The Symbiotic Requirements of Different Medicago Spp. Suggest the Evolution of Sinorhizobium Meliloti and S. Medicae with Hosts Differentially Adapted to Soil pH

Nitrogen fixing rhizobia associated with the Medicago L. genus belong to two closely related species Sinorhizobium medicae and S. meliloti. To investigate the symbiotic requirements of different Medicago species for the two microsymbionts, 39 bacterial isolates from nodules of eleven Medicago species growing in their natural habitats in the Mediterranean basin plus six historical Australian commercial inocula were symbiotically characterized with Medicago hosts. The bacterial species allocation was first assigned on the basis of symbiotic proficiency with M. polymorpha. PCR primers specific for 16S rDNA were then designed to distinguish S. medicae and S. meliloti. PCR amplification results confirmed the species allocation acquired in the glasshouse. PCR fingerprints generated from ERIC, BOXA1R and nif-directed RPO1 primers revealed that the Mediterranean strains were genetically heterogenous. Moreover PCR fingerprints with ERIC and BOX primers showed that these repetitive DNA elements were specifically distributed and conserved in S. meliloti and S. medicae, clustering the strains into two divergent groups according to their species. Linking the Sinorhizobium species with the plant species of origin we have found that S. medicae was mostly associated with medics well adapted to moderately acid soils such as M. polymorpha, M. arabica and M. murex whereas S. meliloti was predominantly isolated from plants naturally growing on alkaline or neutral pH soils such as M. littoralis and M. tornata. Moreover in glasshouse experiments the S. medicae strains were able to induce well-developed nodules on M. murex whilst S. meliloti was not infective on this species. This feature provides a very distinguishing characteristic for S. medicae. Results from the symbiotic, genotypic and cultural characterization suggest that S. meliloti and S. medicae have adapted to different Medicago species according to the niches these medics usually occupy in their natural habitats.     

Nodule morphology,symbiotic specificity and association with unusual rhizobia are distinguishing features of the genus Listia within the southern African crotalarioid clade Lotononis s.l.

Background and Aims

The legume clade Lotononis sensu lato (s.l.; tribe Crotalarieae) comprises three genera: Listia, Leobordea and Lotononis sensu stricto (s.s.). Listia species are symbiotically specific and form lupinoid nodules with rhizobial species of Methylobacterium and Microvirga. This work investigated whether these symbiotic traits were confined to Listia by determining the ability of rhizobial strains isolated from species of Lotononis s.l. to nodulate Listia, Leobordea and Lotononis s.s. hosts and by examining the morphology and structure of the resulting nodules.

Methods

Rhizobia were characterized by sequencing their 16S rRNA and nodA genes. Nodulation and N₂ fixation on eight taxonomically diverse Lotononis s.l. species were determined in glasshouse trials. Nodules of all hosts, and the process of infection and nodule initiation in Listia angolensis and Listia bainesii, were examined by light microscopy.

Key Results

Rhizobia associated with Lotononis s.l. were phylogenetically diverse. Leobordea and Lotononis s.s. isolates were most closely related to Bradyrhizobium spp., Ensifer meliloti, Mesorhizobium tianshanense and Methylobacterium nodulans. Listia angolensis formed effective nodules only with species of Microvirga. Listia bainesii nodulated only with pigmented Methylobacterium. Five lineages of nodA were found. Listia angolensis and L. bainesii formed lupinoid nodules, whereas nodules of Leobordea and Lotononis s.s. species were indeterminate. All effective nodules contained uniformly infected central tissue. Listia angolensis and L. bainesii nodule initials occurred on the border of the hypocotyl and along the tap root, and nodule primordia developed in the outer cortical layer. Neither root hair curling nor infection threads were seen.

Conclusions

Two specificity groups occur within Lotononis s.l.: Listia species are symbiotically specific, while species of Leobordea and Lotononis s.s. are generally promiscuous and interact with rhizobia of diverse chromosomal and symbiotic lineages. The seasonally waterlogged habitat of Listia species may favour the development of symbiotic specificity.     

Rapid In Situ Evolution of Nodulating Strains for Biserrula pelecinus L. through Lateral Transfer of a Symbiosis Island from the Original Mesorhizobial Inoculant

Diverse rhizobia able to nodulate Biserrula pelecinus evolved following in situ transfer of nodA and nifH from an inoculant to soil bacteria. Transfer of these chromosomal genes and the presence of an identical integrase gene adjacent to a Phe tRNA gene in both the inoculant and recipients indicate that there was lateral transfer of a symbiosis island.     

Phenotypic and genetic diversity among rhizobia isolated from three Hedysarum species: H. spinosissimum,H. coronarium and H. flexuosum

Cultural, physiological and biochemical properties of 18 strains of rhizobia isolated from root nodules of the forage legume H. spinosissimum were compared with those of rhizobia from the related species H. coronarium (15 strains) and H. flexuosum (four strains). On the basis of 43 characteristics the 37 strains of Hedysarum rhizobia could be divided into two groups by numerical analysis. The H. spinosissimum rhizobia formed the first group and the second group comprised the strains from H. coronarium and H. flexuosum. The reference Rhizobium leguminosarum bv. viceae strain 250A was clustered with the rhizobia from H. coronarium and H. flexuosum. By contrast Bradyrhizobium sp. (Arachis) reference strain 280A was not clustered with any of the strains tested, indicating that the H. spinosissimum rhizobia differ from both Rhizobium and Bradyrhizobium. Serological data also discriminate between H. spinosissimum and H. coronariumrhizobia but not between the latter and H. flexuosum strains. The strains tested exhibit a high degree of specificity for nodulation and nitrogen fixation. We also determined the16SrRNA gene sequence of H. spinosissimum rhizobia (four strains), H. coronarium (two strains) and H. flexuosum (two strains) and found that the four H. spinosissimum isolates share a 98% identity among each other in this region but they showed less than 92% identity to the H. coronarium and H. flexuosum isolates. The H. spinosissimum isolates were closely related to both Mesorhizobium loti and M. ciceri, sharing 97% identity with each species.     

European Origin of Bradyrhizobium Populations Infecting Lupins and Serradella in Soils of Western Australia and South Africa

We applied a multilocus phylogenetic approach to elucidate the origin of serradella and lupin Bradyrhizobium strains that persist in soils of Western Australia and South Africa. The selected strains belonged to different randomly amplified polymorphic DNA (RAPD)-PCR clusters that were distinct from RAPD clusters of applied inoculant strains. Phylogenetic analyses were performed with nodulation genes (nodA, nodZ, nolL, noeI), housekeeping genes (dnaK, recA, glnII, atpD), and 16S-23S rRNA intergenic transcribed spacer sequences. Housekeeping gene phylogenies revealed that all serradella and Lupinus cosentinii isolates from Western Australia and three of five South African narrow-leaf lupin strains were intermingled with the strains of Bradyrhizobium canariense, forming a well supported branch on each of the trees. All nodA gene sequences of the lupin and serradella bradyrhizobia formed a single branch, referred to as clade II, together with the sequences of other lupin and serradella strains. Similar patterns were detected in nodZ and nolL trees. In contrast, nodA sequences of the strains isolated from native Australian legumes formed either a new branch called clade IV or belonged to clade I or III, whereas their nonsymbiotic genes grouped outside the B. canariense branch. These data suggest that the lupin and serradella strains, including the strains from uncultivated L.cosentinii plants, are descendants of strains that most likely were brought from Europe accidentally with lupin and serradella seeds. The observed dominance of B. canariense strains may be related to this species' adaptation to acid soils common in Western Australia and South Africa and, presumably, to their intrinsic ability to compete for nodulation of lupins and serradella.     

Probing for pH-regulated genes in Sinorhizobium medicae using transcriptional analysis

The low pH sensitivity of Sinorhizobium species is one of the major causes of reduced productivity of Medicago species (such as lucerne) sown in acidic soils. To investigate the pH response of an acid-tolerant Sinorhizobium medicae strain, a pool of random promoter fusions to gusA was created using minitransposon insertional mutagenesis. Acid-activated expression was identified in 11 mutants; rhizobial DNA flanking insertions in 10 mutants could be cloned and the DNA sequences obtained were used to interrogate the genome database of Sinorhizobium meliloti strain 1021. Acid activated expression was detected for fixNO, kdpC, lpiA, and phrR and for genes encoding a putative lipoprotein, two ABC-transporter components, a putative DNA ligase and a MPA1-family protein. These findings implicate cytochrome synthesis, potassium ion cycling, lipid biosynthesis and transport processes as key components of pH response in S. medicae.     

Overcoming non-selective nodulation of Lessertia by soil-borne rhizobium in the presence of inoculant mesorhizobium

Background and aims

Legumes of the South African genus Lessertia, along with their microsymbionts, were introduced into the Western Australia wheatbelt. They achieved poor establishment followed by weak summer survival. This was caused in part by low levels of nodulation with the inoculant strains, and by ineffective nodulation with naturalized strains –an example of non-selective nodulation. The aims of this work were to assess Lessertia spp. symbiotic promiscuity, to study the effect of increased doses of an effective inoculant strain (WSM3565) with L. herbacea, and to study the competitive ability and symbiotic performance of different Mesorhizobium strains nodulating L. diffusa.

Methods

A glasshouse experiment was set up to evaluate the ability of L. diffusa, L. capitata, L. herbacea and L. excisa to nodulate with inoculants under current use in Western Australia. To assess competitive ability two field experiments were set up at Karridale, Western Australia. L. herbacea was inoculated with the strain WSM3565 at different doses and L. diffusa was inoculated with ten different Mesorhizobium strains. Rhizobia were re-isolated from nodules and their identity confirmed through PCR fingerprinting and sequencing of their partial dnaK.

Results

There were differences in promiscuity between different Lessertia spp., where L. herbacea proved to be highly promiscuous under controlled conditions. Increasing the inoculation dose of L. herbacea with WSM3565 did not improve establishment and survival of the legume in the field. Although WSM3565 nodule occupancy improved from 28 to 54 % with higher doses of inoculation, none of the treatments increased L. herbacea yield over the inoculated control. The inoculation of L. diffusa with the strains WSM3598, 3636, 3626 and 3565 resulted in greater biomass production than the uninoculated control. These strains were able to outcompete resident rhizobia and to occupy a high (>60 %) proportion of lateral root nodules. The naturalised strains that achieved nodulation were identified as R. leguminosarum.

Conclusion

The high numbers of resident rhizobia and their ability to rapidly nodulate Lessertia spp. are likely to be the main reasons for the low nodule occupancy achieved by some effective inoculant strains with L. diffusa and L. herbacea. Strains WSM 3636 and 3598 were very competitive on nodule occupancy and together with WSM 3565, WSM 3612 and WSM3626, effective on nodule formation and plant growth of L. diffusa despite the high numbers of resident soil rhizobia. These strains and L. diffusa have potential to be introduced as exotic legumes species and rhizobia strains to Western Australia.