In Situ Phylogenetic Structure and Diversity of Wild Bradyrhizobium Communities

Abstract

Bacteria often infect their hosts from environmental sources, but little is known about how environmental and host-infecting populations are related. Here, phylogenetic clustering and diversity were investigated in a natural community of rhizobial bacteria from the genus Bradyrhizobium. These bacteria live in the soil and also form beneficial root nodule symbioses with legumes, including those in the genus Lotus. Two hundred eighty pure cultures of Bradyrhizobium bacteria were isolated and genotyped from wild hosts, including Lotus angustissimus, Lotus heermannii, Lotus micranthus, and Lotus strigosus. Bacteria were cultured directly from symbiotic nodules and from two microenvironments on the soil-root interface: root tips and mature (old) root surfaces. Bayesian phylogenies of Bradyrhizobium isolates were reconstructed using the internal transcribed spacer (ITS), and the structure of phylogenetic relatedness among bacteria was examined by host species and microenvironment. Inoculation assays were performed to confirm the nodulation status of a subset of isolates. Most recovered rhizobial genotypes were unique and found only in root surface communities, where little bacterial population genetic structure was detected among hosts. Conversely, most nodule isolates could be classified into several related, hyper-abundant genotypes that were phylogenetically clustered within host species. This pattern suggests that host infection provides ample rewards to symbiotic bacteria but that host specificity can strongly structure only a small subset of the rhizobial community.

Epidemic Spread of Symbiotic and Non-Symbiotic Bradyrhizobium Genotypes Across California

“…Collection of Bradyrhizobium Isolates Bradyrhizobium was isolated from the nodules and the soil root interface of A. strigosus, and clonal cultures were grown and archived for genotyping following published protocols [33]. Briefly, whole plants were excavated from native sites and transported in sealed plastic bags to the laboratory where they were washed to remove soil with tap water and sterilized tools were used to remove root nodules.…”

“…Briefly, whole plants were excavated from native sites and transported in sealed plastic bags to the laboratory where they were washed to remove soil with tap water and sterilized tools were used to remove root nodules. Nodules were surface sterilized with bleach and rinsed with sterile water before being crushed with glass rods and the contents plated on a modified arabinose gluconate medium (MAG) [33]. For root surface isolates, the roots were dissected into~1-cm sections, were divided into root tips and Bold^roots, and were vortexed in a sterile solution of 0.01 % Tween 20 (Fisher Scientific Fair Lawn, NJ).…”

“…For root surface isolates, the roots were dissected into~1-cm sections, were divided into root tips and Bold^roots, and were vortexed in a sterile solution of 0.01 % Tween 20 (Fisher Scientific Fair Lawn, NJ). The wash solution was then serially diluted and plated on glucose-based rhizobium-defined medium (GRDM) with cyclohexamide as an antifungal and bromothymol blue as a pH indicator [33]. Among the resultant colonies, we selected for Bradyrhizobium based on growth rate, color, and ability to grow on MAG and GRDM but not on Luria-Bertani (LB) medium [33].…”

“…Bradyrhizobium is the most cosmopolitan rhizobial lineage and is found free-living in soils and in aquatic environments as well as in symbiotic association with plant and animal hosts, including humans [29][30][31][32][33][34][35][36][37]. Recent work suggests that there are~19 species of Bradyrhizobium that form symbiotic associations with legumes [36], although non-symbiotic strains are also common in soils [37].…”

“…(e.g., BTAi1 and ORS278) lack a symbiosis island and use a different mechanism to nodulate hosts [41]. Previous studies have revealed that Bradyrhizobium populations can exhibit patterns consistent with epidemics such as a few genotypes existing at high frequency at a single site [33] or genotypes isolated at multiple locations [34,35,37]. But these patterns remain poorly understood, and it is unclear what role the symbiosis island might play as a driver of increased abundance and epidemic spread.…”