The Effect of Inoculation of a Diazotrophic Bacterial Consortium on the Indigenous Bacterial Community Structure of Sugarcane Apoplast Fluid.

The extracellular space in plants, termed the apoplast, has a pH and sugar content that enables bacterial growth and represents a possible niche for the establishment of endophytic bacteria. Previous studies have investigated the effects of diazotrophic bacteria inoculation in sugarcane varieties, but it has not yet been analyzed how the microbial community of apoplast fluid of sugarcane is affected. High-throughput next generation sequencing of the 16S rRNA gene was used throughout this study to determine the effect of inoculation with a diazotrophic bacteria consortium, previously isolated from sugarcane, on the native bacterial population of sugarcane variety RB867515 grown in the field. The analyses were carried out 450 days after inoculation. The results revealed the presence of 22 phyla, with predominance of Proteobacteria phylum. It was observed that the inoculated consortium changed the indigenous bacterial community structure of sugarcane apoplast fluid by decreasing diversity and evenness, interfering in the composition of rare species. Microbial community composition analysis revealed differences between treatments. The differential abundance test showed there were 43 amplicon sequence variants (ASVs) which were relatively more abundant in the inoculated treatment, with predominance of the Sphingomonas genus. The predicted functions of the most abundant ASVs revealed the presence of genera related to plant growth promotion and protection against phytopathogens. Analysis to evaluate the occurrence of inoculated strains in the recovered data was not conclusive since the ASVs taxonomically close to the inoculated bacteria were observed in low abundance. The present study is the first report to elucidate the bacterial community in sugarcane apoplast fluid using a culture-independent approach. It demonstrated that the diazotrophic bacterial consortium interferes in the natural bacterial community in sugarcane variety RB867515.

Temporal assessment of root and shoot colonization of elephant grass (Pennisetum purpureum Schum.) host seedlings by Gluconacetobacter diazotrophicus strain LP343.

Gluconacetobacter diazotrophicus is a species of great agronomic potential due to its growth-promotion traits. Its colonization process in different plants has been reported. However, there have been no studies regarding its structural colonization in elephant grass. This is a fast-growing C4-Poaceae plant, and its application in Brazil is mainly aimed at feeding dairy cattle, due to its high nutritional value. Also, in the last decade, this grass has been applied in the production of biofuels. The present study aimed to monitor the colonization process of strain LP343 of G. diazotrophicus inoculated in elephant grass seedlings of PCEA genotype, by using a mCherry-tagged bacterium. Samples of roots and shoots collected at different periods were visualized by confocal laser-scanning microscopy. The colony-counting assay was used to compare the number of cells recovered in different niches and a qPCR was performed for the quantification of endophytic cells in root and shoot tissues. Results suggested that the strain LP343 quickly recognized the PCEA roots as host, attached to the elephant grass roots at 6 h, and 7 days after inoculation were able to colonize the xylem vessels of roots and shoots of elephant grass. This study advances our knowledge about the colonization process of G. diazotrophicus species in elephant grass, contributing to future studies involving the plant-bacteria interaction cultivated under gnotobiotic conditions.

A Culture-Independent Approach to Enrich Endophytic Bacterial Cells from Sugarcane Stems for Community Characterization.

Bacterial endophytes constitute a very diverse community and they confer important benefits which help to improve agricultural yield. Some of these benefits remain underexplored or little understood, mainly due to the bottlenecks associated with the plant feature, a low number of endophytic bacterial cells in relation to the plant, and difficulties in accessing these bacteria using cultivation-independent methods. Enriching endophytic bacterial cells from plant tissues, based on a non-biased, cultivation-independent physical enrichment method, may help to circumvent those problems, especially in the case of sugarcane stems, which have a high degree of interfering factors, such as polysaccharides, phenolic compounds, nucleases, and fibers. In the present study, an enrichment approach for endophytic bacterial cells from sugarcane lower stems is described. The results demonstrate that the enriched bacterial cells are suitable for endophytic community characterization. A community analysis revealed the presence of previously well-described but also novel endophytic bacteria in sugarcane tissues which may exert functions such as plant growth promotion and biological control, with a predominance of the Proteobacterial phylum, but also Actinobacteria, Bacteroidetes, and Firmicutes, among others. In addition, by comparing the present and literature data, it was possible to list the most frequently detected bacterial endophyte genera in sugarcane tissues. The presented enrichment approach paves the way for improved future research toward the assessment of endophytic bacterial community in sugarcane and other biofuel crops.