Ross Ballard

Symbiotic performance of Mediterranean Trifolium spp. with naturalised soil rhizobia

Naturalised soil rhizobia that nodulate clover occur in high number and are known to vary in their symbiotic performance (SP) with subterranean clover (Trifolium subterraneum L.). However, the extent of suboptimal fixation across a range of other clover species is not well understood. T. subterraneum and nine other annual clover species of Mediterranean origin were evaluated for their SP in combination with the naturalised clover rhizobia in 71 Australian soils and five strains of Rhizobium leguminosarum bv. trifolii that have been used in the inoculants produced for clovers. The most probable number method, using subterranean clover as the trap plant was used to estimate the number of clover rhizobia in the soils. Ninety-two percent of soils tested contained more than 1000 rhizobia/g. An extract of each soil, or strain of rhizobia was used to inoculate plants growing in N-deficient media in the greenhouse. Plants were grown for 4 weeks after inoculation and shoot dry matter determined and expressed as a percentage of the ‘best’ soil rhizobia treatment, to provide a proportional measure of SP for each clover species. SP (mean of clover species) ranged from 96% with the current inoculant strain for annual clovers (WSM1325) down to 48% with former inoculant strain WU95. When inoculated with soils predominantly from mainland Australia, SP (mean of soil treatments) of the different Trifolium spp. was 55% (resupinatum), 53–47% (subterraneum), 50% (nigrescens), 49% (michelianum), 48% (isthmocarpum), 38% (hirtum), 35% (purpureum), 32% (vesiculosum), 25% (spumosum) and 21% (glanduliferum). Within each of the clover species, SP resulting from individual soil treatments ranged from 100% (by definition for the best soil treatment) down to close to zero. Trifolium glanduliferum formed nodules readily with the inoculant strains but nodulation was erratic with the rhizobia in many soils. It is therefore proposed that the naturalised rhizobia in many soils are unlikely to be inoculant strains. This research demonstrates symbiotic efficiency across annual clover species is compromised where diverse populations of clover rhizobia have naturalised in soils.

Identifying abnormalities in symbiotic development between Trifolium spp. and Rhizobium leguminosarum bv. trifolii leading to sub-optimal and ineffective nodule phenotypes

BACKGROUND AND AIMS Legumes overcome nitrogen limitations by entering into a mutualistic symbiosis with N(2)-fixing bacteria (rhizobia). Fully compatible associations (effective) between Trifolium spp. and Rhizobium leguminosarum bv. trifolii result from successful recognition of symbiotic partners in the rhizosphere, root hair infection and the formation of nodules where N(2)-fixing bacteroids reside. Poorly compatible associations can result in root nodule formation with minimal (sub-optimal) or no (ineffective) N(2)-fixation. Despite the abundance and persistence of strains in agricultural soils which are poorly compatible with the commercially grown clover species, little is known of how and why they fail symbiotically. The aims of this research were to determine the morphological aberrations occurring in sub-optimal and ineffective clover nodules and to determine whether reduced bacteroid numbers or reduced N(2)-fixing activity is the main cause for the Sub-optimal phenotype. METHODS Symbiotic effectiveness of four Trifolium hosts with each of four R. leguminosarum bv. trifolii strains was assessed by analysis of plant yields and nitrogen content; nodule yields, abundance, morphology and internal structure; and bacteroid cytology, quantity and activity. KEY RESULTS Effective nodules (Nodule Function 83-100 %) contained four developmental zones and N(2)-fixing bacteroids. In contrast, Sub-optimal nodules of the same age (Nodule Function 24-57 %) carried prematurely senescing bacteroids and a small bacteroid pool resulting in reduced shoot N. Ineffective-differentiated nodules carried bacteroids aborted at stage 2 or 3 in differentiation. In contrast, bacteroids were not observed in Ineffective-vegetative nodules despite the presence of bacteria within infection threads. CONCLUSIONS Three major responses to N(2)-fixation incompatibility between Trifolium spp. and R. l. trifolii strains were found: failed bacterial endocytosis from infection threads into plant cortical cells, bacteroid differentiation aborted prematurely, and a reduced pool of functional bacteroids which underwent premature senescence. We discuss possible underlying genetic causes of these developmental abnormalities and consider impacts on N(2)-fixation of clovers.

Agronomic and environmental drivers of population size and symbiotic performance of Rhizobium leguminosarum bv. viciae in Mediterranean-type environments

Abstract. The population size and symbiotic performance (ability to fix N2) of rhizobia (Rhizobium leguminosarum bv. viciae) capable of nodulating field pea (Pisum sativum) were assessed in 114 soils from Mediterranean-type environments of southern Australia. All soils were collected in autumn, before the growing season, and had a history of crop legumes including field pea, faba bean, lentil, or vetch. The most probable number (MPN) technique, with vetch as a trap plant, was used to estimate the numbers of pea rhizobia in soils. Of the soils tested, 29% had low numbers of pea rhizobia (<100 rhizobia/g), 38% had moderate numbers (100–1000/g), and the remaining 33% had >1000/g. Soil pH, the frequency of a host crop in the rotation, and the number of summer days with a maximum temperature >35°C were strongly correlated with the pea rhizobia population size. Symbiotic performance (SP) of pea rhizobia in soils was assessed for soils with a MPN >100 rhizobia/g. An extract of the soils was used to inoculate two field pea cultivars growing in a nitrogen-deficient potting media in the greenhouse. Plants were grown for 5 weeks after inoculation and shoot dry matter was expressed as a percentage of the dry matter of plants grown with a commercial strain R. leguminosarum bv. viciae, SU303. Symbiotic performance ranged from 25 to 125%. One-quarter of the soils assessed had suboptimal SP (i.e. <70%). Soil and climatic variables were weakly associated with SP, with pH and average annual rainfall accounting for 17% of the variance. This research highlights the complexity of factors influencing population size and symbiotic performance of pea rhizobia in soils. Options for the improved management of populations of pea rhizobia in Mediterranean environments are discussed. Specifically, our data indicate that inoculation of pea crops is likely to be beneficial where pH(H2O) <6.6, particularly when summers have been hot and dry and when a host has been absent for ≥5 years, as numbers of rhizobia are likely to be below the thresholds needed to optimise nodulation and crop growth. New inoculation technologies and plant breeding will be required to overcome large populations of pea rhizobia with suboptimal SP.

Selecting improved Lotus nodulating rhizobia to expedite the development of new forage species

AimsIn the past decades the increasing focus by Australian pasture development programs on the genus Lotus has seen the evaluation of many species previously untested in Australia. In field trials, nodulation failure was commonplace. This work was undertaken to select effective symbionts for Lotus to ensure further agronomic evaluation of the genus was not compromised. The symbiotic needs of Lotus ornithopodioides were a particular focus of the studies.MethodsGlasshouse experiments were undertaken to evaluate symbiotic relationships between 15 Lotus spp and 23 strains of nodulating Mesorhizobium loti. This was followed by evaluation of elite rhizobial strains for their ability to persist and form nodules under field conditions.ResultsComplex symbiotic interactions were recorded between strains of lotus rhizobia and the different species of Lotus. Notably, the rhizobia that are currently provided commercially in Australia for the inoculation of Lotus corniculatus (strain SU343) and Lotus uliginosus (strain CC829) did not form effective symbioses with the promising species L. ornithopodioides and L. maroccanus. No strain we evaluated was compatible with all the Lotus species, however several strains with a broad host range were identified. WSM1293 and WSM1348 were the most effective strains on L. ornithopodioides and L. peregrinus.These strains were also moderately effective on L. corniculatus (79 and 52% of SU343), less effective on L. maroccanus (26 and 49% of SRDI110) but were ineffective on L. uliginosus. The latter species overall had very specific rhizobial needs. Both WSM1293 and WSM1348 produced adequate levels of nodulation when inoculated on L. ornithopodioides, over two seasons at three field sites.ConclusionsEffective and persistent strains are now available that should allow the un-compromised evaluation of many of the contemporary Lotus species in the field. Selecting a strain for use in commercial inoculants will be more problematic, given the very large host-strain interactions for nitrogen fixation. Here, the balance of Lotus species which are adopted by farmers will have a strong bearing on which rhizobial strains are progressed to commerce.

Host range and saprophytic competence of Sinorhizobium meliloti — a comparison of strains for the inoculation of lucerne, strand and disc medics

Strains of Sinorhizobium meliloti were compared for their saprophytic competence (ability to survive and colonise) in mildly acidic (pHCa 4.8–5.4) soils, using a ‘cross-row’ technique at 3 field locations in the south-east of South Australia. Some strains of rhizobia had greater saprophytic competence than others. Strain WSM879 performed consistently well, nodulating 36% of lucerne seedlings (mean of 3 sites and 4 sampling regions) compared with former inoculant strain WSM826 which nodulated 27% of lucerne seedlings. At one site, strain WSM879 was compared with the former and current Australian inoculant strains (WSM826 and RRI128, respectively). Here, all 3 strains nodulated a similar percentage of lucerne seedlings. However, the addition of 5 t/ha of lime to the soil at this site increased the percentage of lucerne plants nodulated from 23 to 43%. This increase was due to a combination of better strain survival and colonisation and indicates there remains some potential to further improve these aspects of strain performance. The growth of 4 of the rhizobial strains from the field trials was measured on acidified agar media (between pH 4.0 and 7.5). There was virtually no colony growth (<10% of growth at pH 7.0) by strains WSM826, RRI128 and WSM879, at or below pH 6.0. Although strain MSUR52a was still able to grow (40% of potential) at pH 6.0 (in the absence of aluminium) this was not always reflected in better nodulation of lucerne seedlings by this strain in the field. Inclusion of aluminium in the media increased the sensitivity of the strains to acidity. The ability of 6 selected S. meliloti strains to form effective symbioses with 15 plant hosts (from Medicago sativa, Medicago littoralis and Medicago tornata) was compared. All S. meliloti strains formed effective symbioses with all plant hosts. Overall, strain RRI128 was the most effective strain with both the lucernes and the annual medics, resulting in shoot weights similar to those of plants supplied with mineral nitrogen.

Genetic analysis of tolerance to the root lesion nematode Pratylenchus neglectus in the legume Medicago littoralis

BackgroundThe nematode Pratylenchus neglectus has a wide host range and is able to feed on the root systems of cereals, oilseeds, grain and pasture legumes. Under the Mediterranean low rainfall environments of Australia, annual Medicago pasture legumes are used in rotation with cereals to fix atmospheric nitrogen and improve soil parameters. Considerable efforts are being made in breeding programs to improve resistance and tolerance to Pratylenchus neglectus in the major crops wheat and barley, which makes it vital to develop appropriate selection tools in medics.ResultsA strong source of tolerance to root damage by the root lesion nematode (RLN) Pratylenchus neglectus had previously been identified in line RH-1 (strand medic, M. littoralis). Using RH-1, we have developed a single seed descent (SSD) population of 138 lines by crossing it to the intolerant cultivar Herald. After inoculation, RLN-associated root damage clearly segregated in the population. Genetic analysis was performed by constructing a genetic map using simple sequence repeat (SSR) and gene-based SNP markers. A highly significant quantitative trait locus (QTL), QPnTolMl.1, was identified explaining 49% of the phenotypic variation in the SSD population. All SSRs and gene-based markers in the QTL region were derived from chromosome 1 of the sequenced genome of the closely related species M. truncatula. Gene-based markers were validated in advanced breeding lines derived from the RH-1 parent and also a second RLN tolerance source, RH-2 (M. truncatula ssp. tricycla). Comparative analysis to sequenced legume genomes showed that the physical QTL interval exists as a synteny block in Lotus japonicus, common bean, soybean and chickpea. Furthermore, using the sequenced genome information of M. truncatula, the QTL interval contains 55 genes out of which five are discussed as potential candidate genes responsible for the mapped tolerance.ConclusionThe closely linked set of SNP-based PCR markers is directly applicable to select for two different sources of RLN tolerance in breeding programs. Moreover, genome sequence information has allowed proposing candidate genes for further functional analysis and nominates QPnTolMl.1 as a target locus for RLN tolerance in economically important grain legumes, e.g. chickpea.

Symbiotic competence of rose clover (Trifolium hirtum All.)

Rose clover (Trifolium hirtum All.) is a forage plant that is well adapted to acidic and mildly alkaline soils of low natural fertility in southern Australia and to climates with a winter-dominant annual rainfall of 300 mm and above. Reports of low concentrations of nitrogen in rose clover foliage have been attributed to poor N2 fixation and may have discouraged its use in Australia. This investigation, conducted in tube culture, examined the ability of four lines of rose clover to nodulate and fix N2 with effective strains of clover rhizobia (Rhizobium leguminosarum bv. trifolii) and with soils (as a source of naturalised rhizobia) collected from field sites in New South Wales and South Australia. Comparisons with other Trifolium spp. were also made. It was confirmed that there was a low concentration of N in the shoots of the rose clover cvv. SARDI Rose and Hykon. This occurred even where rose clover nodulated and fixed N2 effectively with well known inoculant strains of clover rhizobia and with soil samples collected in the field (provided that the populations of resident clover rhizobia in the soil were at least 150/g). Individual plants were uniform in response to inoculation. Rose clover cv. SARDI Rose was closely related to six of the nine other lines of clover with which it was compared. It was concluded that the registered cultivars of rose clover, cvv. SARDI Rose and Hykon, are symbiotically competent plants. It appears that low N in rose clover foliage is an intrinsic characteristic of the species unconnected with its symbiotic characteristics.

Genome sequence of the Medicago-nodulating Ensifer meliloti commercial inoculant strain RRI128

Ensifer meliloti strain RRI128 is an aerobic, motile, Gram-negative, non-spore-forming rod. RRI128 was isolated from a nodule recovered from the roots of barrel medic (Medicago truncatula) grown in the greenhouse and inoculated with soil collected from Victoria, Australia. The strain is used in commercial inoculants in Australia. RRI128 nodulates and forms an effective symbiosis with a diverse range of lucerne cultivars (Medicago sativa) and several species of annual medic (M. truncatula, Medicago littoralis and Medicago tornata), but forms an ineffective symbiosis with Medicago polymorpha. Here we describe the features of E. meliloti strain RRI128, together with genome sequence information and annotation. The 6,900,273 bp draft genome is arranged into 156 scaffolds of 157 contigs, contains 6,683 protein-coding genes and 87 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.

Genome sequence of the clover-nodulating Rhizobium leguminosarum bv. trifolii strain SRDI565.

Rhizobium leguminosarum bv. trifolii SRDI565 (syn. N8-J) is an aerobic, motile, Gram-negative, non-spore-forming rod. SRDI565 was isolated from a nodule recovered from the roots of the annual clover Trifolium subterraneum subsp. subterraneum grown in the greenhouse and inoculated with soil collected from New South Wales, Australia. SRDI565 has a broad host range for nodulation within the clover genus, however N2-fixation is sub-optimal with some Trifolium species and ineffective with others. Here we describe the features of R. leguminosarum bv. trifolii strain SRDI565, together with genome sequence information and annotation. The 6,905,599 bp high-quality-draft genome is arranged into 7 scaffolds of 7 contigs, contains 6,750 protein-coding genes and 86 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.

Complete genome sequences of the endophytic Streptomyces sp. strains LUP30 and LUP47B, isolated from lucerne plants

ABSTRACT The complete genome sequences of two endophytic Streptomyces sp. strains, LUP30 and LUP47B, were analyzed. These strains were isolated from surface-sterilized roots of lucerne plants from South Australia and were found to promote the growth of the rhizobial partner in vitro and significantly increased nodulation and nitrogen fixation in lucerne plants.