA indigènes induite par le SST3 de C7R12 s'est traduite par une amélioration de la croissance de la plante. En revanche, l'inactivation du SST3 chez C7SM7 a eu un impact délétère sur la colonisation de la racine de M. truncatula par les CMA du sol étudié et sur la croissance de la plante. L'observation d'effets quantitatifs opposés entre C7R12 et C7SM7, nous a conduits à nous interroger sur l'existence d'un effet différentiel de l'inoculation de ces bactéries sur la structure et la diversité des communautés des microorganismes associés. Dans une étude présentée chapitre IV, le suivi dynamique en parallèle de la structure des communautés totales bactériennes (B-RISA) et fongiques (F-RISA) et de la colonisation de la racine par les CMA a été réalisée. Aucun effet de l'inoculation n'a été observé sur la structure des communautés fongiques de la rhizosphère ou des racines. En revanche, la structure des communautés bactériennes a varié selon que les plantes aient été inoculées ou non et selon la souche inoculée. Néanmoins, ces différences ont été observées plusieurs semaines après les effets de l'inoculation de C7R12 ou de C7SM7 sur la colonisation de la racine par les CMA. Ce décalage dans le temps, suggère que les différences observées dans la structure des communautés bactériennes pourraient être une conséquence plutôt qu'une cause des variations observées sur la mycorhization de M. truncatula. Nos résultats n'ont pas permis de mettre en évidence d'effets de l'inoculation sur la diversité des populations des bactéries fixatrices d'azote présentes dans les nodosités de M. truncatula. L'analyse des séquences de la grande sous-unité de l'ADN ribosomique (LSU rDNA) amplifiées à partir d'ADN extrait des racines, a montré pour les plantes inoculées et non inoculées, que les populations de CMA étaient majoritairement apparentées à Glomus intraradices. Un groupe d'isolats spécifiquement associé aux racines inoculées avec C7R12 et apparenté à G. claroideum a été décrit. Le groupe spécifique pourrait être associé à l'amélioration de la mycorhization observée dans les racines inoculées avec C7R12. Néanmoins, compte tenu de sa faible représentation numérique (8%), il semble probable que l'inoculation de C7R12 ait aussi un effet quantitatif sur la colonisation de la racine de M. truncatula par les CMA. etc

Machair is a habitat found predominantly in coastal areas of the north–west of Scotland and is characterised by a low-input, rotational arable system interspersed with semi-natural grasslands. Land management is an essential part of the machair system as both intensification and abandonment threaten its future. Little is known about this system in terms of its below ground communities. A survey was conducted over a three season, two year period for molecular determination of bacterial, AM fungal and nematode community structure. Cropped, fallow and undisturbed grassland were sampled; soil and roots associated with two commonly occurring species and composite soil samples were collected. In addition the effect of edaphic factors and vegetation composition on soil communities was assessed, both in the field and in greenhouse experiments. Results show that although all of the examined factors impacted the community structure of the three soil groups to some degree, the strength of this effect varied between groups. Bacterial communities were mainly affected by temporal factors, particularly year, possibly due to variation in soil moisture content. AM fungal community structure also varied considerably with season, but the mechanisms behind this were less obvious, with both abiotic and plant factors playing a role. AM fungal community structure varied with different plant hosts but also with soil moisture content. In contrast to both bacteria and AM fungi, nematode communities showed a strong response to land use although temporal factors and differences between locations were also observed. Grasslands harboured a distinctly different community structure from cropped and fallow, largely as a result of variation in bacterivores and carnivores. Vegetation composition and soil moisture content were also found to affect nematode community structure. This study has revealed that machair soil communities seem to be highly complex, dynamic and adapted to the changeable conditions that persist.

Maize (Zea mays L.) is a direct staple food crop in Africa and remains an essential component of global food security, with maize crops accounting for over 60% of the total harvested area of annual food crops. Stress caused by drought and high soil salinity limits crop growth and productivity more than any other single environmental factor, with grain yield reductions up to 76% depending on the severity of the drought and the plant growth stage. Arbuscular mycorrhizal (AM) fungi and Plant Growth Promotion Rhizobacteria (PGPR) have previously been shown to improve tolerance of plants to drought stress through a number of chemical and physiological processes. The aim of this investigation was to determine whether mycorrhizal fungi and rhizobacteria adapted to drought and saline conditions and possessing plant growth promoting (PGP) traits were able to stimulate plant growth responses when applied to Zea mays seeds growing under greenhouse conditions Bacterial isolates selected were tolerant to concentrations of NaCl up to 600 mM and maintained 50% growth at low water potentials (-1.44 MPa). They were positive for Indole Acetic Acid (IAA) production, phosphate solubilisation and secretion of siderophores. Bacterial isolates showing plant growth promoting potential were identified using 16S rDNA gene sequencing as Achromobacter xylosoxidans strains A8 and C54 and Klebsiella oxytoca strain M1. Mixed inoculum was prepared from indigenous communities of mycorrhizas in soils sampled from the Cerebos Salt Pan and the Kalahari Desert. Mycorrhizal diversity was investigated using 454-Pyrosequencing which revealed that the community composition was dominated by species in the Ambispora, Glomus and Paraglomus genera with a rare component represented by species in the Redeckera, Archaeospora and Geosiphon genera. Microscopic examination of plant roots at the end of the trial revealed the presence of diagnostic mycorrhizal structures within the root cells, confirming that colonization was successful. Plant growth response to microbial inoculation was assessed by monitoring changes in plant photosynthetic capacity over the duration of a 7 week pot trial. A significant difference in photosynthetic and biomass data was observed between drought and well-watered groups but no mycorrhizal or bacterial treatment effect was evident within the groups, despite the high levels of colonization by mycorrhizas. These results suggest that the beneficial effects of mycorrhizal colonization may be primarily attributed to improved nutrient and mineral uptake in conditions where nutrients are limiting, resulting in improved growth. The improved growth may then have secondary effects on the plant‟s ability to withstand drought. Having controlled for nutrient deficiency, it was not evident in this study that mycorrhizal fungi were able to stimulate a change in plant physiology and confer drought tolerance under the conditions imposed.

Empresa Brasileira de Pesquisa Agropecuária
The knowledge of the level of association that occurs between diazotrophic bacteria and fungi arbusculares mycorrhizae (AMF) in grassland ecosystems may be important for the understanding of the changes caused by the addition of fertilizers containing phosphorus (P) and nitrogen (N), in the production and botanical composition of natural pastures. The objective of this study was to evaluate. (a) the occurrence of three genera of diazotrophic bacterial in the root system under fertilization with N and P, and determine the contribution of N via BNF, and (b) evaluate the behavior of the association between AMF and native grasses. Four most abundant grasses in natural grasslands of the Southern Campos in Rio Grande do Sul , Axonopus affinis, Paspalum notatum, Andropogon lateralis and Aristida laevis were grown in pots of 5 kg, in a greenhouse, two studies being conducted (A e B ). In A, two treatments were applied: 50 mg kg-1 soil P and 100 mg kg-1 of soil N (NP) and a control, being evaluated, the number of diazotrophic bacteria of the genera Azotobacter, Azospirillum and Herbaspirillum, and the contribution of BNF was determined by the technique of natural 15N abundance. In B, the treatments consisted of applying 50 mg kg-1 soil P (P), application of 50 mg kg-1 soil P and 100 mg kg-1 of soil N (NP), and a control, in both treatments mycorrhizal colonization was determined. For A, A. laevis demonstrate to be more dependent on biological N fixation than the other species. The grass P. notatum compared with other species demonstrated to be more efficient to absorb available soil N. The dry matter accumulation in shoots of the native species was higher with the application of NP. In B the mycorrhizal colonization was similar between the control, P and NP to the roots of A. lateralis and A. laevis, thus presenting a greater dependence on the mycorrhizal association. Differently, in A. affinis and P. notatum, the mycorrhizal colonization was lower when subjected to fertilization with P and NP, thus presenting a lower dependence. It is concluded for A that fertilization with N and P reduces diazotrophic colonization, increasing the production of dry matter and N content of the tissue. A. laevis showed the highest contribution of biological nitrogen fixation, since P. notatum showed higher N accumulation in soil. In relation to B, A. laevis and A. lateralis have a higher dependence on the mycorrhizal than A. affinis and P. notatum.
O conhecimento do nível de associação que ocorre entre bactérias diazotróficas e fungos micorrízicos arbusculares (FMA) nos ecossistemas campestres, pode ser importante para a o entendimento das alterações provocadas pela adição de fertilizantes, contendo fósforo (P) e nitrogênio (N), na produção e na composição botânica das pastagens naturais. O objetivo do presente trabalho foi avaliar (a) a ocorrência de três gêneros de bactérias diazotróficas no sistema radicular sob fertilização com N e P, e determinar a contribuição de N via FBN, e (b) avaliar o comportamento da associação entre FMAs e gramíneas nativas. Foram utilizadas quatro gramíneas de maior abundância nas pastagens naturais dos Campos Sulinos no Rio Grande do Sul, Axonopus affinis, Paspalum notatum, Andropogon lateralis e Aristida laevis, cultivadas em casa de vegetação, sendo conduzidos dois estudos (A e B). Em A, foram aplicados dois tratamentos: 50 mg kg-1 de solo de P + 100 mg kg-1 de N solo (NP) e uma testemunha, sendo avaliados, o número de bactérias diazotróficas dos gêneros Azotobacter, Azospirillum e Herbaspirillum, e a contribuição da FBN através da técnica da abundância natural de 15N. Em B, os tratamentos consistiram na aplicação de 50 mg kg-1 de solo de P (P); aplicação de 50 mg kg-1 de solo de P + 100 mg kg-1 de N solo (NP), e uma testemunha, em ambos foi determinada a colonização micorrízica. A espécie A. laevis demonstrou ser mais dependente da fixação biológica de N que as demais espécies. A espécie P. notatum em comparação as demais espécies, demostrou ser mais hábil em absorver o N disponível no solo. A matéria seca acumulada da parte aérea das espécies nativas foi maior com a aplicação de NP. A colonização micorrízica foi semelhante entre a testemunha, P e NP para as raízes de A. lateralis e A. laevis, apresentando assim uma maior dependência da associação micorrízica. Diferentemente, nas espécies A. affinis e P. notatum, a colonização micorrízica foi menor quando submetidas a adubação com P e NP, apresentando assim uma menor dependência. Conclui-se assim, que a adubação com N e P reduz a colonização de bactérias diazotróficas, aumentando a produção de matéria seca e teor de N no tecido. A. laevis apresentou a maior contribuição da fixação biológica de nitrogênio, já P. notatum apresentou maior acúmulo de N do solo. As espécies A. laevis e A. lateralis apresentam maior dependência da micorrização que as espécies A. affinis e P. notatum.

This thesis was performed within the research project “TrophinOak”, which addresses the impact of multitrophic interactions on the pedunculate oak (Quercus robur) clone DF159. In this frame, the present work focuses on the genetic and physiological mechanisms ruling the interaction of the mycorrhiza helper bacterium (MHB) Streptomyces sp. AcH 505 with microcuttings of DF159 either alone or in presence of the ectomycorrhizal fungus Piloderma croceum or the fungal leaf pathogen oak powdery mildew Microsphaera alphitoides. The work consists of 3 chapters. Chapter 1 characterises the growth of AcH 505 and P. croceum in a soil-based culture system used within the TrophinOak project. Besides the establishment and evaluation of quantification methods of these microorganisms by quantitative real-time PCR, the impact of the soil microbial community and the oak on the bacterium-fungus interaction was investigated, and AcH 505 and P. croceum were visualized by scanning electron microscopy. It was observed that the presence of the soil microorganisms and the oak both affect the bacterium-fungus interaction, and that P. croceum enhances the growth of AcH 505. Chapter 2 presents a study with the oak, AcH 505 and the EM fungus P. croceum, enabling to disentangle the direct effect of the MHB on the oak from the indirect one via the EM symbiosis. The used approach was transcriptomic based on RNA sequencing. It was shown that i) differential gene expression occurred between root and the distant leaf tissues (local vs. systemic effects), different developmental stages and treatments, suggesting that oak specifically coordinates its gene expression patterns, and ii) that genes related to plant growth, defence and DNA modification were dominant among the differential expressed genes, suggesting that these processes play essential roles in both symbiotic interactions investigated. Chapter 3 represents a second transcriptome study, addressing how AcH 505 suppresses powdery mildew infection in oak by analysing RNA Sequencing data from singly- and coinoculated oaks. This study combined the systemic impact of the root associated bacterium with local effects of the leaf pathogen, thereby linking belowground and aboveground interactions. Systemic defence response is induced by the bacterium and further enhanced upon pathogen challenge, suggesting that on the leaf level, some bacterial effectors are recognized as harmful for the plant.

A cana-de-açúcar é uma das principais culturas do sistema agrícola brasileiro, e apresenta-se atualmente em plena expansão. Porém o uso do solo e a implementação de diferentes tecnologias de manejo têm originado alterações no equilíbrio ambiental, onde importantes interações microbianas ocorrem de forma essencial para o desenvolvimento vegetal. Dentre a vasta diversidade de microrganismos do solo, destacam-se os fungos micorrízicos, organismos intimamente associados as raízes das plantas, auxiliando a mesma, dentre outras formas, na obtenção de água e nutrientes. Estes fungos, no entanto, interagem também com outros organismos do solo, como por exemplo, com a comunidade bacteriana presente neste ambiente. Desta forma, o presente trabalho buscou estudar a dinâmica de interação entre cana-de-açúcar e o fungo micorrízico arbuscular (FMA) G.clarum em solos com diferentes composições da comunidade bacteriana. A metodologia utilizada foi a \'diluição para extinção\', onde diluições seriadas (10-1; 10-3; 10-6 e 10-9) de um solo natural foram usadas para inocular o solo estéril. Sobre esta base, foi monitorada pelo período de 60 dias, a colonização da planta pelo FMA e a estruturação das comunidades bacterianas. Como resultado, foi observada uma maior colonização das raízes de cana-de-açúcar para os tratamentos inoculada com menores diluições da comunidade original (solo natural e diluições 10-1 e 10-3), sendo da mesma forma observada uma distinção entre as comunidades bacterianas destes tratamentos para os demais. Estabelecendo correlações entre os grupos microbianos e as taxas de colonização micorrízica, foi possível nomear, com base no sequenciamento massivo da região V6 do gene ribossomal 16S DNAr, a alteração conjunta da micorrização com mudanças nos grupos de Actinobacteria,Bacteriodetes,Firmicutes,Proteobacteria,Verrucomicrobiae Acidobacteria. Concluindo, este trabalho demonstra a dependência que um processo importante, como a micorrização, possui da comunidade bacteriana do solo, e indica que em áreas degradadas, com menores níveis de diversidade bacteriana, tal processo pode ocorrer com menor eficiência.
Sugarcane is an important Brazilian agricultural system crop and presents currently booming. Nevertheless, land use, and implementation of different management technologies have originated changes in environmental balance, where important microbial interactions occur as essential for plant development. Among the wide diversity of soil microorganisms, the mycorrhizal fungi is highilighted as organisms closely associated with plant roots, helping plants, in any way, to obtain water and nutrients. These fungi however, also interact with other soil organisms, such as for example, bacterial community in these environments. Thus, the present work aimed to study the dynamics of interaction between sugarcane and arbuscularmycorrhizal fungi (AMF) Glomusclarum in soils with different compositions of the bacterial community. The methodology used was \"dilution to extinction\", where serial dilutions (10-1, 10-3, 10-6 and 10-9) of a natural soil were used to inoculate a sterile soil. On this basis, were monitored along a period of 60 days, plant colonization by AMF, and structure of bacterial communities. As a result, we observed a higher colonization of roots of cane sugar for treatments inoculated with lower dilutions of the original community (natural soil and dilutions 10-1 and 10-3), and likewise observed a distinction between these bacterial communities treatments to others. Establishing correlations between microbial groups with observed rates of colonization, it was possible to name, based on the massive sequencing of the region V6 ribosomal gene 16S rDNA, the joint amendment of mycorrhiza with changes in groups of Actinobacteria; Bacteriodetes; Firmicutes, Proteobacteria; Verrucomicrobia and Acidobacteria. In conclusion, this work demonstrates the dependence of an important process, as the AMF, has tosoil bacterial community, and indicates that degraded areas, with lower levels of bacterial diversity, such a process can occur with lower efficiency.

A pesquisa sobre bactérias diazotróficas associadas à cultura do trigo tem demonstrado a necessidade de associar bactérias eficientes a genótipos responsivos ao nitrogênio, os quais mais se beneficiariam dessa associação. Em um experimento com parcelas subdivididas instalado em condições de campo, em Mococa (SP), empregando os tratamentos: 3 doses de N (0, 60 e 120 kg ha -1 ) x 3 genótipos de trigo (IAC-24, ITD-19 e IAC-355), foi avaliada a ocorrência de microrganismos diazotróficos endofíticos em raízes desinfestadas superficialmente, utilizando-se 3 meios de cultivo distintos, NFb, semi-específico para Azospirillum spp, JNFb, semi-específico para Herbaspirillum spp., e LGI-P, semi-específico Gluconacetobacter diazotrophicus. O genótipo IAC-355 apresentou a menor quantidade de bactérias diazotróficas endofíticas. Além disso, para este genótipo, houve um ajuste linear ascendente da quantidade de bactérias diazotróficas com o aumento na quantidade de N adicionada, o que comprova a influência do genótipo da planta na associação com essas bactérias. Nenhuma bactéria pertencente aos gêneros Azospirillum ou Herbaspirillum foi isolada do genótipo IAC-355. Nas condições estudadas, não foi identificado nenhum isolado de Gluconacetobacter diazotrophicus nas raízes do trigo. Foram obtidos 8 isolados do gênero Azospirillum e 12 do gênero Herbaspirillum. Esses isolados foram testados "in vitro", no genótipo do qual foram originalmente isolados. Todos os isolados testados no genótipo ITD-19 causaram maior crescimento radicular que a testemunha e apenas 1 isolado de Herbaspirillum sp. propiciou aumento significativo do teor de N na parte aérea. A colonização micorrízica no genótipo IAC-355 foi maior que nos demais genótipos independente da dose de N, comprovando a influência do genótipo na colonização. A colonização micorrízica se correlacionou com a massa da matéria seca, e com o teor e a quantidade acumulada de P e N na parte aérea, assim como com a produtividade. A atividade da biomassa microbiana foi alterada na ausência de N, obtendo-se correlação entre o qCO2 e a relação Cmic:Corg, indicando que na ausência de N, houve perdas de C no solo cultivado com o genótipo IAC-24, enquanto que na presença do IAC-355, houve maior eficiência na utilização do C do solo pelos microrganismos. Na contagem de bactérias nitrificadoras obteve-se um ajuste linear ascendente em relação à quantidade de N adicionada, provavelmente devido à maior disponibilidade de substrato. A quantidade de microrganismos nitrificadores se correlacionou com a massa da matéria seca, e com o teor e a quantidade acumulada de N na parte aérea, assim como com a produtividade. A interação FMA-bactéria diazotrófica não propiciou benefícios para cultura do trigo. A interação FMA-bactéria diazotrófica demonstrou ser bastante especifica. As plantas associadas a Glomus, quando em presença dos isolados bacterianos apresentaram maior crescimento, acúmulo e aproveitamento dos nutrientes. Confirmou-se que o fungo micorrízico realmente é um agente transmissor de bactérias diazotróficas endofíticas, sendo que Acaullospora causou maior colonização radicular. As plantas dos tratamentos em que somente a bactéria foi inoculada apresentaram o dobro da produção de matéria seca, da quantidade acumulada e do índice de eficiência de utilização de N e P na parte aérea em relação à testemunha. A especificidade da interação planta-bactéria diazotrófica associativa indica que é possível obter benefícios desta associação, explorando bactérias e cultivares locais.
The research on diazotrophic bacteria associated to wheat has demonstrated the need to associate efficient bacteria to N-responsive genotypes, which would be more benefited from this association. A field experiment was carried out in Mococa, state of São Paulo-Brazil, with 3 genotypes of wheat (IAC-24, ITD-19 and IAC-355) under 3 nitrogen doses (0, 60 e 120 kg ha -1 ). The occurrence of diazotrophic bacteria was evaluated in three media, namely, NFb semi-specific for Azospirillum spp., JNFb semi-specific for Herbaspirillum spp., and LGI-P semi-specific for Gluconacetobacter diazotrophicus, using surface-sterilized roots. Regardless the nitrogen dose, the population of diazotrophic bacteria established poorly in the genotype IAC-355, but the infection increased with the addition of nitrogen for the same genotype, proving the influence of the host genotype for its association with these bacteria. Azospirillum spp. or Herbaspirillum spp. could not be isolated from the surface-sterilized roots of IAC-355. In the field experiment G. diazotrophicus was not found in any of the wheat-genotype roots. It was obtained 12 Herbaspirillum spp. isolates and 8 Azospirillum spp isolates. These strains were tested under gnotobiotic conditions, using the genotype from which they had been originally isolated. Inoculated ITD-19 plants showed an increase in root length, even though, only one strain showed a significant increase on shoot N accumulation. In the genotype IAC-355 mycorrhizal colonization was higher, proving the influence of the plant genotype. Mycorrhizal colonization showed significant correlation to shoot dry matter, shoot N and shoot P concentration and accumulation, as well as to the grain yield. In the absence of added N, the activity of microbial biomass was affected. The correlation between the qCO2 and biomass C-to-N ratio, showed that in absence of N, soil-C loss under IAC-24 cultivation, but a greater efficiency in the use of the soil-C by the microorganisms under IAC-355 cultivation. The populations of nitrifying bacteria increased with N addition, probably due to the N-rich substrate availability. The nitrifying bacteria showed significant correlation to shoot dry matter, shoot N concentration and accumulation, as well as to grain yield. There was no benefit from the AMF-diazotrophic bacteria co-inoculation on wheat plants. The specificity of AMF-diazotrophic bacteria interaction was demonstrated, it was confirmed that AMF indeed is a transmitting agent of endophytic diazotrophic bacteria, Acaullospora caused higher endophytic-bacteria root colonization. Plants inoculated with single strain doubled shoot dry matter, shoot N and P concentration and accumulation as compared to the control. Plant-bacteria interaction specificity demonstrates the possibility of getting benefits from this association by exploring both bacterial strains and plant genotypes from the same location.

L'influence de bactéries solubilisant les phosphates (agrobactérium) et de champignons éctomycorhizogènes sur l'altération d'un mica, la phlogopite et de phosphates insolubles, a été étudiée : en cultures liquides, en l'absence de racine ; dans la rhizosphère du pin sylvestre et du hêtre, en relation avec les composés exsudés. Les thizobactéries favorisent la solubilisation des éléments minéraux par production d'acides organiques. Les champignons ectomycorhizogènes solubilisent les phosphates en culture pure, mais, en conditions symbiotiques, ils jouent surtout un rôle par l'augmentation du volume de sol exploré par les racines, donc des surfaces d'échnage et d'adhésion avec les minéraux

Les activités anthropiques et la pollution qu’elles engendrent peuvent entraîner des changements drastiques dans les structures des écosystèmes et de leurs services écologiques. Le phytomanagement basé sur l’utilisation d’espèces ligneuses peut contribuer à la restauration des sols et de la diversité microbienne, tout en permettant la production de biomasses d’intérêt. Dans le cadre des projets PROLIPHYT (2013-2018, ADEME), BIOFILTREE (2010-2014, ANR) et PHYTOPOP (2007-2011, ANR) gérés par le laboratoire Chrono-environnement, des parcelles de phytomanagement ont été installées sur sites pollués par les éléments traces métalliques (ETMs), pour déterminer le potentiel de croissance d’espèces ligneuses, ainsi que les voies de valorisation des biomasses produites. Dans le cadre de mon projet de thèse, différentes approches ont été mises en œuvre afin d’identifier et de caractériser les microorganismes inféodés aux végétaux ligneux implantés, avec pour finalité la production de ressources microbiennes permettant d’améliorer la reprise et la croissance des arbres sur sols pollués. La première approche a consisté à étudier les communautés microbiennes en association avec les espèces ligneuses par une approche innovante de séquençage à haut débit associée à l’analyse physico-chimique des sols. Cette méthode a été mise au point et testée sur trois sites expérimentaux contaminés par des ETMs. Elle a permis de révéler les changements dans la structure et la composition des communautés microbiennes dus à la nature du couvert végétal et aux caractéristiques du sol (Zappelini et al., 2015; Foulon et al., 2016a; b). La deuxième approche a consisté à isoler et caractériser les champignons mycorhiziens et endophytes associés à des peupliers (Berthelot et al., 2016; Lacercat-Didier et al., 2016). Ces champignons sont connus pour favoriser la croissance des végétaux qu’ils colonisent mais également pour conférer une meilleure tolérance face à un stress métallique. Plusieurs campagnes de prélèvements de racines et de champignons sur différents sites pollués ont permis l’isolement de souches fongiques tolérantes aux ETMs (comme Serendipita vermifera) et améliorant la croissance (Phialophora sp. et Leptodontidium sp.). Dans une troisième approche, les mécanismes impliqués dans la résistance aux ETMs ont été étudiés i) chez Paxillus involutus pour le Hg (Foulon et al. manuscrit en préparation) ii) par métatranscriptomique fonctionnelle pour le Zn et Cd (Lehembre et al., 2013). L’ensemble de ces travaux a permis i) d’améliorer nos connaissances sur l’impact du couvert ligneux sur la structure et la composition des communautés microbiennes, et sur la compréhension des mécanismes physiologiques mis en jeu ii) de constituer une collection de souches fongiques d’intérêt pour le phytomanagement de sols contaminés par les ETMs
Anthropogenic activities can cause dramatic changes in ecosystem structures and their ecological services. Phytomanagement based on the use of woody species can contribute to soil and microbial diversity restoration, while allowing the production of biomass of interest. As part of PROLIPHYT (2013-2018, ADEME), BIOFILTREE (2010-2014, ANR) and PHYTOPOP (2007-2011, ANR) projects, managed by the Chrono-environment laboratory, phytomanagement plots were installed on trace elements (TE) polluted sites to determine the growth potential of woody species and the recovery methods of biomass produced. In my PhD project, different approaches have been implemented to identify and characterize microorganisms subservient to implanted woody plants, with the aim of producing microbial resources to improve the recovery and growth of trees on polluted soil. The first approach was to study microbial communities in association with woody species by innovative high throughput sequencing approach associated with the physical and chemical analysis of soils. This method was developed and tested on three experimental sites contaminated by TE. It revealed changes in the structure and composition of microbial communities due to the nature of the vegetation cover and the soil characteristics (Zappelini et al., 2015; Foulon et al., 2016a; b). The second approach was to isolate and characterize mycorrhizal fungi and endophytes associated with poplars (Berthelot et al., 2016; Lacercat-Didier et al., 2016). These fungi are known to promote the growth of the plants they colonize but also to increase their TE tolerance. Several sampling campaigns of roots and sporocarps on different polluted sites have allowed the isolation of fungal strains tolerant to TE (like Serendipita vermifera) and enhancing growth (Phialophora sp. and Leptodontidium sp.). In a third approach, the mechanisms involved in TE resistance were studied i) in Paxillus involutus for Hg (Foulon et al. In prep) ii) by functional metatranscriptomics for Zn and Cd (Lehembre et al., 2013). In conclusion, this work allowed to i) improve our understanding of the impact of a woody cover on the structure and composition of microbial communities, and on the understanding of the physiological mechanisms involved ii) to constitute a collection of fungal strains that will be of great interest for future phytomanagement projects

Le phytomanagement compte parmi les méthodes innovantes de gestion des sites et sols pollués en raison de son adéquation avec le développement durable. Pour répondre à la double exigence de performances environnementales et économiques, les phytotechnologies sont désormais combinées à la valorisation de la biomasse produite sur sols pollués. Parmi les filières éco-innovantes et non-alimentaires de valorisation de cette biomasse, la culture de plantes à parfums, aromatiques et médicinales (PPAM) pour la production d’huiles essentielles (HE), substances biosourcées à haute valeur ajoutée, a été proposée dans le cadre du projet PhytEO, financé par l’ADEME. Ainsi, ma contribution à ce projet a permis d‘évaluer la pertinence d’un mode de gestion reposant sur la culture de la sauge sclarée et de la coriandre, en présence ou non d’un amendement mycorhizien, en s’appuyant sur des démonstrateurs in situ à l’échelle de l’hectare, parcelles historiquement polluées ou non par les éléments traces (ET). La coriandre ainsi que la sauge sclarée ont toutes deux montré une bonne capacité à s’installer, à se développer et à produire une quantité importante de biomasse sur des sols présentant une forte pollution historique par les ET. Bien que l’ajout d’un amendement biologique à base de champignons mycorhiziens à arbuscules ait permis d’augmenter de façon significative les taux de mycorhization des racines de coriandre et de sauge sclarée, aucun gain n’a été observé ni au niveau de la croissance de ces deux PPAM, ni au niveau de la quantité et de la qualité des HE. Un effet positif en termes d’immobilisation des ET dans le sol, en particulier du Pb, et une réduction des transferts des ET (Cd et Pb) dans les parties aériennes a toutefois été montré chez la sauge sclarée. D’autre part, les HE distillées à partir de la biomasse de sauge sclarée (inflorescences) ou de coriandre (parties aériennes ou graines) cultivées sur sols pollués présentent une qualité très satisfaisante, caractérisée par leur non contamination en ET ou en résidus de pesticides (traces). Leurs compositions chimiques, non altérées par la pollution en ET et l’inoculation mycorhizienne, affichent divers principes actifs dont le linalol, l’acétate de linalyle, le germacrène, l’α-pinène, le γ-terpinène, le 2-décénal, le décanal et le 2-dodécénal. Ainsi, plusieurs propriétés biologiques potentiellement valorisables dans des domaines à vocation non-alimentaire, tels que la protection des cultures (effets fongistatiques, anti-germinatifs et herbicides) ou la santé humaine (effets anti-inflammatoires et antioxydants) ont été mises en évidence. En revanche, en dépit de son fort pouvoir complexant des HE, la β-cyclodextrine n’a montré aucun effet positif sur les propriétés biologiques testées. Par ailleurs, la végétalisation du sol pollué par la sauge sclarée permet une modulation de la structure des communautés bactériennes et fongiques, telluriques et racinaires. Enfin, l’ensemble des résultats de cette étude et de l’analyse technico-économique de la filière PPAM-HE soulignent le haut potentiel de la sauge sclarée dans une démarche de phytomanagement de sols pollués par les ET
To cope with the soil trace element (TE) pollution, phytomanagement was brought forward as an eco-friendly and feasible approach. To meet the dual requirements of environmental and economic performances, phytotechnologies are now combined with the valorisarion of biomass produced on polluted soils. Among the eco-innovative channels intended for the non-food valorisation of the produced biomass, the cultivation of aromatic and medicinal plants producing essential oils (EO), high-added value biosourced products, has been suggested within the framework of PhytEO project, funded by ADEME. Thus, this thesis contributed to evaluate the relevance of a management approach based on clary sage and coriander cultivation, combined or not with a mycorrhizal inoculum, on in situ experimental plots polluted or not with TE. Both clary sage and coriander have shown a good ability to settle in, to grow and to produce high amounts of biomass, despite the presence of high TE concentrations in the soil. The addition of an amendment based on arbuscular mycorrhizal fungi enhanced the mycorrhizal colonisation rates of both coriander and clary sage roots. However, no significant improvement was observed in terms of plants’ growth, EO amounts and quality. Moreover, mycorrhizal inoculation allowed TE immobilisation in soils, in particular Pb, and reduced TE (Cd and Pb) transfers in aerial parts of clary sage. Besides, the EO distilled from clary sage inflorescences and coriander (aerial parts orseeds) grown on TE polluted soils, displayed a highly satisfactory quality, regarding the absence of contamination by TE or pesticide residues (trace amounts). Their chemical compositions were not altered by both TE pollution and mycorrhizal inoculation and were characterized by several active principles, such as linalool, linalyl acetate, germacrene, α-pinene, γ-terpinene, 2-decenal, decanal and 2-dodecenal. Furthermore, several biological properties with potential applications in non-food fields such as crop protection (antifungal, antigerminative and herbicidal) or human healthcare (antioxidant and anti-inflammatory) have been brought forward. However, despite its ability to form inclusion complexes with EO components, β-cyclodextrin has shown no significant improvement of the tested EO biological activities. On another note, the soil revegetation with clary sage displayed a clear shaping of the bacterial and fungal communities, in both the rhizospheric soil and the roots of the aromatic plant species. Altogether, our results combined with the techno-economic feasibility assessment of the channel “aromatic and medicinal plants/EO” have emphasised clary sage as a good candidate for the phytomanagement of TE-polluted soils

碩士
國立中興大學
土壤學研究所
79
Not much data are available regarding the distribution and its affecting factors of vesicular-arbuscular mycorrhizal fungi (VAMF) and phosphorus-solubilizing bacteria (PSB) in tea rhizospheric soils. Even works on applications of VAMF and PSB as bio-fertilizer for tea have not been reported. Effects of season, soil sampling position, soil properties and P fertilizer on VAMF and PSB population in tea soils were investigated. Effects of inoculation with VAMF and PSB on tea seedlings were also studied. Based on data of a surface soil sampling, the dominant mycorrhizal fungi in tea soils were Glomus sp., with population desity 40-150 spores/lOOg dry soil, and infection rate 4-32%. The dominant PSB species were Pseudomonas sp. and Bacillus sp., with bacterial number, 0.01-8x10(5)/g dry soil. Changes in number of VAMF spores in tea rhizospheric soils could affected by precipitation, temperature, tea pruning and tea dormancy. Lower spore countings were obtained in winter season (December to February). There was no significant correlation between number of VAMF spores and soil properties such as pH, organic matter and available P (Bray No.1). Mycorrhizal infection rate was the highest in April of spring season. This might be due to better soil temperature and moisture which resulted in better root activity, especially better carbohydrate content in roots as a consequences of better shoot growth in spring. PSB population was apparently associated with preciptitation, air temperature and root density of tea. However, no significant relation between PSB spore counting and soil properties was found. Number of VAMF sopres in soil decreased with increasing soil depth; so as the VAMF infection rate. There were positively correlation among number of VAMF spores, infection rates and soil organic matter contents. Similar relationships were obtained based on soil samples taken at different distance from tea trunks. Soils with high dispersity, high bulk density, low organic content(lower than 10 g/kg) or low water holding capacity decreased VAMF and PSB population. Based on the results of a long-term pot experiment on phosphorus, application of P fertilizer (P2O5) more than 192 kg/ha apparently decreased number of VAMF spores, mycorrhizal infection rate, and PSB spot number. Appropriate amounts of P fertilizers (48-96 kg/ha) effectively increased VAMF and PSB population as well as leaf P concentration, and upgraded tea quality. Uncultivated soils inoculated respectively with VAMF and PSB or both microorganisms had no promoting effects on the growth of tea cuttings of cvs. Chin-Hsin Oolong and TTES No.12, However,in tea cultivated soils inoculation of VAMF or PSB alone obviously enhanced seedling growth of cuttings, cv. TTES No.12. No similar effects were found in cv. Chin-Hsin Oolong. Additive effects on seedling growth of cvs. Chin-Hsin Oolong and TTES No.12. were obtained if both VAMF and PSB were inoculated simultaneously in tea cultivated soils.

碩士
國立中興大學
園藝學系
89
This study is trying to understand the relationship between plantlet, culture medium and microorganism by using wax apple (Syzygium samarangense, Merret Perry) micropropagated plantlets derived from tissue culture with treatments of arbuscular mycorrhizal fungi (AMF), and phosphate solublizing bacteria (PSB) inoculation, and NAA treatment. The results showed that plantlet inoculated with AMF alone, PSB alone, and AMF and PSB mixed could all promoted the morphological change of the root by increasing main root and side root quantity and ratio. Although the results from inoculation treatments and NAA treatment had a little bit difference but not significant, rooting effect of NAA treatment seemed could be substituted by inoculation treatments. However, owing to microorganism population not fully developed, early stage of plantlet growth was slow after inoculation period. Plantlet growth speed up after 12 weeks of inoculation. On the ground part uptake element content, NAA treatment had higher N, K, Ca content than those of other treatments. AMF inoculated alone had higher N, P, K content than that of control set and PSB inoculated alone. P, K, Ca, Mg, Fe, Mn, Cu, and Zn of inoculated PSB alone are higher than that of control set. Mixed microorganism inoculation is better than single microorganism inoculation alone in every aspect. In NAA treatment, however, P content of ground part was not significant with control set and was higher than that of other treatments. Ground part uptake Fe amount was not significant with that of AMF and PSB inoculated alone, but higher than other treatments. Cu content had positive accumulative effect in comparison with others. When wax apple plantlets were inoculated with AMF and PSB mixed, water content variation was small. Organic substance effect on plant growth of PSB inoculated one was better than that of AMF inoculated one significantly. Wax apple grew well when inoculated with PSB. However, mixed microorganism inoculation had even greater effect. Therefore, plant growth and nutrition uptake could be promoted as mixed microorganism inoculation was treated on wax apple plantlet production.

博士
國立中山大學
海洋環境及工程學系研究所
97
Heavy metals are one of the most important environmental pollutants. In recent years, many low cost stretages of bioremediation for contaminated sites by heavy metals, such as fungi, bacteria and plants have been investigated for their biosorption capacity towards heavy metals. The uses of plant species for remediate contaminated sites by heavy metals are so called phytoremediation. The purpose of the first parts of this study are to (1) evaluate bioavailability of Cadmium (Cd) in contaminated soil and phytoremediation potential by three plant species, Vetiveria zizanioides, Pteris multifida, and Alternanthera philoxeroides (Mart.), and (2) realized the influence of photosynthetic bacteria (PSB) on the uptake of Cd in the three species. The results showed that the Alternanthera philoxeroides (Mart.) could accumulate the highest concentration of Cd among the three species, in which the Cd concentration of plant tissue increased with the concentration in soil. The highest concentration of Cd (164.9 mg kg-1) was found in the below-ground parts of Alternanthera philoxeroides (Mart.) at the 8th week of culturing period. However, the species of Vetiveria zizanioides could accumulate the largest total Cd, up to 547.5 μg/ plant, which thus extracted the greatest amounts of Cd from the soil. Therefore, in the first part of this study the species of Vetiveria zizanioides was concluded to be the best accumulator among the three plant species. In addition, the concentration of Cd in the species of Pteris multifida was found significantly increased after PSB was added into the soil, but the plants died later due to Cd stress. The experimental results also showed that PSB seemed to be not suitable for each species used in this study to accumulate Cd from Cd-contaminated soil. In the second part of this research, both pot and field experiments were conducted to (1) evaluate bioavailability of copper (Cu), lead (Pb) and zinc (Zn) in contaminated soil and phytoremediation potential by domesticated plants, Bidens pilosa and Passiflora foetida inoculated with arbuscular mycorrhizal (AM) fungi, and to (2) compare the results of pot and field experiments. The plant species of Bidens pilosa inoculated with AM fungi had significantly higher Cu concentrations in the shoots and roots than non-inoculated plants. The plant species of Passiflora foetida inoculated with AM fungi also had significantly higher Cu and Pb concentrations in the roots than non-inoculated plants. When we found that the root dry weight of Passiflora foetida inoculated with AM fungi dramatically increased, the concentrations of Cu, Pb and Zn in the root of the plant species increased by 9-14 times, comparing with the plants without inoculation of AM fungi. The AM fungi have potential either to promot plant growth or to increase heavy metal accumulation. The values of element translocation proportion from root to shoot was Zn>Cu>Pb for the plant species in both pot and field experiments. For both experiments, the results of pot test and field test were significantly different. The concentrations of pot tests were found higher than the field tests, and some values of pot tests were even found significantly greater than those in the field tests. In the third part of this study, the field experiments were conducted to test the feasibility of using domesticated vegetations for phytoremediation of the contaminated farmland. The objectives of this study were (1) to acquire information about the ability of five plant species growing wild in the polluted area to accumulate Cu, Pb and Zn, (2) to investigate the season effects on phytoremediaton of five plant species and evaluate the total uptake of heavy metal, and (3) to run both pot tests and a field trial of phytotremediation to compare their differences. The experimental results showed that three maximum toxic elements in a pot were 3020 mg kg-1 Pb, 232 mg kg-1 Cu and 1012 mg kg-1 Zn respectively. The Cu concentrations of the five plant species collected from the polluted plots ranged from 0.7 to 17.43 mg kg-1. The range of variation of Pb in plant tissues was measured varied from 2.29 to 81.65 mg kg−1, while a wide range of Zn concentrations was found from 12.84 to 192.85 mg kg-1 among the plants collected at the contaminated plots. In comparison to winter season, the Zn concentrations in Broussonetia papyrifera, Passiflora foetida and Saccharum sinensis collected in summer season was significantly higher. The higher Cu concentrations were obtained in both plant species of Bidens pilosa and Mimosa diplotricha in summer season. However, Pb concentrations in Saccharum sinensis collected in winter were significantly higher than those in the same plant species collected in summer. Bidens pilosa was also found having the highest total amount of Cu and Zn. The highest total amount of Pb was found in Mimosa diplotricha. For both plant species, both of the pot and field tests were different.

碩士
國立屏東科技大學
熱帶農業研究所
88
Inoculation of six species of vesicular-arbuscular mycorrhiza (VAMF) fungi and the grafting of different cultivars of tomato (Lycopersicon esculentum Mills.) onto three eggplants (Solanum melongena L.) were studied to determine their effects on the bacterial wilt incidence and yield of tomato. Among the eggplant cultivars, EG203 had the highest rate of colonization (22.8%). However, there was no significant difference in plant height and stem diameter among the eggplants inoculated with the different VAMF species. The interaction between cultivars and mycorrhizal fungi was significant in colonization as well as shoot and root fresh weight. There was a significant interaction between the R. solanacearum inoculum density and VAM species on the 39-day old seedlings with respect to disease index and the progress of the disease (AUDPC). Treatments inoculated with the mycorrhizal fungi species Glomus etunicatum had lower disease index than the check although the difference was not significant. For the 28-day-old seedlings, only the disease index showed difference among the inoculum density treatment. There was no significant difference in the interaction between the inoculation of different mycorrhizal species and the bacterial wilt inoculum density. The resistant to bacterial wilt in five tomato cultivars where the ranking of severity (disease index) and progress (AUDPC) among them was not changed after VAMF colonization. Using the soil infestation of the Ralstonia. solanacearum pathogen there was significant difference in the disease index and progress (AUDPC) among the plant materials but not in the inoculation of different mycorrhizal fungi species. The field investigation revealed no significant interaction among the three factors of the inoculation mycorrhizal fungi, phosphorus application level and grafting. The treatment with mycorrhizal inoculation recorded higher in fruit number, fruit weight, yield and phosphorus content. However, there was no significant difference between the inoculated and non-inoculated treatments in fruit, fruit weight, yield and phosphorus content. Application of the higher P-level (160kg P/ha) resulted in significantly higher fruit number and leaf phosphorus content than that of the lower P-level (40kg P/ha) but the difference was not significant for fruit weight and yield. Tomatoes grafted to eggplant showed a significant reduction in yield but an increase in leaf phosphorus content as compared to tomatoes that were not grafted onto eggplants. There was no significant difference among the treatments in the screenhouse

Some microorganisms are known to form mutualistic symbiosis with plant roots and by their impact they can improve some plant parameters. These symbiotic microorganisms, which are able to improve some plant parameters, include especially mycorrhizal fungi, plant growth promoting bacteria and some saprotrophic mycoparasitical fungi. Mechanisms of changes of these parameters, as influenced by symbiotic microorganisms, are known only in part and nowadays are being actively researched. Aims of this work were to find out if selected microbial treatments influence selected growth, physiological and yield parameters of plants and contents of selected substances in fruits. Within this work were made three pot greenhouse experiments (experiments 1, 2 and 3) and three pilot greenhouse experiments (experiments 4, 5 and 6), performed on tomato (Solanum lycopersicum) and cucumber (Cucumis sativus) plants. Plants were grown in hydroponics using a carrier of rockwool and they were watered by nutrient solution. As microbial treatments for plants in experiments have been used a mixture of arbuscular mycorrhizal fungi (AM), mixture of plant growth promoting bacteria (PGPB), saprotrophic mycoparasitical fungus Trichoderma harzianum (Th) and various mutual combinations of these treatments. There have been observed...

Les champignons mycorhiziens à arbuscules (CMA) forment un groupe de champignons qui appartient à l'embranchement des Gloméromycètes (Glomeromycota). Les CMA forment des associations symbiotiques, connus sous le nom des mycorhizes à arbuscules avec plus de 80 % des plantes vasculaires terrestres. Une fois que les CMA colonisent les racines de plantes, ils améliorent leurs apports nutritionnels, notamment le phosphore et l'azote, et protègent les plantes contre les différents pathogènes du sol. En contrepartie, les plantes offrent un habitat et les ressources de carbone nécessaires pour le développement et la reproduction des CMA. Des études plus récentes ont démontré que les CMA peuvent aussi jouer des rôles clés dans la phytoremédiation des sols contaminés par les hydrocarbures pétroliers (HP) et les éléments traces métaliques. Toutefois, dans les écosystèmes naturels, les CMA établissent des associations tripartites avec les plantes hôtes et les microorganismes (bactéries et champignons) qui vivent dans la rhizosphère, l'endosphère (à l'intérieur des racines) et la mycosphère (sur la surface des mycéliums des CMA), dont certains d'entre eux jouent un rôle dans la translocation, l’immobilisation et/ou la dégradation des polluants organiques et inorganiques présents dans le sol. Par conséquent, la diversité des CMA et celle des microorganismes qui leur sont associés sont influencées par la concentration et la composition des polluants présents dans le sol, et aussi par les différents exsudats sécrétés par les trois partenaires (CMA, bactéries et les racines de plantes). Cependant, la diversité des CMA et celle des microorganismes qui leur sont associés demeure très peu connue dans les sols contaminés. Les interactions entre les CMA et ces microorganismes sont aussi méconnus aussi bien dans les aires naturelles que contaminées. Dans ce contexte, les objectifs de ma thèse sont: i) étudier la diversité des CMA et les microorganismes qui leur sont associés dans des sols contaminés par les HP, ii) étudier la variation de la diversité des CMA ainsi que celle des microorganismes qui leur sont associés par rapport au niveau de concentration en HP et aux espèces de plantes hôtes, iii) étudier les correlations (covariations) entre les CMA et les microorganismes qui leur sont associés et iv) comparer les communautés microbiennes trouvées dans les racines et sols contaminés par les HP avec celles trouvées en association avec les CMA. Pour ce faire, des spores et/ou des propagules de CMA ont été extraites à partir des racines et des sols de l'environnement racinaire de trois espèces de plantes qui poussaient spontanément dans trois bassins de décantation d'une ancienne raffinerie de pétrole située dans la Rive-Sud du fleuve St-Laurent, près de Montréal. Les spores et les propagules collectées, ainsi que des échantillons du sol et des racines ont été soumis à des techniques de PCR (nous avons ciblés les genes 16S de l'ARNr pour bactéries, les genes 18S de l'ARNr pour CMA et les régions ITS pour les autres champignons), de clonage, de séquençage de Sanger ou de séquençage à haut débit. Ensuite, des analyses bio-informatiques et statistiques ont été réalisées afin d'évaluer les effets des paramètres biotiques et abiotiques sur les communautés des CMA et les microorganismes qui leur sont associés. Mes résultats ont montré une diversité importante de bactéries et de champignons en association avec les spores et les propagules des CMA. De plus, la communauté microbienne associée aux spores des CMA a été significativement affectée par l'affiliation taxonomique des plantes hôtes et les niveaux de concentration en HP. D'autre part, les corrélations positives ou négatives qui ont été observées entre certaines espèces de CMA et microorganismes suggérèrent qu’en plus des effets de la concentration en HP et l'identité des plantes hôtes, les CMA peuvent aussi affecter la structure des communautés microbiennes qui vivent sur leurs spores et mycéliums. La comparaison entre les communautés microbiennes identifiées en association avec les spores et celles identifiées dans les racines montre que les communautés microbiennes recrutées par les CMA sont différentes de celles retrouvées dans les sols et les racines. En conclusion, mon projet de doctorat apporte de nouvelles connaissances importantes sur la diversité des CMA dans un environnement extrêmement pollué par les HP, et démontre que les interactions entre les CMA et les microorganismes qui leur sont associés sont plus compliquées que ce qu’on croyait précédemment. Par conséquent, d'autres travaux de recherche sont recommandés, dans le futur, afin de comprendre les processus de recrutement des microorganismes par les CMA dans les différents environnements.
Arbuscular mycorrhizal fungi (AMF) are an important soil fungal group that belongs to the phylum Glomeromycota. AMF form symbiosic associations known as arbuscular mycorrhiza with more than 80% of vascular plants on earth. Once AMF colonize plant roots, they promote nutrient uptake, in particular phosphorus and nitrogen, and protect plants against soil-borne pathogens. In turn, plants provide AMF with carbon resources and habitat. Furthermore, more recent studies demonstrated that AMF may also play key roles in phytoremediation of soils contaminated with petroleum hydrocarbon pollutants (PHP) and trace elements. Though, in natural ecosystems, AMF undergo tripartite associations with host plants and micoorganisms (Bacteria and Fungi) living in rhizosphere (the narrow region of soil surounding the plant roots), endosphere (inside roots) and mycosphere (on the surface AMF mycelia), which some of them play a key role on translocation, immobilization and/or degradation of organic and inorganic pollutants. Consequently, the diversity and community structures of AMF and their associated microorganisms are influenced by the composition and concentration of pollutants and exudates released by the three partners (AMF, bacteria and plant roots). However, little is known about the diversity of AMF and their associated microorganisms in polluted soils and the interaction between AMF and these microorganisms remains poorly understood both in natural and contaminated areas. In this context, the objectives of my thesis were to: i) study the diversity of AMF and their associated microorganisms in PHP contaminated soils, ii) study the variation in diversity and community structures of AMF and their associated microorganisms across plant species identity and PHP concentrations, iii) study the correlations (covariations) between AMF species and their associated microorganisms and iv) compare microbial community structures of PHP contaminated soils and roots with those associated with AMF spores in order to determine if the microbial communities shaped on the surface of AMF spores and mycelia are different from those identified in soil and roots. To do so, AMF spores and/or their intraradical propagules were harvested from rhizospheric soil and roots of three plant species growing spontaneously in three distinct waste decantation basins of a former petrochemical plant located on the south shore of the St-Lawrence River, near Montreal. The harvested spores and propagules, as well as samples of soils and roots were subjected to PCR (we target 16S rRNA genes for bacteria, 18S rRNA genes for AMF and ITS regions for the other fungi), cloning, Sanger sequencing or 454 high throughput sequencing. Then, bioinformatics and statistics were performed to evaluate the effects of biotic and abiotic driving forces on AMF and their associated microbial communities. My results showed high fungal and bacterial diversity associated with AMF spores and propagules in PHP contaminated soils. I also observed that the microbial community structures associated with AMF spores were significantly affected by plant species identity and PHP concentrations. Furthermore, I observed positive and negative correlations between some AMF species and some AMF-associated microorganisms, suggesting that in addition to PHP concentrations and plant species identity, AMF species may also play a key role in shaping the microbial community surrounding their spores. Comparisons between the AMF spore-associated microbiome and the whole microbiome found in rhizospheric soil and roots showed that AMF spores recruit a microbiome differing from those found in the surrounding soil and roots. Overall, my PhD project brings a new level of knowledge on AMF diversity on extremely polluted environment and demonstrates that interaction of AMF and their associated microbes is much complex that we though previously. Further investigations are needed to better understand how AMF select and reward their associated microbes in different environments.