Matthew D. Denton

Distribution, abundance and symbiotic effectiveness of Rhizobium leguminosarum bv. trifolii from alkaline pasture soils in South Australia

The current dissatisfaction with low productivity of annual medic (Medicago spp.) pastures has highlighted the need to seek alternative legumes to provide efficient N2 fixation in low rainfall, alkaline soil environments of southern Australia. Clover species adapted to these environments will have limited N2 fixation if effective rhizobia are not present in sufficient quantities. A survey of 61 sites was conducted across South Australia to determine the size, distribution and effectiveness of Rhizobium leguminosarum bv. trifolii (clover rhizobia) populations resident in these low rainfall, alkaline soil environments. Clover rhizobia were detected at 56 of the sites, with a median density of 230–920 rhizobia/g soil. Most rhizobial populations were poor in their capacity to fix nitrogen. Rhizobial populations from fields provided 11–89% and 10–85% of the shoot biomass of commercial reference strains when inoculated onto host legumes T. purpureum (purple clover) and T. resupinatum (persian clover), respectively. Rhizobial population size was correlated negatively to pH and the percentage of CaCO3 in the soil, and was significantly increased in the rhizospheres of naturalised clover, found at 17 sites. Management options for rhizobial populations to improve legume diversity and productivity are discussed in terms of rhizobial population dynamics and likely soil constraints to successful rhizobial colonisation.

Complete genome sequence of Rhizobium leguminosarum bv. trifolii strain WSM1325, an effective microsymbiont of annual Mediterranean clovers.

Rhizobium leguminosarum bv trifolii is a soil-inhabiting bacterium that has the capacity to be an effective nitrogen fixing microsymbiont of a diverse range of annual Trifolium (clover) species. Strain WSM1325 is an aerobic, motile, non-spore forming, Gram-negative rod isolated from root nodules collected in 1993 from the Greek Island of Serifos. WSM1325 is produced commercially in Australia as an inoculant for a broad range of annual clovers of Mediterranean origin due to its superior attributes of saprophytic competence, nitrogen fixation and acid-tolerance. Here we describe the basic features of this organism, together with the complete genome sequence, and annotation. This is the first completed genome sequence for a microsymbiont of annual clovers. We reveal that its genome size is 7,418,122 bp encoding 7,232 protein-coding genes and 61 RNA-only encoding genes. This multipartite genome contains 6 distinct replicons; a chromosome of size 4,767,043 bp and 5 plasmids of size 828,924 bp, 660,973 bp, 516,088 bp, 350,312 bp and 294,782 bp.

Complete genome sequence of Rhizobium leguminosarum bv trifolii strain WSM2304, an effective microsymbiont of the South American clover Trifolium polymorphum.

Rhizobium leguminosarum bv trifolii is the effective nitrogen fixing microsymbiont of a diverse range of annual and perennial Trifolium (clover) species. Strain WSM2304 is an aerobic, motile, non-spore forming, Gram-negative rod, isolated from Trifolium polymorphum in Uruguay in 1998. This microsymbiont predominated in the perennial grasslands of Glencoe Research Station, in Uruguay, to competitively nodulate its host, and fix atmospheric nitrogen. Here we describe the basic features of WSM2304, together with the complete genome sequence, and annotation. This is the first completed genome sequence for a nitrogen fixing microsymbiont of a clover species from the American center of origin. We reveal that its genome size is 6,872,702 bp encoding 6,643 protein-coding genes and 62 RNA only encoding genes. This multipartite genome was found to contain 5 distinct replicons; a chromosome of size 4,537,948 bp and four circular plasmids of size 1,266,105 bp, 501,946 bp, 308,747 bp and 257,956 bp.

Root distributions of Australian herbaceous perennial legumes in response to phosphorus placement

Many Australian plant species have specific root adaptations for growth in phosphorus-impoverished soils, and are often sensitive to high external P concentrations. The growth responses of native Australian legumes in agricultural soils with elevated P availability in the surface horizons are unknown. The aim of these experiments was to test the hypothesis that increased P concentration in surface soil would reduce root proliferation at depth in native legumes. The effect of P placement on root distribution was assessed for two Australian legumes, Kennedia prorepens F. Muell. and Lotus australis Andrews, and the exotic Medicago sativa L. Three treatments were established in a low-P loam soil: amendment of 0.15 g mono-calcium phosphate in either (i) the top 50 mm (120 µg P g-1) or (ii) the top 500 mm (12 µg P g-1) of soil, and an unamended control. In the unamended soil M. sativa was shallow rooted, with 58% of the root length of in the top 50 mm. K. prorepens and L. australis had a more even distribution down the pot length, with only 4 and 22% of their roots in the 0-50 mm pot section, respectively. When exposed to amendment of P in the top 50 mm, root length in the top 50 mm increased 4-fold for K. prorepens and 10-fold for M. sativa, although the pattern of root distribution did not change for M. sativa. L. australis was relatively unresponsive to P additions and had an even distribution of roots down the pot. Shoot P concentrations differed according to species but not treatment (K. prorepens 2.1 mg g-1, L. australis 2.4 mg g-1, M. sativa 3.2 mg g-1). Total shoot P content was higher for K. prorepens than for the other species in all treatments. In a second experiment, mono-ester phosphatases were analysed from 1-mm slices of soil collected directly adjacent to the rhizosphere. All species exuded phosphatases into the rhizosphere, but addition of P to soil reduced phosphatase activity only for K. prorepens. Overall, high P concentration in the surface soil altered root distribution, but did not reduce root proliferation at depth. Furthermore, the Australian herbaceous perennial legumes had root distributions that enhanced P acquisition from low-P soils.

Evaluation of Perennial Forage Legumes and Herbs in Six Mediterranean Environments

There is an absence of drought tolerant herbaceous perennial forage legume and herb options other than lucerne (Medicago sativa L.) for environments with Mediterranean-like climates common in extensive areas of Southern Australia, the Mediterranean basin, and Chile. Therefore, a collection of 174 forage perennial legume and herb entries from 103 species and 32 genera was evaluated for adaptation in a diverse range of Mediterranean climatic environments in Southern Australia. The seasonal rainfall distribution varied from moderately to highly winter dominant with long term average annual rainfall ranging from 318 to 655 mm. The entries were rated for productivity and persistence over 3 yr. The 12 entries identified as the most promising for winter, summer, or all-year round production included Bituminaria bituminosa (L.) C.H. Stirt. var. albomarginata; Cichorium intybus L.; Cullen australasicum (Schltdl.) J.W. Grimes; Dorycnium hirsutum (L.) Ser.; Kennedia prostrata R. Br.; Lotononis bainesii Baker, Lotus pedunculatus Cav.; L. corniculatus L.; L. cytisoides L.; Medicago sativa subsp. sativa L.; Medicago sativa subsp. caerulea (Less. ex Ledeb.) Schmalh., and M. sativa subsp. falcata (L.) Arcang. These entries maintained production and persisted for the period of the evaluation, with the exception of C. intybus and L. corniculatus that declined in persistence over time. The potential role of these species in extensive grazing systems in Mediterranean climatic zones, their attributes and limitations, and current progress in developing them as useful forage plants was discussed.

Response of juvenile Melaleuca halmaturorum to flooding: Management implications for a seasonal wetland, Bool Lagoon, South Australia

The response of M. halmaturorum was measured to determine the ability of juvenile plants to survive flooding and to examine how this influenced plant growth. Seedlings and one- and two-year-old plants were flooded to 0% (control), 50% or 100% of their initial height for periods of three to 14 weeks, followed by an eight-week recovery period. Only 22% of seedlings survived five weeks of 100% coverage, but these died during the recovery period. The older plants survived the flooding treatments well, but as duration increased so survival through the recovery period declined. After 100% coverage for six weeks followed by the recovery period, only 29% survived. Stem linear extension rates corresponding to these survival rates were used to predict the survival of juvenile M. halmaturorum in Bool Lagoon, South Australia. The results showed that, with the current water regime, natural recruitment would occur only at the highest elevation but that this could be improved by planting older, taller plants at lower elevations. Indices of growth showed that flooded plants performed poorly compared with the controls as duration and percentage of coverage increased. The data suggested that coverage inhibited plant growth during the flood period, but there was a further, interactive response to duration that became apparent after the post-flood recovery period. An interpretation of these results is that M. halmaturorum in its juvenile stages is intermediate between a flood-sensitive and a flood-tolerant species because it is able to recover from short floods of three weeks or less but performs poorly if floods exceed six to nine weeks.

Competitive abilities of common field isolates and a commercial strain of Rhizobium leguminosarum bv. trifolii for clover nodule occupancy

We previously reported that commercial Rhizobium leguminosarum bv. trifolii inoculants failed to outcompete naturalized strains for nodule occupation of clover sown into an alkaline soil [Aust. J. Agric. Res. 53 (2002) 1019]. Two field isolates that dominated nodule occupancy at the field site were labeled with a PnifH-gusA marker. Marked strains were chosen on the basis that they were equally competitive and fixed similar amounts of nitrogen in comparison to their parental strain. The minitransposon insertions were cloned and sequence analysis revealed that neither lesion disrupted the integrity of any known gene. The marked strains were then used to follow nodule occupancy of Trifolium alexandrinum in competition against the commercial inoculant TA1 under a range of experimental conditions. In co-inoculation experiments in sand–vermiculite, TA1 outcompeted each marked field isolate for nodule occupancy. However, using TA1-inoculated seed sown into alkaline soil containing a marked field strain, it was demonstrated that by increasing the cell number of marked rhizobia in the soil and reducing the cell number of the commercial inoculant, the proportion of nodules occupied by TA1 was reduced. These studies indicate that the ability of the field isolates to dominate nodule occupancy in the alkaline field soils was most likely caused by poor commercial inoculant survival providing the advantage for naturalized soil rhizobia to initiate nodulation.

Transgenic alfalfa secretes a fungal endochitinase protein to the rhizosphere

Transgenic plants containing a chimeric gene construct that facilitates the exudation of proteins from roots offer novel approaches for modification of the rhizosphere and production of relatively pure recombinant proteins. The aim of this study was to develop alfalfa (Medicago sativa L.) plants that exude a heterologous recombinant protein into the rhizosphere. Alfalfa transformed with a fungal endochitinase (ech42) cDNA fused in frame to the signal peptide of a white lupin acid phosphatase and under the control of the cassava vein mosaic virus (CsVMV) promoter expressed increased chitinase activity in vegetative organs and root exudates. Chitinase activity in root exudates of transgenic alfalfa was 7.5–25.7 times higher than in the untransformed Regen-SY plants. Chitinase enzyme activity was accompanied by increased synthesis of mRNA and protein in transformed plants. By comparison, untransformed and vector only transformed plants displayed no expression of recombinant protein and mRNA. A single band of the expected molecular weight was present only in western blots of root exudates of transgenic alfalfa plants. The secreted endochitinase enzyme not only retained its lytic activity against glycol chitin but also showed antifungal activity by inhibition of spore germination of two fungal pathogens. Exudation of recombinant proteins from roots may offer alternative uses for alfalfa in the production of value-added biopharmaceuticals and may influence microbes or modify soil nutrient availability near plant roots.

The diversity of arbuscular mycorrhizas of selected Australian Fabaceae

Abstract Members of the Australian native perennial Fabaceae have been little explored with regard to their root biology and the role played by arbuscular mycorrhizal (AM) fungi in their establishment, nutrition and long-term health. The ultimate goal of our research is to determine the dependency of native perennial legumes on their co-evolved AM fungi and conversely, the impact of AM fungal species in agricultural fields on the productivity of sown native perennial legume pastures. In this paper we investigate the colonisation morphology in roots and the AMF, identified by spores extracted from rhizosphere soil, from three replicate plots of each of the native legumes, Cullen australasicum, C. tenax and Lotus australis and the exotic legumes L. pedunculatus and Medicago sativa. The plants were grown in an agricultural field. The level and density of colonisation by AM fungi, and the frequency of intraradical and extraradical hyphae, arbuscules, intraradical spores and hyphal coils all differed between host plants and did not consistently differ between native and exotic species. However, there were strong similarities between species in the same genus. The three dominant species of AM fungi in rhizosphere soil also differed with host plant, but one fungus (Glomus mosseae) was always the most dominant. Sub-dominant AM species were the same between species in the same genus. No consistent differences in dominant spores were observed between the exotic and native Fabaceae species. Our results suggest that plant host influences the mycorrhizal community in the rhizosphere soil and that structural and functional differences in the symbiosis may occur at the plant genus level, not the species level or due to provenance.

Soil phosphorus supply affects nodulation and N : P ratio in 11 perennial legume seedlings

Developing new perennial pasture legumes for low-P soils is a priority for Australian Mediterranean agro-ecosystems, where soil P availability is naturally low. As legumes tend to require higher P inputs than non-legumes, the ability of these plants to fix N2 under varying soil P levels must be determined. Therefore, the objective of this study was to investigate the influence of soil P supply on plant N status and nodule formation in 11 perennial legumes, including some novel pasture species. We investigated the effect of applying soil P, ranging from 0 to 384 μg P/g dry soil, on plant N status and nodulation in a glasshouse. Without exogenous P supply, shoot N concentration and N : P ratio were higher than at 6 μg P/g soil. Shoot N concentration and N : P ratio then changed little with further increase in P supply. There was a close positive correlation between the number of nodules and shoot P concentration in 7 of the 11 species. Total nodule dry weight and the percentage of plant dry weight that consisted of nodules increased when P supply increased from 6 to 48 μg P/g. Without exogenous P addition, N : P ratios partitioned into a two-group distribution, with species having a N : P ratio of either >70 or <50 g/g. We suggest that plants with a high N : P ratio may take up N from the soil constitutively, while those with a low N : P ratio may regulate their N uptake in relation to internal P concentration. The flexibility of the novel pasture legumes in this study to adjust their leaf N concentrations under different levels of soil P supplements other published evidence of good growth and high P uptake and P-use efficiency under low soil P supply and suggests their potential as pasture plants in low-P soils in Australian Mediterranean agro-ecosystems warrants further attention.