Sugarcane (Saccharum officinarum L.) is one of the world's highly significant commercial crops. The amounts of synthetic nitrogen (N2) fertilizer required to grow the sugarcane plant at its initial growth stages are higher, which increases the production costs and adverse environmental consequences globally. To combat this issue, sustainable environmental and economic concerns among researchers are necessary. The endophytic diazotrophs can offer significant amounts of nitrogen to crops through the biological nitrogen fixation mediated nif gene. The nifH gene is the most extensively utilized molecular marker in nature for studying N2 fixing microbiomes. The present research intended to determine the existence of novel endophytic diazotrophs through culturable and unculturable bacterial communities (EDBCs). The EDBCs of different tissues (root, stem, and leaf) of five sugarcane cultivars (Saccharum officinarum L. cv. Badila, S. barberi Jesw.cv Pansahi, S. robustum, S. spontaneum, and S. sinense Roxb.cv Uba) were isolated and molecularly characterized to evaluate N2 fixation ability. The diversity of EDBCs was observed based on nifH gene Illumina MiSeq sequencing and a culturable approach. In this study, 319766 operational taxonomic units (OTUs) were identified from 15 samples. The minimum number of OTUs was recorded in leaf tissues of S. robustum and maximum reads in root tissues of S. spontaneum. These data were assessed to ascertain the structure, diversity, abundance, and relationship between the microbial community. A total of 40 bacterial families with 58 genera were detected in different sugarcane species. Bacterial communities exhibited substantially different alpha and beta diversity. In total, 16 out of 20 genera showed potent N2-fixation in sugarcane and other crops. According to principal component analysis (PCA) and hierarchical clustering (Bray-Curtis dis) evaluation of OTUs, bacterial microbiomes associated with root tissues differed significantly from stem and leaf tissues of sugarcane. Significant differences often were observed in EDBCs among the sugarcane tissues. We tracked and validated the plethora of individual phylum strains and assessed their nitrogenase activity with a culture-dependent technique. The current work illustrated the significant and novel results of many uncharted endophytic microbial communities in different tissues of sugarcane species, which provides an experimental system to evaluate the biological nitrogen fixation (BNF) mechanism in sugarcane. The novel endophytic microbial communities with N2-fixation ability play a remarkable and promising role in sustainable agriculture production.
Keywords: endophytic diazotrophs; high-throughput sequencing; microbial diversity; nifH gene; nitrogen fixation; sugarcane.