Yvette Hill

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.

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 are soil bacteria capable of forming N2-fixing symbioses with legumes, with highly effective strains often selected in agriculture as inoculants to maximize symbiotic N2 fixation. When rhizobia in the genus Mesorhizobium have been introduced with exotic legumes into farming systems, horizontal transfer of symbiosis integrative and conjugative elements (ICEs) from the inoculant strain to soil bacteria has resulted in the evolution of ineffective N2-fixing rhizobia that are competitive for nodulation with the target legume. In Australia, Cicer arietinum (chickpea) has been inoculated since the 1970s with Mesorhizobium ciceri symbiovar ciceri CC1192, a highly effective strain from Israel. Although the full genome sequence of this organism is available, little is known about the mobility of its symbiosis genes and the diversity of cultivated C. arietinum-nodulating organisms. Here, we show that the CC1192 genome harbors a 419-kb symbiosis ICE (ICEMcSym1192) and a 648-kb repABC-type plasmid (pMC1192) carrying putative fix genes. We sequenced the genomes of 11 C. arietinum nodule isolates from a field site exclusively inoculated with CC1192, and we showed that they were diverse unrelated Mesorhizobium strains carrying ICEMcSym1192, which indicated that they had acquired the ICE by environmental transfer. No exconjugants harbored pMc1192, and the plasmid was not essential for N2 fixation in CC1192. Laboratory conjugation experiments confirmed that ICEMcSym1192 is mobile, integrating site specifically within the 3′ end of one of the four Ser--tRNA genes in the R7ANS recipient genome. Strikingly, all ICEMcSym1192 exconjugants were as efficient as CC1192 at fixing N2 with C. arietinum, demonstrating that ICE transfer does not necessarily yield ineffective microsymbionts as observed previously.

The integration of plant available nitrogen (N) into the nutrient cycles of dryland ecosystems is integral to the establishment and persistence of the flora in these regions. Much of this available N is due to the conversion of atmospheric dinitrogen (N2) by legumes and their bacterial microsymbionts, root nodule bacteria (RNB). There are numerous environmental constraints in dryland areas that impede the growth and interactions of both symbiotic partners. At Shark Bay Salt Pty. Ltd., a solar salt facility in Western Australia, the associations between provenant RNB and the key over-story species Acacia ligulata Benth. and Acacia tetragonophylla F.Muell. were investigated in situ and in glasshouse conditions. This was done to determine whether the selection of provenant RNB that effectively fix nitrogen, and their inoculation onto these two species, could improve plant establishment at degraded pit sites within the Shark Bay Salt lease area (SBSLA). The effect that mining processes has had on the biological, chemical and physical characteristics of the remaining substrate of selected borrow pit soils was evaluated. The removal of the soil, subsoil and regolith had altered the chemical characteristics of these sites in comparison to adjacent undisturbed areas. This activity had been deleterious to the biota, with no established floral community and reduced populations of RNB that nodulate A. ligulata Benth. and A. tetragonophylla F.Muell. in the pit areas. There was reduced organic carbon, nitrate and phosphorus concentrations in the pit soils in comparison to the adjacent undisturbed soils and at one pit site, soil salinity was at toxic levels. There were marked differences in the floristic structure and diversity between the different undisturbed sites, with A. ligulata Benth. and A. tetragonophylla F.Muell. identified at all the selected sites. The RNB in the soils was assessed in 2007 and 2008, years with contrasting annual rainfalls of 79.3 mm and 513.6 mm and it was found that the RNB population increased with the higher rainfall in all pit and undisturbed site soils, with the exception of the toxic saline pit soil where RNB were not detected. In both years, the most probable number (MPN) of RNB that nodulated A. ligulata Benth. and A. tetragonophylla F.Muell. were reduced in the pit soils compared to the adjacent undisturbed soils. Provenant isolates of RNB from the soils of SBSLA were collected and assessed for the effectiveness of these RNB isolates as well as Wattle Grow™ in promoting the growth of selected host species in glasshouse conditions for 56 day post inoculation (dpi). Many of the RNB isolated from A. ligulata Benth. and A. tetragonophylla F.Muell. readily cross-infected these two species and a number of strains also nodulated with Acacia rostellifera Benth. and Templetonia retusa (Vent.)R.Br.. There was a significant growth response of A. ligulata Benth., A. rostellifera Benth. and A. tetragonophylla F.Muell. to inoculation with a number of the RNB in comparison to uninoculated plants, with some producing foliage weights greater than 100% of the nitrogen-fed control. A. ligulata Benth. and A. rostellifera Benth. produced significantly increased growth when inoculated with Wattle Grow™ (containing Bradyrhizobium spp.). The nitrogen concentrations of A. ligulata Benth. and A. tetragonophylla F.Muell. foliage of selected treatments showed a weakly positive, non-significant relationship when correlated to the plant dry foliage weights of these treatments. While only nine RNB isolates were obtained from nodules collected from A. ligulata Benth. plants growing within the SBSLA, 78% produced a significant growth response. In contrast, only 22% of 32 A. ligulata Benth. isolates trapped from soil collected from SBSLA produced a significant growth response in comparison to the uninoculated control. This indicates a possible selection pressure and bias when trapping RNB from soils in glasshouse conditions opposed to collecting RNB directly from nodules formed on legumes at the field site. No RNB symbionts of A. ligulata Benth. and A. tetragonophylla F.Muell. have previously been described and the phenotypic characteristics, phylogenetic relationships and the genetic diversity of 25 SBSLA RNB isolates of these Acacia spp. were assessed. The RNB showed tolerance of alkaline, saline and high temperature conditions. All grew at pH 11.0 and the majority tolerated up to 750 mM NaCl. With the exception of two isolates, all grew at 37°C and five isolates were able to grow at 42°C. Based on RPO1-PCR fingerprints, there were indications of considerable genetic diversity among the RNB isolates. The 16s rDNA restriction patterns produced by Alul, Mspl and Sau3Al digestions grouped the isolates into one of six RFLP type groups. On determining the phylogeny of ten of the isolates, the 16s rDNA sequences aligned within the Ensifer, Rhizobium and Neorhizobium genera. Eight of the isolates aligned within Ensifer, six of which formed a distinct cluster. A multi-locus approach of conserved gene regions would need to be examined to more confidently assess the phylogeny of these RNB. Based on the effectiveness results, a number of RNB were selected to be re-introduced into selected pit sites in seeding and inoculation trials. Coupled with these trials, different carriers for the RNB were also evaluated to determine their efficacy in relation to the nodulation and growth response of A. ligulata Benth. and A. tetragonophylla F.Muell. in the field conditions. There was increased nodulation of A. ligulata Benth. and A. tetragonophylla F.Muell. plants that had been inoculated. The number of germinated plants and the inoculant treatment indicated no significant relationship. However, seeds inoculated with the peat treatment did generally have a greater number of plants that were growing at the assessment periods compared to the other carriers and uninoculated treatments. This itself is noteworthy as reducing seed loss is one of the major impediments to successful rehabilitation of dryland areas. The nodules on the Acacia spp. grown in the pits were occupied by RNB whose RPO1-PCR fingerprints were identical to selected RNB and an additional novel isolate. It was found that inoculation of RNB into the pit soils increased and stabilised the RNB population, with MPN comparable to the population in the surrounding undisturbed soils at 4 months post inoculation. So as to maintain the provenance of the RNB population in the SBSLA soils and avoid introducing genetic material that could transfer into the resident RNB, Wattle Grow™ could not be included in the seeding and inoculation trials. In a glasshouse experiment, growth tanks containing pit soil were used to compare the nitrogen fixation efficacy and competitive ability of Wattle Grow™ to nodulate A. ligulata Benth. and A. tetragonophylla F.Muell. against the background RNB in the pit soils and with selected SBSLA isolates over successive sowing periods. No Bradyrhizobium spp. were isolated from the nodules of the Acacia spp. from any of the treatments over the different sowing periods. The majority of the RPO1-PCR fingerprints of the nodule occupants corresponded to SBSLA isolates and an additional three unique fingerprints were identified. The occupancy of the nodules of A. ligulata Benth. and A. tetragonophylla F.Muell. subtly changed with each successive sowing. A number of RNB occurred with greater frequency at the different sowing periods, however, there was a trend towards increased diversity of nodule occupants with each successive sowing, particularly of the RNB nodulating A. tetragonophylla F.Muell.. There was a difference in the response of the two Acacia spp. to the treatments and conditions of the growth tanks. The plant foliage nitrogen concentrations and foliage mass of A. ligulata Benth. were negatively correlated. In contrast, the A. tetragonophylla F.Muell. foliage nitrogen concentrations were positively correlated to the foliage production of these plants. The use of provenant RNB shows potential in improving the germination and establishment of selected legume species in the degraded areas within SBSLA. However, it was shown that different growth conditions for A. ligulata Benth. and A. tetragonophylla F.Muell. alters the symbiotic relationships, nitrogen fixation and growth response of these plants. This illustrates the caution to be exercised when screening for effective symbionts of legumes for the purpose of rehabilitation.

The effect of soil pH on the competitive abilities of two rhizobial strains was investigated in two sterile systems. The two strains come from wild plants of the endemic shrubby legumes Cytisus multiflorus (cmu) and Cytisus balansae (cba). Strains were used to infect seedlings of C. multiflorus, C. balansae, and Ornithopus compressus, grown in soil and hydroponics with acidic and neutral pH. All seedlings were inoculated with a single-strain inoculum containing 106 total cells of one of the two test strains or with a mixed inoculum (1:1 cmu:cba). Controls consisted of non-inoculated seedlings. At harvest, nodule occupants were determined by PCR. The majority of nodules (>95 %) formed on plants grown in acidic soil were occupied by cmu strain. This pattern of nodule occupancy changed in neutral pH both in soil and in hydroponics. When cmu was paired with cba, the former formed 78 % of the nodules in the acidic media and the number of nodules formed by cba was higher in neutral media (8 %). When nodule occupancy was dominated by cmu, the total nitrogen and biologically fixed nitrogen were higher in C. multiflorus and O. compressus. The different nodule occupancy percentages indicate a correlation between the preferred growing conditions of both host plants and Bradyrhizobia strains. Results indicate that soil pH can influence which symbiotype will competitively nodulate C. multiflorus and C. balansae in the field which accounts for the current distribution of these two plants.

Ensifer medicae Di28 is an aerobic, motile, Gram-negative, non-spore-forming rod that can exist as a soil saprophyte or as a legume microsymbiont of Medicago spp. Di28 was isolated in 1998 from a nodule recovered from the roots of M. polymorpha growing in the south east of Sardinia (Italy). Di28 is an effective microsymbiont of the annual forage legumes M. polymorpha and M. murex and is capable of establishing a partially effective symbiotic association with the perennial M. sativa. Here we describe the features of E. medicae Di28, together with genome sequence information and its annotation. The 6,553,624 bp standard draft genome is arranged into 104 scaffolds of 104 contigs containing 6,394 protein-coding genes and 75 RNA-only encoding genes. This rhizobial genome is one of 100 sequenced as part of the DOE Joint Genome Institute 2010 Genomic Encyclopedia for Bacteria and Archaea-Root Nodule Bacteria (GEBA-RNB) project.

Ensifer meliloti WSM1022 is an aerobic, motile, Gram-negative, non-spore-forming rod that can exist as a soil saprophyte or as a legume microsymbiont of Medicago. WSM1022 was isolated in 1987 from a nodule recovered from the roots of the annual Medicago orbicularis growing on the Cyclades Island of Naxos in Greece. WSM1022 is highly effective at fixing nitrogen with M. truncatula and other annual species such as M. tornata and M. littoralis and is also highly effective with the perennial M. sativa (alfalfa or lucerne). In common with other characterized E. meliloti strains, WSM1022 will nodulate but fixes poorly with M. polymorpha and M. sphaerocarpos and does not nodulate M. murex. Here we describe the features of E. meliloti WSM1022, together with genome sequence information and its annotation. The 6,649,661 bp high-quality-draft genome is arranged into 121 scaffolds of 125 contigs containing 6,323 protein-coding genes and 75 RNA-only encoding genes, and is one of 100 rhizobial genomes sequenced as part of the DOE Joint Genome Institute 2010 Genomic Encyclopedia for Bacteria and Archaea-Root Nodule Bacteria (GEBA-RNB) project.

Ensifer medicae WSM1369 is an aerobic, motile, Gram-negative, non-spore-forming rod that can exist as a soil saprophyte or as a legume microsymbiont of Medicago. WSM1369 was isolated in 1993 from a nodule recovered from the roots of Medicago sphaerocarpos growing at San Pietro di Rudas, near Aggius in Sardinia (Italy). WSM1369 is an effective microsymbiont of the annual forage legumes M. polymorpha and M. sphaerocarpos. Here we describe the features of E. medicae WSM1369, together with genome sequence information and its annotation. The 6,402,557 bp standard draft genome is arranged into 307 scaffolds of 307 contigs containing 6,656 protein-coding genes and 79 RNA-only encoding genes. This rhizobial genome is one of 100 sequenced as part of the DOE Joint Genome Institute 2010 Genomic Encyclopedia for Bacteria and Archaea-Root Nodule Bacteria (GEBA-RNB) project.

Ensifer sp. TW10 is a novel N2-fixing bacterium isolated from a root nodule of the perennial legume Tephrosia wallichii Graham (known locally as Biyani) found in the Great Indian (or Thar) desert, a large arid region in the northwestern part of the Indian subcontinent. Strain TW10 is a Gram-negative, rod shaped, aerobic, motile, non-spore forming, species of root nodule bacteria (RNB) that promiscuously nodulates legumes in Thar Desert alkaline soil. It is fast growing, acid-producing, and tolerates up to 2% NaCl and capable of growth at 40°C. In this report we describe for the first time the primary features of this Thar Desert soil saprophyte together with genome sequence information and annotation. The 6,802,256 bp genome has a GC content of 62% and is arranged into 57 scaffolds containing 6,470 protein-coding genes, 73 RNA genes and a single rRNA operon. This genome is one of 100 RNB genomes sequenced as part of the DOE Joint Genome Institute 2010 Genomic Encyclopedia for Bacteria and Archaea-Root Nodule Bacteria (GEBA-RNB) project.