MacLean Kohlmeier

Rhizobia are soil bacteria capable of establishing symbiosis within legume root nodules, where they reduce atmospheric N2 into ammonia and supply it to the plant for growth. Australian soils often lack rhizobia compatible with introduced agricultural legumes, so inoculation with exotic strains has become a common practice for over 50 years. While extensive research has assessed the N2-fixing capabilities of these inoculants, their genomics, taxonomy, and core and accessory gene phylogeny are poorly characterized. Furthermore, in some cases, inoculant strains have been developed from isolations made in Australia. It is unknown whether these strains represent naturalized exotic organisms, native rhizobia with a capacity to nodulate introduced legumes, or recombinant strains arising from horizontal transfer between introduced and native bacteria. Here, we describe the complete, closed genome sequences of 42 Australian commercial rhizobia. These strains span the genera, Bradyrhizobium, Mesorhizobium, Methylobacterium, Rhizobium, and Sinorhizobium, and only 23 strains were identified to species level. Within inoculant strain genomes, replicon structure and location of symbiosis genes were consistent with those of model strains for each genus, except for Rhizobium sp. SRDI969, where the symbiosis genes are chromosomally encoded. Genomic analysis of the strains isolated from Australia showed they were related to exotic strains, suggesting that they may have colonized Australian soils following undocumented introductions. These genome sequences provide the basis for accurate strain identification to manage inoculation and identify the prevalence and impact of horizontal gene transfer (HGT) on legume productivity.

IMPORTANCE: Inoculation of cultivated legumes with exotic rhizobia is integral to Australian agriculture in soils lacking compatible rhizobia. The Australian inoculant program supplies phenotypically characterized high-performing strains for farmers but in most cases, little is known about the genomes of these rhizobia. Horizontal gene transfer (HGT) of symbiosis genes from inoculant strains to native non-symbiotic rhizobia frequently occurs in Australian soils and can impact the long-term stability and efficacy of legume inoculation. Here, we present the analysis of reference-quality genomes for 42 Australian commercial rhizobial inoculants. We verify and classify the genetics, genome architecture, and taxonomy of these organisms. Importantly, these genome sequences will facilitate the accurate strain identification and monitoring of inoculants in soils and plant nodules, as well as enable detection of horizontal gene transfer to native rhizobia, thus ensuring the efficacy and integrity of Australia’s legume inoculation program.

Background and aims
Mesorhizobium ciceri CC1192 is the commercial inoculant strain for Cicer arietinum (chickpea) cultivation in Australia, including in the Ord River Irrigation Area (ORIA), where C. arietinum cropping began in 1985. Mesorhizobium strains are known to gain the capacity to nodulate legumes through acquisition of symbiosis Integrative and Conjugative Elements (ICEs), leading to the evolution of novel rhizobia. Here, we assess the impact of symbiosis ICE transfer and compare the genomic diversity and symbiotic effectiveness of C. arietinum nodulating rhizobia from the ORIA.

Methods
Nodule isolates collected from field cultivated C. arietinum were genotyped by RAPD-PCR, and representative strains from each genotype were whole genome sequenced and symbiotically phenotyped in glasshouse conditions to assess N2 fixation effectiveness against CC1192.

Results
A total of 68 nodule isolates, all harbouring the CC1192 symbiosis ICE (ICEMcSym1192), were analysed, with 12 identified as the inoculant strain, and 56 novel strains clustering into ten genotypes. These novel genotypes dominated as nodule occupants across the majority of sites sampled. Nine of the ten representative strains were as effective at N2 fixation as CC1192, with WSM4904 the only ineffective strain. Core genome phylogeny showed the ten strains represent four novel Mesorhizobium genospecies. Novel strains WSM4904 and WSM4906 shared 98.7% sequence identity, yet exhibited very different symbiotic phenotypes.

Conclusions
The CC1192 symbiosis ICE has transferred to a wide diversity of Mesorhizobium spp. in the ORIA. These evolved strains are competitive against CC1192 at nodulating C. arietinum, and the majority are effective symbiotic N2 fixers.

Rhizobia are a diverse group of α- and β-proteobacteria that boost soil fertility by forming a nitrogen-fixing symbiosis with legumes, which is why legumes are grown in rotation with cereals in agriculture. Rhizobia that naturally populate Australian soils are largely incompatible with exotic agricultural legumes, therefore, compatible strains have been imported from all over the world for use as inoculants. An amalgamated collection of these strains, called the International Legume Inoculant Genebank (ILIG), has been established at Murdoch University, to provide a centralised strain storage facility and support rhizobial research and inoculant development (see http://ilig.murdoch.edu.au). The ILIG contains 11,558 strains representing 96 bacterial species from 778 legume species collected from >1200 locations across 100 countries. New and sometimes inefficient rhizobia evolve in the field following legume inoculation, through horizontal symbiosis gene transfer from inoculants to soil bacteria. To provide a benchmark to monitor and assess the impact of this evolution, all commercial Australian inoculant strains were genome sequenced and these data made available (PRJNA783123, see https://www.ncbi.nlm.nih.gov/bioproject/PRJNA783123/). These data, and the further sequencing of the >11,000 historical strains in the ILIG, will increase our understanding of rhizobial evolution and diversity and provide the backbone for efforts to safeguard Australia’s legume inoculation program.

Mesorhizobia are soil bacteria that establish nitrogen-fixing symbioses with various legumes. Novel symbiotic mesorhizobia frequently evolve following horizontal transfer of symbiosis-gene-carrying integrative and conjugative elements (ICESyms) to indigenous mesorhizobia in soils. Evolved symbionts exhibit a wide range in symbiotic effectiveness, with some fixing nitrogen poorly or not at all. Little is known about the genetic diversity and symbiotic potential of indigenous soil mesorhizobia prior to ICESym acquisition. Here we sequenced genomes of 144 Mesorhizobium spp. strains cultured directly from cultivated and uncultivated Australian soils. Of these, 126 lacked symbiosis genes. The only isolated symbiotic strains were either exotic strains used previously as legume inoculants, or indigenous mesorhizobia that had acquired exotic ICESyms. No native symbiotic strains were identified. Indigenous nonsymbiotic strains formed 22 genospecies with phylogenomic diversity overlapping the diversity of internationally isolated symbiotic Mesorhizobium spp. The genomes of indigenous mesorhizobia exhibited no evidence of prior involvement in nitrogen-fixing symbiosis, yet their core genomes were similar to symbiotic strains and they generally lacked genes for synthesis of biotin, nicotinate and thiamine. Genomes of nonsymbiotic mesorhizobia harboured similar mobile elements to those of symbiotic mesorhizobia, including ICESym-like elements carrying aforementioned vitamin-synthesis genes but lacking symbiosis genes. Diverse indigenous isolates receiving ICESyms through horizontal gene transfer formed effective symbioses with Lotus and Biserrula legumes, indicating most nonsymbiotic mesorhizobia have an innate capacity for nitrogen-fixing symbiosis following ICESym acquisition. Non-fixing ICESym-harbouring strains were isolated sporadically within species alongside effective symbionts, indicating chromosomal lineage does not predict symbiotic potential. Our observations suggest previously observed genomic diversity amongst symbiotic Mesorhizobium spp. represents a fraction of the extant diversity of nonsymbiotic strains. The overlapping phylogeny of symbiotic and nonsymbiotic clades suggests major clades of Mesorhizobium diverged prior to introduction of symbiosis genes and therefore chromosomal genes involved in symbiosis have evolved largely independent of nitrogen-fixing symbiosis.