Jason Terpolilli

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.

Genomic islands are regions of the bacterial genome (usually the chromosome) that appear to have been acquired through horizontal gene transfer. Genomic islands can harbor genes for pathogenicity, metabolism, antibiotic resistance and symbiosis. Rhizobia are soil bacteria that can establish nitrogen-fixing symbioses with leguminous plants. In Bradyrhizobium and Mesorhizobium spp., symbiosis genes are commonly located within genomic islands termed “symbiosis islands”. Symbiosis islands in Mesorhizobium are members of a class of mobile genetic elements called integrative and conjugative elements (ICEs) that horizontally transfer to other mesorhizobia via conjugation. Symbiosis ICEs (ICESyms) confer symbiotic ability to the recipient bacterium and carry a wide range of other genes that may contribute to the plant-symbiont interaction. Individual ICESyms integrate at a specific tRNA gene or other housekeeping gene, and their integration, excision, and transfer are controlled by ICE-encoded genes whose expression is under complex regulation involving quorum sensing. In agriculture, acquisition of an ICESym from an inoculant strain can convert saprophytic soil bacteria into novel symbionts that then outcompete the inoculant for nodulation of the legume, although not all the newly evolved symbionts effectively fix nitrogen. ICESyms likely evolved from a common ICE ancestor, which itself evolved from a large family of ICEs distributed throughout the proteobacteria.

Background and Aims

Inoculation of legumes with effective N2-fixing rhizobia is a common practice to improve farming profitability and sustainability. To succeed, inoculant rhizobia must overcome competition for nodulation by resident soil rhizobia that fix N2 ineffectively. In Kenya, where Phaseolus vulgaris (common bean) is inoculated with highly effective Rhizobium tropici CIAT899 from Colombia, response to inoculation is low, possibly due to competition from ineffective resident soil rhizobia. Here, we evaluate the competitiveness of CIAT899 against diverse rhizobia isolated from cultivated Kenyan P. vulgaris.

Methods

The ability of 28 Kenyan P. vulgaris strains to nodulate this host when co-inoculated with CIAT899 was assessed. Rhizosphere competence of a subset of strains and the ability of seed inoculated CIAT899 to nodulate P. vulgaris when sown into soil with pre-existing populations of rhizobia was analyzed.

Results

Competitiveness varied widely, with only 27% of the test strains more competitive than CIAT899 at nodulating P. vulgaris. While competitiveness did not correlate with symbiotic effectiveness, five strains were competitive against CIAT899 and symbiotically effective. In contrast, rhizosphere competence strongly correlated with competitiveness. Soil rhizobia had a position-dependent numerical advantage, outcompeting seed-inoculated CIAT899 for nodulation of P. vulgaris, unless the resident strain was poorly competitive.

Conclusion

Suboptimally effective rhizobia can outcompete CIAT899 for nodulation of P. vulgaris. If these strains are widespread in Kenyan soils, they may largely explain the poor response to inoculation. The five competitive and effective strains characterized here are candidates for inoculant development and may prove better adapted to Kenyan conditions than CIAT899.

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.

Rhizobia induce nodule formation on legume roots and differentiate into bacteroids, which catabolize plant-derived dicarboxylates to reduce atmospheric N2 into ammonia. Despite the agricultural importance of this symbiosis, the mechanisms that govern carbon and nitrogen allocation in bacteroids and promote ammonia secretion to the plant are largely unknown. Using a metabolic model derived from genome-scale datasets, we show that carbon polymer synthesis and alanine secretion by bacteroids facilitate redox balance in microaerobic nodules. Catabolism of dicarboxylates induces not only a higher oxygen demand but also a higher NADH/NAD+ ratio than sugars. Modeling and 13C metabolic flux analysis indicate that oxygen limitation restricts the decarboxylating arm of the tricarboxylic acid cycle, which limits ammonia assimilation into glutamate. By tightly controlling oxygen supply and providing dicarboxylates as the energy and electron source donors for N2 fixation, legumes promote ammonia secretion by bacteroids. This is a defining feature of rhizobium-legume symbioses.

The availability of effective inoculant rhizobia is often critical to the successful development of productive forage legumes. Biserrula pelecinus L. is a legume with potential as forage in Ethiopia to improve livestock feed quality and soil fertility. B. pelecinus can form N2‐fixing symbiosis with rhizobia in the genus Mesorhizobium. This study investigated the N2 fixation effectiveness of 15 B. pelecinus‐nodulating Mesorhizobium strains on two subspecies of B. pelecinus (B. pelecinus ssp. leiocarpa, native to Ethiopia, and the introduced B. pelecinus ssp. pelecinus). The most effective strain (WSM3873) on both subspecies was assessed at two sites; one with pre‐existing populations of B. pelecinus‐nodulating rhizobia (Modjo), and one without (Holeta). No inoculation response was observed at Modjo when B. pelecinus ssp. pelecinus was inoculated with WSM3873 alone, however, biomass yield was greatest (11.5 tonne DM/ha) following inoculation along with co‐application of phosphorus and nitrogen. At Holeta, a strong inoculation response was achieved with WSM3873 alone on B. pelecinus ssp. pelecinus. In contrast, B. pelecinus ssp. leiocarpa did not show any response at Modjo and failed to emerge after sowing at Holeta. While the native legume B. pelecinus ssp. leiocarpa appears poorly suited to development as a forage, B. pelecinus ssp. pelecinus and WSM3873 represents a promising legume‐rhizobia symbiosis that could benefit farming systems of the central Ethiopian highlands.