abstract: The aboveground surfaces of plants (i.e. the phyllosphere) comprise the largest biological interface on Earth (over 108 km2). The phyllosphere is a diverse microbial environment where bacterial inhabitants have been shown to sequester and degrade airborne pollutants (i.e. phylloremediation). However, phyllosphere dynamics are not well understood in urban environments, and this environment has never been studied in the City of Phoenix, which maintains roughly 92,000 city trees. The phyllosphere will grow if the City of Phoenix is able to achieve its goal of 25% canopy coverage by 2030, but this begs the question: How and where should the urban canopy expand? I addressed this question from a phyllosphere perspective by sampling city trees of two species, Ulmus parvifolia (Chinese Elm) and Dalbergia sissoo (Indian Rosewood) in parks and on roadsides. I identified characteristics of the bacterial community structure and interpreted the ecosystem service potential of trees in these two settings. I used culture-independent methods to compare the abundance of each unique bacterial lineage (i.e. ontological taxonomic units or OTUs) on the leaves of park trees versus on roadside tree leaves. I found numerous bacteria (81 OTUs) that were significantly more abundant on park trees than on roadside trees. Many of these OTUs are ubiquitous to bacterial phyllosphere communities, are known to promote the health of the host tree, or have been shown to degrade airborne pollutants. Roadside trees had fewer bacteria (10 OTUs) that were significantly more abundant when compared to park trees, but several have been linked to the remediation of petroleum combustion by-products. These findings, that were not available prior to this study, may inform the City of Phoenix as it is designing its future urban forests. / Dissertation/Thesis / Masters Thesis Sustainability 2016

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第17792号 / 農博第2013号 / 新制||農||1016(附属図書館) / 学位論文||H25||N4783(農学部図書室) / 30599 / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 阪井 康能, 教授 小川 順, 教授 梅澤 俊明 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

The microorganisms that live in and on a host (the microbiome) influence host phenotype, health, and behavior in plants and animals. However, the effects of the host and environment on the composition of microbiome communities are unclear. This is especially true in arid and semi-arid environments such as deserts that filter many microorganisms. This study investigated variation in phyllosphere microbiotal bacterial assemblages of prickly pear cactus (Opuntia) across differing spatial scales, ecoregions, and taxa throughout Texas at two timepoints. I expected bacterial assemblages to differ significantly among Texas ecoregions and among species. Results support significant influences on bacterial assemblage composition including ecoregion, taxonomy, and potentially seasonal time of sampling. The dry season timepoint yielded high microbial diversity and abundance across species and ecoregions, with different species and ecoregions harboring unique microbial communities. The rainy season timepoint yielded significantly lower levels of microbial diversity and abundance across species and ecoregions.

Plant-associated microbes (collectively the microbiome) are important contributors to plant health. They are known to play roles in increasing yield via improving stress tolerance, promoting growth, and suppressing the activity of plant pathogens. We investigated the wheat seed-head microbiome (phytomicrobiome) as a potential source of resistance to Fusarium head blight (FHB), or scab. FHB is a devastating disease in wheat, and other cereal grains, that causes losses in both quantity, through reduced yield, and quality of grain, through the production of toxins such as Deoxynivalenol. Efforts to combat FHB have focused primarily on breeding cultivars with resistance and applying fungicides. However, new resources for combatting FHB may lie in microbiome-plant interactions. To explore host-microbiome-pathogen interactions, we used field trials to characterize the seed head bacterial community (16S rRNA gene amplicons) across planting locations, host resistance genotypes, varieties, and plant development stages. We identified bacterial amplicon sequence variants (ASVs) present in each sample and then examined ASV community composition based on our variables. Characterizing bacterial relative abundance across samples, we identified 9,063 ASVs. These ASVs clustered according to plant developmental stages or maturity plant, location, and host genotype, but not by variety or maturity group. First, comparing plants at the pre-flowering versus mature grain-head stage, we found that both bacterial community richness and evenness changed significantly. In addition to these developmental changes, we found that bacterial community structure changes across locations, even between locations. Finally, we found that, in the presence of the pathogen, ASVs cluster by host resistance genotype, and that there are important taxonomic groups that are differentially abundant in the presence of the pathogen. Overall, we found that the wheat grain-head microbiome is shaped by environment-host-pathogen interactions, and that these interactions lead to differential abundance of particular community members that may be important in the management of FHB. / Master of Science in Life Sciences / Plant associated microbes are important contributors to plant health. They are known to play roles in increasing yield via improved stress tolerance, promoting growth, and suppressing plant disease. We investigated the wheat grain-head microbial communities as a source of disease resistance. The disease is called Fusarium Head Blight (FHB) and is caused by Fusarium graminaerum. FHB is a devastating disease in wheat and other cereals, causing losses, through reduced yield and quality through the production of toxins that prohibit use of the grain. To combat FHB, research has focused on developing plants that have resistance and the application of chemical fungicides. However, new resources for combating FHB may lie in the interactions between plants and microbes. This research is focused on identifying microbes that naturally interact with the plant, and how the pathogen, Fusarium, interacts with these beneficial microbes. In field trials, we characterized the microbial community by DNA sequencing technologies across locations, wheat with varying levels of genetic resistance, and wheat developmental stages. First, between the wheat kernel samples of pre-flowering and maturity, we found significant differences in microbial community. Consistent with other studies we found that the largest changes in microbial community composition across different growing locations. Finally, we found an interaction between the grain head microbiome and host resistance state when plants were exposed to the pathogen. Overall, we find that the wheat grain head microbiome is shaped by growing location and through interactions with the plant host and pathogen.

Secreted surface proteins are an innate immune defense component employed by animals to inhibit invading microbes. Surface proteins have not been documented in plants, even though the aerial leaf surface, or phylloplane, is a major site of pathogen ingress. We have discovered novel proteins, termed phylloplanins, which accumulate on leaf surfaces of Nicotiana tabacum, and we have isolated the gene Phylloplanin that is unique in gene databases. Natural and E. coli-expressed phylloplanins inhibit spore germination and limit leaf infection by the oomycete pathogen Peronospora tabacina. We investigated the site of phylloplanin biosynthesis using biochemical techniques. These techniques included radiolabeling of detached trichome glands, radiolabeling of epidermal peels, analysis of leaf water washes of various Nicotiana plants, and examination of guttation fluid, leaf vein contents, and extracellular fluid. From these experiments, we tentatively conclude that phylloplanins are produced by hydathodes, or an unknown surface secreting system, but not by glandular secreting trichomes. Future experiments with the phylloplanin promoter, whose elucidation is described herein, and its fusion to a reporter gene (GUS or GFP), will undoubtedly provide further insight into the location of phylloplanin biosynthesis and deposition. We suggest that the hydrophobic nature of phylloplanins aids in their dispersal over the leaf surface. Phylloplanins constitute a first-point-of-contact, rapid response, innate immune deterrent to pathogen establishment on N. tabacum leaf surfaces, and are the first studied representatives of a novel protein class in the plant kingdom. Further study of leaf surface proteins is justified to understand further their roles in plant defense, and to investigate their potential in agricultural biotechnology. Additionally, we describe miscellaneous observations we have made during the course of this research. Low molecular mass proteins (as yet uncharacterized) are washed from leaf surfaces of sunflower, soybean, and other plants. Pathogenesis-related (PR-)-5a, a known antifungal protein, was found to be present on the leaf surfaces of healthy plants, although its function there remains unknown. A phylloplanin homologue from Arabidopsis appears to be antibacterial. Further study of this protein is warranted. We note that proteins can also be recovered from N. tabacum root surfaces, or the rhizoplane, but we have not further characterized these proteins. In summary, novel surface-accumulated proteins, termed phylloplanins, and the gene encoding these have been discovered in N. tabacum. An antifungal function for phylloplanins is reported, and evidence was found for a unique mechanism of surface deposition.

付記する学位プログラム名: 京都大学大学院思修館 / 京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第20424号 / 農博第2209号 / 新制||農||1047(附属図書館) / 学位論文||H29||N5045(農学部図書室) / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 阪井 康能, 教授 矢﨑 一史, 教授 栗原 達夫 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

O processo de fixação biológica de nitrogênio (FBN) é a principal forma de entrada de nitrogênio (N) em ecossistemas naturais e é intermediado por microorganismos diazotróficos simbióticos ou de vida livre. A produção de biofertilizantes com bactérias diazotróficas é a principal alternativa ao uso de fertilizantes nitrogenados solúveis. Apesar da simbiose Rhizobium-leguminosas ser eficiente em promover o crescimento das plantas, a inoculação de bactérias diazotróficas em gramíneas vem apresentando resultados questionáveis, principalmente devido à baixa eficiência e incompatibilidade entre os atuais inoculantes bacterianos e as plantas. Os biofertilizantes para gramíneas utilizam bactérias endofíticas as quais desenvolvem interações pouco conhecidas com as plantas. Uma possibilidade para melhorar a eficiência da FBN em gramíneas é a aplicação de bactérias epifíticas de menor seletividade em relação às bactérias endofíticas. Os objetivos deste trabalho foram isolar novos genótipos de bactérias diazotróficas da filosfera de bambu da Mata Atlântica, verificar seu potencial biotecnológico in vitro e avaliar sua eficiência agronômica como biofertilizante em cana-de-açúcar em casa-de-vegetação. Foram obtidos 120 isolados bacterianos os quais foram caracterizados morfológica e filogenéticamente. Com relação ao potencial biotecnológico, 48 isolados bacterianos apresentaram resposta positiva nos testes in vitro de redução de acetileno (ARA) e para produção in vitro de ácido indol-3-acético (AIA) na presença de L-triptofano. Desses 48 isolados, 50% apresentaram capacidade de solubilizar fosfato de cálcio, 8% de produzir quitinases, 16% de produzir ACC desaminase e 17% de produzir sideróforos. O sequenciamento do gene rRNA 16S indicou que 75% dos isolados da filosfera de bambu foram similares à classe Gammaproteobacteria (Proteobacteria) , e a família Enterobacteriaceae. Dentre eles, 32% dos isolados apresentaram sequências similares a Klebsiella sp. e 2% foram similares a Serratia e Enterobacter. Das 48 sequências analisadas, 37% delas não foram classificadas quanto ao gênero, podendo então representar novos gêneros ou espécies. Dez isolados contendo o gene nifH foram selecionados para experimentos de eficiência agronômica em casa-de-vegetação usando bactérias inoculadas via pulverização foliar ou encapsuladas em micro-esferas de alginato e associados a diferentes níveis de adubação nitrogenada. A inoculação por meio de pulverização nas folhas de cana-de-açúcar resultou em aumento significativo de massa seca de parte aérea e concentração de N na parte aérea das plantas na sua fase inicial de desenvolvimento. A maior taxa de fixação nas folhas inoculadas foi observada sete dias após a inoculação no tratamento sem adição de N mineral. A inoculação no solo com os mesmos 10 isolados encapsulados em matriz polimérica em conjunto com diferentes níveis de adubação nitrogenada mostrou aumentos significativos de massa seca da parte aérea, raízes e concentração de N na parte aérea da cana-de-açúcar em comparação aos controles, principalmente nas fases tardias do desenvolvimento das plantas. O solo que recebeu as bactérias encapsuladas apresentaram elevadas taxas de FBN, oscilando entre 0 e 4 g de N g-1 h-1, 7 dias após a inoculação. Os resultados sugerem que as bactérias selecionadas possuem alto potencial biotecnológico para promover o crescimento das plantas em momentos diferentes do seu ciclo de desenvolvimento, dependendo do tipo de abordagem para inoculação. / The process of biological nitrogen fixation (BNF) is the most important form of nitrogen (N) input in natural ecosystems and is mediated by symbiotic or free-living diazotrophic microorganisms. The production of biofertilizers containing diazotrophic bacteria is the main alternative to the use of soluble nitrogen fertilizers, improving plant growth by nitrogen fixation or other plant growth promoting mechanisms. Despite the efficiency of the symbioses Rhizobium-legumes in promoting plant growth, the inoculation of diazotrophic bacteria in grasses, such as sugarcane, has shown variable results, mainly due to the low efficiency and incompatibility between the current bacterial strains used in inoculants and plant genotypes. Most of the biofertilizers for grasses uses endophytic bacteria that develop complexes interactions with the host plant, which are not totally understood. A possibility to improve the efficiency of BNF in grasses is the application of epiphytic diazotrophic bacteria that are less selective as compared to endophytes. The aims of this work were to isolate new genotypes of diazotrophic bacteria from the phyllosphere of bamboo from Atlantic Forest, determine their biotechnological potential based on in vitro assays, and evaluate their agronomic efficiency as biofertilizer for sugarcane under greenhouse conditions. A total of 120 bacterial isolates were obtained and characterized morphologically and phylogenetically. Regarding the biotechnological potential, 48 isolates showed positive responses under in vitro the acetylene reduction assay (ARA) and indole-acetic-acid (IAA) production in vitro assay in the presence of L-tryptophan. Among the 48 isolates evaluated, 50% were able to solubilize calcium phosphate, 8% produced chitinases, 16% were able to produce ACC deaminase, and 17% produced siderophores. The sequencing of the rRNA 16S gene revealed that 75% of the isolates were phylogenetically related to the family Enterobacteriaceae (Gammaproteobacteria) . The genus Klebsiella accounted for 32% of the isolates, whereas Serratia and Enterobacter accounted for 2%. Aproximatelly 37% of the isolates were assembled unclassified Bacteria. Ten isolates containing the nifH gene were selected for agronomic efficiency test under greenhouse conditions, using bacteria inoculated via foliar spraying, or encapsulation in alginate beads and inoculation in the soil, associated with different doses of nitrogen fertilizer. The inoculation on the sugarcane leaf surfaces resulted in significant increases in root biomass and N concentration in the shoots at the early stage of plant development. The highest N fixation rates in inoculated leaves were observed 7 days after inoculation in the absence of mineral N. The soil inoculation with the same 10 isolates immobilized in polymeric matrix in addiction to different rates of nitrogen fertilization showed significant increases in shoot and root biomass and N concentration in the shoots of sugarcane, when compared to the controls, mostly at later stages of plant development. The soil inoculated with encapsulated bacteria showed high rates of BNF even when nitrogen fertilizer was applied, ranging between 0 and 4 g of N g-1 h-1 seven days after the inoculation. The results suggest that the selected bacteria have high biotechnological potential to promote sugarcane growth at different stages of development, depending on the inoculation approach.

A Mata Atlântica é uma floresta tropical úmida considerada um \"hotspot\" de biodiversidade e endemismo. É uma das florestas mais antigas do mundo e uma das maiores florestas da América, abrangendo aproximadamente 150 milhões de hectares em condições ambientais altamente heterogêneas. Estudos em diferentes ambientes da Mata Atlântica, nos núcleos de Picinguaba e Santa Virginia no Parque Estadual da Serra do Mar (PESM), têm sido realizados para determinar a diversidade de espécies e alterações da estrutura das comunidades de bactérias, tanto na filosfera, quanto na dermosfera e solo rizosférico. No entanto, pouco se sabe sobre as funções ecológicas dessas bactérias, e sobre as interações ecológicas entre as comunidades microbianas e os ambientes onde se desenvolvem. Assim o objetivo desse trabalho foi explorar as interações entre as comunidades microbianas da filosfera, dermosfera e solo coletado sobre a projeção da copa de duas espécies arbóreas da Mata Atlântica ao longo de um gradiente altitudinal, usando análises de co-ocorrência, a partir dos dados obtidos por pirosequenciamento da região V4 do gene rRNA 16S de bactérias, para determinar padrões de associações de bactérias em diferentes níveis taxonômicos em cada microambiente. Para esse estudo, foi proposta a hipótese de que mesmo que as condições ambientais sejam diferentes em cada tipo de floresta (gradiente altitudinal), pode existir grupos de bactérias específicos que co-ocorrem na filosfera, dermosfera ou solo das plantas, funcionando como taxons chaves na estruturação das comunidades bacterianas. Com base do sequenciamento dos genes rRNA 16S, as comunidades bacterianas associadas à filosfera e dermosfera de E. edulis e G. opposita nas diferentes florestas foram mais similares entre si do que as do solo. Actinobacteria, Firmicutes, Bacteroidetes e Proteobacteria foram mais abundantes em todos os microambientes estudados. Diferenças nas estruturas das comunidades bacterianas na filosfera, dermosfera e solo foram observadas ao longo do gradiente altitudinal, independente da espécie de planta. Na floresta de terras baixas, a comunidade bacteriana associada à filosfera foi mais similar entre E. edulis e G. opposita. No solo, a comunidade bacteriana foi mais similar dentro de cada tipo de floresta do que entre florestas, sugerindo um efeito da fisionomia da floresta nas comunidades de bactérias dos solos. Explorando as redes de co-ocorrência das comunidades bacterianas em cada microambiente observou-se que no nível de UTOs, cada microambiente têm diferentes táxons chaves que podem regular as interações ecológicas da comunidade. Embora táxons chaves não representam as UTOs mais abundantes em cada microambiente, eles pertencem, predominantemente às classes Alphaproteobacteria e Gammaproteobacteria, sugerindo que na filosfera, dermosfera e solo o core microbioma não pode ser definido ao nível de UTO, mas possivelmente a níveis taxonômicos mais elevados representando grandes grupos microbianos que apresentam funções redundantes. / The Atlantic Forest is a rainforest considered a hotspot of biodiversity and endemism. It is one of the oldest forests in the world and one of the largest forests of America, covering approximately 150 million hectares in highly heterogeneous environmental conditions. Studies in different environments of the Atlantic forest, in the Picinguaba and Santa Virginia areas in the Serra do Mar State Park (PESM) have been conducted to determine the species diversity and changes in the structure of the bacterial communities in the phyllosphere, dermosphere and rhizosphere. However, little is known on the ecological functions of these bacteria, and on the ecological interactions between microbial communities and the environment in which they develop. The aim of this study was to explore the interactions between the microbial communities of the phyllosphere, dermosphere and rhizosphere of two tree species of the Atlantic Forest along an altitudinal gradient. Co-occurrence analysis based on data obtained by pyrosequencing of the 16S rRNA gene V4 region of bacteria to determine patterns of bacterial associations in different taxonomic levels in each microenvironment. For this study, the hypothesis that even if the environmental conditions are different in each type of forest (altitudinal gradient), there may be specific groups of bacteria that co-occur in the phyllosphere, dermosphere or rhizosphere, functioning as keystone taxa in the bacterial communities. Based on the sequencing of 16S rRNA genes, bacterial communities associated with the E. edulis and G. opposita phyllosphere and dermosphere in different forests were more similar to each other than the rhizosphere. Actinobacteria, Firmicutes, Proteobacteria and Bacteroidetes were the more abundant taxa in all studied microenvironments. Differences in the bacterial community structures in the phyllosphere, dermosphere and rhizosphere were observed along the altitudinal gradient, regardless of the plant species. In the lowland forest, the bacterial community associated with the phyllosphere was more similar between E. edulis and G. opposita. The rhizosphere bacterial community was more similar within each forest type than between forests, suggesting an effect of the forest physiognomy on the bacterial communities of the rhizosphere. Exploring the co-occurrence networks in the bacterial communities of each microenvironment it was observed that at the OTU level each microenvironment has different keystoine taxa that may regulate the ecological interactions in the community. Although the keystone taxa do not represent the most abundant OTUs in each microenvironment, they belong predominantly to Alphaproteobacteria and Gammaproteobacteria classes, suggesting that in the phyllosphere, dermosphere and rhizosphere the core microbiome cannot be determined at the OTU level, but possibly at higher taxonomic levels representing microbial groups having redundant functions.

A Mata Atlântica é uma floresta tropical úmida considerada um \"hotspot\" de biodiversidade e endemismo. É uma das florestas mais antigas do mundo e uma das maiores florestas da América, abrangendo aproximadamente 150 milhões de hectares em condições ambientais altamente heterogêneas. Estudos em diferentes ambientes da Mata Atlântica, nos núcleos de Picinguaba e Santa Virginia no Parque Estadual da Serra do Mar (PESM), têm sido realizados para determinar a diversidade de espécies e alterações da estrutura das comunidades de bactérias, tanto na filosfera, quanto na dermosfera e solo rizosférico. No entanto, pouco se sabe sobre as funções ecológicas dessas bactérias, e sobre as interações ecológicas entre as comunidades microbianas e os ambientes onde se desenvolvem. Assim o objetivo desse trabalho foi explorar as interações entre as comunidades microbianas da filosfera, dermosfera e solo coletado sobre a projeção da copa de duas espécies arbóreas da Mata Atlântica ao longo de um gradiente altitudinal, usando análises de co-ocorrência, a partir dos dados obtidos por pirosequenciamento da região V4 do gene rRNA 16S de bactérias, para determinar padrões de associações de bactérias em diferentes níveis taxonômicos em cada microambiente. Para esse estudo, foi proposta a hipótese de que mesmo que as condições ambientais sejam diferentes em cada tipo de floresta (gradiente altitudinal), pode existir grupos de bactérias específicos que co-ocorrem na filosfera, dermosfera ou solo das plantas, funcionando como taxons chaves na estruturação das comunidades bacterianas. Com base do sequenciamento dos genes rRNA 16S, as comunidades bacterianas associadas à filosfera e dermosfera de E. edulis e G. opposita nas diferentes florestas foram mais similares entre si do que as do solo. Actinobacteria, Firmicutes, Bacteroidetes e Proteobacteria foram mais abundantes em todos os microambientes estudados. Diferenças nas estruturas das comunidades bacterianas na filosfera, dermosfera e solo foram observadas ao longo do gradiente altitudinal, independente da espécie de planta. Na floresta de terras baixas, a comunidade bacteriana associada à filosfera foi mais similar entre E. edulis e G. opposita. No solo, a comunidade bacteriana foi mais similar dentro de cada tipo de floresta do que entre florestas, sugerindo um efeito da fisionomia da floresta nas comunidades de bactérias dos solos. Explorando as redes de co-ocorrência das comunidades bacterianas em cada microambiente observou-se que no nível de UTOs, cada microambiente têm diferentes táxons chaves que podem regular as interações ecológicas da comunidade. Embora táxons chaves não representam as UTOs mais abundantes em cada microambiente, eles pertencem, predominantemente às classes Alphaproteobacteria e Gammaproteobacteria, sugerindo que na filosfera, dermosfera e solo o core microbioma não pode ser definido ao nível de UTO, mas possivelmente a níveis taxonômicos mais elevados representando grandes grupos microbianos que apresentam funções redundantes. / The Atlantic Forest is a rainforest considered a hotspot of biodiversity and endemism. It is one of the oldest forests in the world and one of the largest forests of America, covering approximately 150 million hectares in highly heterogeneous environmental conditions. Studies in different environments of the Atlantic forest, in the Picinguaba and Santa Virginia areas in the Serra do Mar State Park (PESM) have been conducted to determine the species diversity and changes in the structure of the bacterial communities in the phyllosphere, dermosphere and rhizosphere. However, little is known on the ecological functions of these bacteria, and on the ecological interactions between microbial communities and the environment in which they develop. The aim of this study was to explore the interactions between the microbial communities of the phyllosphere, dermosphere and rhizosphere of two tree species of the Atlantic Forest along an altitudinal gradient. Co-occurrence analysis based on data obtained by pyrosequencing of the 16S rRNA gene V4 region of bacteria to determine patterns of bacterial associations in different taxonomic levels in each microenvironment. For this study, the hypothesis that even if the environmental conditions are different in each type of forest (altitudinal gradient), there may be specific groups of bacteria that co-occur in the phyllosphere, dermosphere or rhizosphere, functioning as keystone taxa in the bacterial communities. Based on the sequencing of 16S rRNA genes, bacterial communities associated with the E. edulis and G. opposita phyllosphere and dermosphere in different forests were more similar to each other than the rhizosphere. Actinobacteria, Firmicutes, Proteobacteria and Bacteroidetes were the more abundant taxa in all studied microenvironments. Differences in the bacterial community structures in the phyllosphere, dermosphere and rhizosphere were observed along the altitudinal gradient, regardless of the plant species. In the lowland forest, the bacterial community associated with the phyllosphere was more similar between E. edulis and G. opposita. The rhizosphere bacterial community was more similar within each forest type than between forests, suggesting an effect of the forest physiognomy on the bacterial communities of the rhizosphere. Exploring the co-occurrence networks in the bacterial communities of each microenvironment it was observed that at the OTU level each microenvironment has different keystoine taxa that may regulate the ecological interactions in the community. Although the keystone taxa do not represent the most abundant OTUs in each microenvironment, they belong predominantly to Alphaproteobacteria and Gammaproteobacteria classes, suggesting that in the phyllosphere, dermosphere and rhizosphere the core microbiome cannot be determined at the OTU level, but possibly at higher taxonomic levels representing microbial groups having redundant functions.

O processo de fixação biológica de nitrogênio (FBN) é a principal forma de entrada de nitrogênio (N) em ecossistemas naturais e é intermediado por microorganismos diazotróficos simbióticos ou de vida livre. A produção de biofertilizantes com bactérias diazotróficas é a principal alternativa ao uso de fertilizantes nitrogenados solúveis. Apesar da simbiose Rhizobium-leguminosas ser eficiente em promover o crescimento das plantas, a inoculação de bactérias diazotróficas em gramíneas vem apresentando resultados questionáveis, principalmente devido à baixa eficiência e incompatibilidade entre os atuais inoculantes bacterianos e as plantas. Os biofertilizantes para gramíneas utilizam bactérias endofíticas as quais desenvolvem interações pouco conhecidas com as plantas. Uma possibilidade para melhorar a eficiência da FBN em gramíneas é a aplicação de bactérias epifíticas de menor seletividade em relação às bactérias endofíticas. Os objetivos deste trabalho foram isolar novos genótipos de bactérias diazotróficas da filosfera de bambu da Mata Atlântica, verificar seu potencial biotecnológico in vitro e avaliar sua eficiência agronômica como biofertilizante em cana-de-açúcar em casa-de-vegetação. Foram obtidos 120 isolados bacterianos os quais foram caracterizados morfológica e filogenéticamente. Com relação ao potencial biotecnológico, 48 isolados bacterianos apresentaram resposta positiva nos testes in vitro de redução de acetileno (ARA) e para produção in vitro de ácido indol-3-acético (AIA) na presença de L-triptofano. Desses 48 isolados, 50% apresentaram capacidade de solubilizar fosfato de cálcio, 8% de produzir quitinases, 16% de produzir ACC desaminase e 17% de produzir sideróforos. O sequenciamento do gene rRNA 16S indicou que 75% dos isolados da filosfera de bambu foram similares à classe Gammaproteobacteria (Proteobacteria) , e a família Enterobacteriaceae. Dentre eles, 32% dos isolados apresentaram sequências similares a Klebsiella sp. e 2% foram similares a Serratia e Enterobacter. Das 48 sequências analisadas, 37% delas não foram classificadas quanto ao gênero, podendo então representar novos gêneros ou espécies. Dez isolados contendo o gene nifH foram selecionados para experimentos de eficiência agronômica em casa-de-vegetação usando bactérias inoculadas via pulverização foliar ou encapsuladas em micro-esferas de alginato e associados a diferentes níveis de adubação nitrogenada. A inoculação por meio de pulverização nas folhas de cana-de-açúcar resultou em aumento significativo de massa seca de parte aérea e concentração de N na parte aérea das plantas na sua fase inicial de desenvolvimento. A maior taxa de fixação nas folhas inoculadas foi observada sete dias após a inoculação no tratamento sem adição de N mineral. A inoculação no solo com os mesmos 10 isolados encapsulados em matriz polimérica em conjunto com diferentes níveis de adubação nitrogenada mostrou aumentos significativos de massa seca da parte aérea, raízes e concentração de N na parte aérea da cana-de-açúcar em comparação aos controles, principalmente nas fases tardias do desenvolvimento das plantas. O solo que recebeu as bactérias encapsuladas apresentaram elevadas taxas de FBN, oscilando entre 0 e 4 g de N g-1 h-1, 7 dias após a inoculação. Os resultados sugerem que as bactérias selecionadas possuem alto potencial biotecnológico para promover o crescimento das plantas em momentos diferentes do seu ciclo de desenvolvimento, dependendo do tipo de abordagem para inoculação. / The process of biological nitrogen fixation (BNF) is the most important form of nitrogen (N) input in natural ecosystems and is mediated by symbiotic or free-living diazotrophic microorganisms. The production of biofertilizers containing diazotrophic bacteria is the main alternative to the use of soluble nitrogen fertilizers, improving plant growth by nitrogen fixation or other plant growth promoting mechanisms. Despite the efficiency of the symbioses Rhizobium-legumes in promoting plant growth, the inoculation of diazotrophic bacteria in grasses, such as sugarcane, has shown variable results, mainly due to the low efficiency and incompatibility between the current bacterial strains used in inoculants and plant genotypes. Most of the biofertilizers for grasses uses endophytic bacteria that develop complexes interactions with the host plant, which are not totally understood. A possibility to improve the efficiency of BNF in grasses is the application of epiphytic diazotrophic bacteria that are less selective as compared to endophytes. The aims of this work were to isolate new genotypes of diazotrophic bacteria from the phyllosphere of bamboo from Atlantic Forest, determine their biotechnological potential based on in vitro assays, and evaluate their agronomic efficiency as biofertilizer for sugarcane under greenhouse conditions. A total of 120 bacterial isolates were obtained and characterized morphologically and phylogenetically. Regarding the biotechnological potential, 48 isolates showed positive responses under in vitro the acetylene reduction assay (ARA) and indole-acetic-acid (IAA) production in vitro assay in the presence of L-tryptophan. Among the 48 isolates evaluated, 50% were able to solubilize calcium phosphate, 8% produced chitinases, 16% were able to produce ACC deaminase, and 17% produced siderophores. The sequencing of the rRNA 16S gene revealed that 75% of the isolates were phylogenetically related to the family Enterobacteriaceae (Gammaproteobacteria) . The genus Klebsiella accounted for 32% of the isolates, whereas Serratia and Enterobacter accounted for 2%. Aproximatelly 37% of the isolates were assembled unclassified Bacteria. Ten isolates containing the nifH gene were selected for agronomic efficiency test under greenhouse conditions, using bacteria inoculated via foliar spraying, or encapsulation in alginate beads and inoculation in the soil, associated with different doses of nitrogen fertilizer. The inoculation on the sugarcane leaf surfaces resulted in significant increases in root biomass and N concentration in the shoots at the early stage of plant development. The highest N fixation rates in inoculated leaves were observed 7 days after inoculation in the absence of mineral N. The soil inoculation with the same 10 isolates immobilized in polymeric matrix in addiction to different rates of nitrogen fertilization showed significant increases in shoot and root biomass and N concentration in the shoots of sugarcane, when compared to the controls, mostly at later stages of plant development. The soil inoculated with encapsulated bacteria showed high rates of BNF even when nitrogen fertilizer was applied, ranging between 0 and 4 g of N g-1 h-1 seven days after the inoculation. The results suggest that the selected bacteria have high biotechnological potential to promote sugarcane growth at different stages of development, depending on the inoculation approach.