Diazotroph Diversity and Nitrogen Fixation in Summer Active Perennial Grasses in a Mediterranean Region Agricultural Soil

ORIGINAL RESEARCH published: 05 November 2019 doi: 10.3389/fmolb.2019.00115 Diazotroph Diversity and Nitrogen Fixation in Summer Active Perennial Grasses in a Mediterranean Region Agricultural Soil Vadakattu V. S. R. Gupta 1*, Bangzhou Zhang 2,3 , Christopher Ryan Penton 4,5 , Julian Yu 4,5 and James M. Tiedje 3 1 CSIRO Agriculture and Food, Waite Campus, Urrbrae, SA, Australia, 2 Institute for Microbial Ecology, School of Medicine, Xiamen University, Xiamen, China, 3 Center for Microbial Ecology, Michigan State University, East Lansing, MI, United States, 4 College of Integrative Sciences and Arts, Arizona State University, Mesa, AZ, United States, 5 Center for Fundamental and Applied Microbiomics, Biodesign Institute, Arizona State University, Tempe, AZ, United States Edited by: Christopher Staley, University of Minnesota Twin Cities, United States Reviewed by: Gregorio Peron, University of Barcelona, Spain Michael Kertesz, University of Sydney, Australia *Correspondence: Vadakattu V. S. R. Gupta Specialty section: This article was submitted to Metabolomics, a section of the journal Frontiers in Molecular Biosciences Received: 28 June 2019 Accepted: 11 October 2019 Published: 05 November 2019 Citation: Gupta VVSR, Zhang B, Penton CR, Yu J and Tiedje JM (2019) Diazotroph Diversity and Nitrogen Fixation in Summer Active Perennial Grasses in a Mediterranean Region Agricultural Soil. Front. Mol. Biosci. 6:115. doi: 10.3389/fmolb.2019.00115 Summer-growing perennial grasses such as Panicum coloratum L. cv. Bambatsi (Bambatsi panic), Chloris gayana Kunth cv. Katambora (Rhodes grass) and Digitaria eriantha Steud. cv. Premier (Premier digit grass) growing in the poor fertility sandy soils in the Mediterranean regions of southern Australia and western Australia mainly depend upon soil N and biological N inputs through diazotrophic (free living or associative) N fixation. We investigated the community composition and diversity (nifH-amplicon sequencing), abundance (qPCR) and functional capacity (15 N incubation assay) of the endophytic diazotrophic community in the below and above ground plant parts of field grown and unfertilized grasses. Results showed a diverse and abundant diazotrophic community inside plant both above and below-ground and there was a distinct diazotrophic assemblage in the different plant parts in all the three grasses. There was a limited difference in the diversity between leaves, stems and roots except that Panicum grass roots harbored greater species richness. Nitrogen fixation potentials ranged between 0.24 and 5.9 mg N kg−1 day−1 and N fixation capacity was found in both the above and below ground plant parts. Results confirmed previous reports of plant species-based variation and that Alpha-Proteobacteria were the dominant group of nifH-harboring taxa both in the belowground and aboveground parts of the three grass species. Results also showed a well-structured nifH-harboring community in all plant parts, an example for a functional endophytic community. Overall, the variation in the number and identity of module hubs and connectors among the different plant parts suggests that co-occurrence patterns within the nifH-harboring community specific to individual compartments and local environments of the niches within each plant part may dictate the overall composition of diazotrophs within a plant. Keywords: diazotrophs, N fixation, endosphere, phyllosphere, nifH, perennial grasses Frontiers in Molecular Biosciences | www.frontiersin.org 1 November 2019 | Volume 6 | Article 115 Gupta et al. Diazotrophs in Perennial Grass Endosphere INTRODUCTION 2013), perennial grasses grown during the summer in southern and western Australia contain dense, N-limited rhizospheres that supply significant inputs of C through rhizodeposition contributing to deep soil C inputs and providing a C-rich environment for the maintenance of diazotrophic metabolism. For example, Panicum species has been shown to allocate a large portion of photosynthetically fixed C belowground that may be assimilated into the microbial component within a short-period of time (Roper et al., 2013). Compared to cropped soils, it has been suggested that the combination of higher total rhizosphere volume, increased C input, and a greater diversity of rhizosphere bacteria results in significant impacts to N-cycling processes such as N mineralization, with a further impact on soil C turnover (Roper et al., 2013; Sanderman et al., 2013; Gupta et al., 2014). Diazotrophic N2 fixation by roots of Panicum spp., Rhodes grass and Digitaria was reported to range between 0.92 and 2.35 mg 15 N/kg root/day (Gupta et al., 2014). Reis et al. (2001) reported that grass species Urochloa brizantha and Panicum maximum obtain up to 41% of their N through biological N2 fixation and suggested that this is achieved by allocating large quantities of C through root exudates. Until recently, the diversity of diazotrophic communities was mostly based on cultivated members of the Proteobacteria, green sulfur Bacteria, Cyanobacteria, and Firmicutes (Roper and Gupta, 2016). Conversely, molecular approaches for characterizing the N-fixing microbial community target the catalyst for this process, the nitrogenase enzyme, which is widely distributed among prokaryotic phyla (Gaby and Buckley, 2011). The nitrogenase enzyme complex consists of two multi-subunit metallo-proteins encoded by the nifH, nif D and nif K genes, with nifH gene generally used as the marker gene (Zehr et al., 2003; Gaby and Buckley, 2011). The nifH gene, which encodes the iron nitrogenase unit of the nitrogenase complex, is considered advantageous over others due to a well-conserved amino acid sequence and the presence of extensive reference sequence databases (Zehr et al., 2003; Izquierdo and Nusslein, 2006; Penton et al., 2016). Cultivation independent approaches utilizing this target gene have indicated a high level of diversity in agricultural, forest, rhizosphere and other soil environments (Zehr et al., 2003; Buckley et al., 2007; Kumar et al., 2017) as well as in plants (Hsu and Buckley, 2009). It has been suggested that the varying growth requirements of the phylogenetically heterogeneous diazotrophs may have precluded the cultivation of a substantial proportion of these bacteria. Therefore, it is not surprising that molecular approaches have revealed the presence of a wide diversity of uncultured diazotrophs (Lovell et al., 2000; Buckley et al., 2007). Research on diazotrophic N fixation has been principally concentrated on bulk and rhizosphere soils, plant residues and roots. Since plant roots are colonized by a subset of microorganisms recruited from the bulk soil, soil characteristics influence the composition of the rhizosphere microbial community (Regan et al., 2014; Bulgarelli et al., 2015), as does plant type (Bulgarelli et al., 2013). For example, a significant effect of plant species and varieties on diazotroph abundance and the num (...truncated)