In Situ Cultivation Approach to Increase the Culturable Bacterial Diversity in the Rhizobiome of Plants

The use of high-throughput DNA sequencing (HTS) has revealed the great diversity of rhizobacteria in plant rhizospheres; however, only a minor portion (<= 1%) of rhizobacteria belonging to few taxa can be cultured under laboratory conditions. In recent years, in situ cultivation has opened a window to explore a greater diversity of bacterial taxa in the environment. Here, we explored the total and culturable rhizobacterial communities associated with the rhizosphere of wheat plants by using 16S rRNA-based HTS and in situ cultivation with microwell chambers (MWCs), respectively. Results by HTS revealed to phyla Proteobacteria (29-39%), Acidobacteria (17%), Actinobacteria (11-15%), and Bacteroidetes (5-12%) as the most abundant rhizobacterial taxa in rhizosphere samples. A total of 206 isolates (26 genera) were obtained with MWCs, where coincidentally with HTS, the most abundant phyla were Proteobacteria (70.4%), Firmicutes (24%), Actinobacteria (4%), and Bacteroidetes (1.5%). At the genus level, the most of isolates (72%) belonged to Pseudomonas, followed by Bacillus, Stenotrophomonas, Delftia, and Herbaspirillum. Members of rare taxa (Lelliottia, Rhodococcus, Micrococcus, Variovorax, and Bosea) also were isolated by MWCs. In addition, a high proportion (82%) of isolates showed high similarity with plant beneficial and environmental non-pathogenic bacteria whereas a minor proportion (18%) of isolates showed high similarity to human and plant pathogenic bacteria. This study demonstrates that in situ cultivation represents a useful tool to isolate a greater number of rhizobacterial taxa, which can be investigated under laboratory conditions in bioprospecting (e.g., plant growth-promoting bacteria) and public health (e.g., human opportunist and plant pathogens) studies