Plant microbiome and its manipulation herald a new era for plant biotechnology with the potential to benefit sustainable crop production. However, studies evaluating the diversity, structure and impact of the microbiota in economic important crops are still rare. Here we describe a comprehensive inventory of the structure and assemblage of the bacterial and fungal communities associated with sugarcane. Our analysis identified 23,811 bacterial OTUs and an unexpected 11,727 fungal OTUs inhabiting the endophytic and exophytic compartments of roots, shoots, and leaves. These communities originate primarily from native soil around plants and colonize plant organs in distinct patterns. The sample type is the primary driver of fungal community assemblage, and the organ compartment plays a major role in bacterial community assemblage. We identified core bacterial and fungal communities composed of less than 20% of the total microbial richness but accounting for over 90% of the total microbial relative abundance. The roots showed 89 core bacterial families, 19 of which accounted for 44% of the total relative abundance. Stalks are dominated by groups of yeasts that represent over 12% of total relative abundance. The core microbiome described here comprise groups whose biological role underlies important traits in plant growth and fermentative processes.

The soil-plant ecosystem harbors an immense microbial diversity that challenges investigative approaches to study traits underlying plant-microbe association. Studies solely based on culture-dependent techniques have overlooked most microbial diversity. Here we describe the concomitant use of culture-dependent and -independent techniques to target plant-beneficial microbial groups from the sugarcane microbiome. The community-based culture collection (CBC) approach was used to access microbes from roots and stalks. The CBC recovered 399 unique bacteria representing 15.9% of the rhizosphere core microbiome and 61.6–65.3% of the endophytic core microbiomes of stalks. By cross-referencing the CBC (culture-dependent) with the sugarcane microbiome profile (culture-independent), we designed a synthetic community comprised of naturally occurring highly abundant bacterial groups from roots and stalks, most of which has been poorly explored so far. We then used maize as a model to probe the abundance-based synthetic inoculant. We show that when inoculated in maize plants, members of the synthetic community efficiently colonize plant organs, displace the natural microbiota and dominate at 53.9% of the rhizosphere microbial abundance. As a result, inoculated plants increased biomass by 3.4-fold as compared to uninoculated plants. The results demonstrate that abundance-based synthetic inoculants can be successfully applied to recover beneficial plant microbes from plant microbiota.

Microbiome analysis using metagenomic sequencing has revealed a vast microbial diversity associated with plants. Identifying the molecular functions associated with microbiome-plant interaction is a significant challenge concerning the development of microbiome-derived technologies applied to agriculture. An alternative to accelerate the discovery of the microbiome benefits to plants is to construct microbial culture collections concomitant with accessing microbial community structure and abundance. However, traditional methods of isolation, cultivation, and identification of microbes are time-consuming and expensive. Here we describe a method for identification of microbes in culture collections constructed by picking colonies from primary platings that may contain single or multiple microorganisms, which we named community-based culture collections (CBC). A multiplexing 16S rRNA gene amplicon sequencing based on two-step PCR amplifications with tagged primers for plates, rows, and columns allowed the identification of the microbial composition regardless if the well contains single or multiple microorganisms. The multiplexing system enables pooling amplicons into a single tube. The sequencing performed on the PacBio platform led to recovery near-full-length 16S rRNA gene sequences allowing accurate identification of microorganism composition in each plate well. Cross-referencing with plant microbiome structure and abundance allowed the estimation of diversity and abundance representation of microorganism in the CBC.

Overexpression of a tomato TCTP impacts plant biomass production and performance under stress. These phenotypic alterations were associated with the up-regulation of genes mainly related to photosynthesis, fatty acid metabolism and water transport. The translationally controlled tumor protein (TCTP) is a multifaceted and highly conserved eukaryotic protein. In plants, despite the existence of functional data implicating this protein in cell proliferation and growth, the detailed physiological roles of many plant TCTPs remain poorly understood. Here we focused on a yet uncharacterized TCTP from tomato (SlTCTP). We show that, when overexpressed in tobacco, SlTCTP may promote plant biomass production and affect performance under salt and osmotic stress. Transcriptomic analysis of the transgenic plants revealed the up-regulation of genes mainly related to photosynthesis, fatty acid metabolism and water transport. This induced photosynthetic gene expression was paralleled by an increase in the photosynthetic rate and stomatal conductance of the transgenic plants. Moreover, the transcriptional modulation of genes involved in ABA-mediated regulation of stomatal movement was detected. On the other hand, genes playing a pivotal role in ethylene biosynthesis were found to be down-regulated in the transgenic lines, thus suggesting deregulated ethylene accumulation in these plants. Overall, these results point to a role of TCTP in photosynthesis and hormone signaling.

Os membros da Comissão Examinadora acima assinaram a Ata de Defesa, que se encontra no processo de vida acadêmica da aluna. RESUMOO ecossistema formado pela interação entre plantas e solo é composto por uma imensa diversidade de microorganismos que desafiam abordagens de estudos quanto as suas características e mecanismos de associação. Métodos tradicionais de isolamento, cultivo e identificação de microorganismos em coleções são laboriosos, de alto custo e consumem grande parcela de tempo. Neste trabalho utilizamos amostras de raiz, rizosfera e colmo da cana-deaçúcar para obtenção dos microorganismos associados. A partir disso, descrevemos uma nova metodologia para construção de uma coleção de microorganismos representativa do microbioma da cana-de-açúcar baseado em um sistema de obtenção de culturas. Este método é baseado em picar colônias de placas de cultura que podem conter um ou múltiplos microorganismos, armazenando-as em placas de 96 poços. Foram usados diferentes meios de cultura, suplementação com xarope de cana-de-açúcar e diferentes temperaturas de incubação em estufa para obtenção de uma ampla diversidade de microorganismos. A coleção completa é composta de 56 placas de 96 poços. Para identificação das bactérias presentes na coleção, foi desenvolvido um sistema multiplex para a amplificação do gene correspondente ao RNA ribossomal 16S constituído por duas etapas de amplificação por PCR com primers contendo barcodes para placas, linhas e colunas que permitiram identificar e rastrear o conteúdo bacteriano de cada poço independentemente do mesmo conter um ou múltiplos microorganismos. O sistema multiplex permite o agrupamento dos amplicons em um único tubo. O sequenciamento foi feito através da plataforma PacBio e permitiu a obtenção de sequências de tamanho quase completo do gene 16S que permitiu uma identificação acurada de cada poço. Comparando os dados obtidos para a coleção de microorganismos (dependente de cultivo) com o perfil do microbioma da cana (independente de cultivo) conseguimos obter 399 bactérias cultivadas que representam 15,9% do microbioma core da rizosfera e 61,6-65,3% dos representantes do microbioma core de colmo. Os resultados mostram que a metodologia utilizada para construção da coleção de microorganismos teve êxito em nosso objetivo de propor uma nova abordagem para obtenção de microorganismos benéficos para plantas a partir de seu microbioma.