Key messages

Aim

Evaluate strain WSM4643 for the replacement of the present Australian commercial inoculant group E/F that will enable associated legumes to achieve improved N fixation on infertile, acidic soils.

Introduction

Legumes can contribute to the sustainability of agriculture by improving crop diversity, suppressing pests and diseases, increasing soil fertility and, importantly, by providing biologically fixed nitrogen (N) for subsequent crops (Howieson et al 2008; Peoples et al 2009). For many legumes achieving optimal yields requires soils with pH CaCl₂ in the range 6-8, good soil moisture retention (adequate clay content), structural attributes to facilitate drainage and root growth, and balanced mineral fertility (Howieson and Ballard 2004). Such conditions allow ideal performance of the legume plants, survival and proliferation of their associated root nodule bacteria (rhizobia), optimal nodulation and therefore the formation of an effective N fixing symbiosis. However, in southern Australian dryland agriculture there is a high proportion of soils that are not conducive to successful legume farming (Moore, 2001). These soils are characterised by low organic matter, clay mineral fertility and pH, and remain hot with negligible plant available moisture for more than half of the year (Van Gool 2011). These conditions, particularly when in combination, are unfavourable to legume nodulation (Atieno and Lesueur 2019). To overcome these limitations, research has focused on selecting superior rhizobial strains that can endure these soil constraints to optimise biological N fixation (Howieson et al 2000; 2008).

All agricultural legumes grown in Australia are exotic and are not able to nodulate with Australian native rhizobia. Their preferred rhizobial symbionts arrived unintentionally, transported on either contaminated soil or seeds. Strains of rhizobia are not all equal in the amount of atmospheric N they can fix when they form symbiosis with a suitable legume (termed effectiveness). Since 1953, agricultural researchers have been tasked with screening and identifying the most effective rhizobia to develop the most elite strains into commercial inoculants (Bullard et al 2005). Putative inoculant rhizobial strains go through an extensive selection process, in which they are screened for their ability to maintain high N fixation over a broad host range, their adaptation to the anticipated soil niche(s) of the target legume(s), their ability to be manufactured efficiently, genetic stability and their aptitude to survive the inoculation process (i.e., when placed on seed before sowing) (Drew et al 2012). Rhizobial strains should also be screened for high levels of saprophytic competence, which estimates the ability of the rhizobia cells to persist and colonise in the soil over summer when there is no legume host present. This trait increases the chances of successful nodulation, particularly when legumes are either self-regenerating or sown into marginal moisture conditions (Yates et al 2021a). Once commercialised, it is recommended to growers that legumes are inoculated with these improved rhizobia (marketed as rhizobial Groups) to optimise N fixation.

The micro-symbiont Rhizobium leguminosarum biovar viciae forms a symbiosis with many grain legumes including Pisum sativum (field peas), Lens culinaris (lentils) and Vicia faba (faba beans), and the forage species of Vicia spp. (e.g. vetch) and Lathyrus spp. (e.g. grass pea). Currently this group of legumes is inoculated with the Australian commercial strain WSM1455 (Group F). However, there is evidence that legumes inoculated with the commercial strain perform poorly in infertile, acidic soils in southern Australia (Evans 2005). For this reason, a research project was initiated to identify new strains better suited to these soils. Additional strain germplasm was required as there were a limited number of strains fitting the basic criteria in the international Western Soil Microbiology (WSM) Rhizobium Genebank (at the LRS group, Murdoch University). This deficiency was addressed by collecting nodules from field pea plants grown in low pH soils from southern Italy (a recognised centre of origin) (Yates et al 2021a). As a result of a thorough evaluation, a new elite strain WSM4643 was selected displaying superior symbiotic performance and saprophytic competence (Yates et al 2021b). The agronomic field trials reported in this paper were undertaken to assess if WSM4643 would form a more effective symbiosis with field peas, vetch and lentils, and produce higher biomass, increased N fixation and higher seed yields than the current commercially available rhizobial strain (WSM1455) when applied to infertile, acidic soils.

Method

Trial establishment

Five randomised and replicated small plot cone seeder inoculant trials were undertaken in Gibson (2019, 4 reps) and Muresk (2021, 6 reps) in low pH soils. Strict hygiene protocols were observed to avoid cross contamination of rhizobial strains (as described by Yates et al 2016). Soil samples from the two locations were cored in March of the year of establishment of the experiment (Table 1). The soil type at Gibson was a grey shallow sandy duplex (Brown sodosol) and evaluated field peas (CVs Butler PBR symbol), vetch (CVs RM4 PBR symbol) and lentils (CVs Bolt PBR symbol) sown in 20m-long plots at a seeding rate of 140, 35 and 60kg/ha, respectively. The Muresk site was a red shallow loamy duplex (Red Chromosol) (Table 1) and assessed field peas (CVs Butler PBR symbol) and lentils (CVs Bolt PBR symbol ) sown in 15m-long plots at a seeding rate of 105 and 50kg/ha, respectively. All seed was surface sterilised and coated with fungicide (co-formulated thiram and thiabendazole), dried, then inoculated with peat containing a specific rhizobial strain at the recommended label rate (250g peat containing a minimum 1 x 10⁹ cells/g applied to 50 kg seed) adhered by a 2% PVA solution for less than 24 hours before being sown into moist soil.

Assessment

Legumes at all field experiments were assessed after 10-12 weeks of growth for nodulation using the methods outlined in Yates et al (2016). At early to mid-pod set, the percentage of N in the tops derived from the atmosphere (%Ndfa) was evaluated via 15N natural abundance (Unkovich et al 2008). To do this, mixed plant leaf samples from each plot were randomly collected, dried (4 days at 60^oC), finely ground and analysed using a mass spectrometer. Total dry weight biomass cuts were measured by cutting three random 1m² quadrats (3m²) per plot, dried at 60°C for four days then weighed to estimate t/ha of above-ground biomass and the amount of biomass N fixed kg/ha. Entire plots were harvested by machine or measured by collecting seed from three random 1m² quadrats within the plots to provide grain yields (t/ha).

Statistical analysis

All analyses and plots were performed in R (Team 2019). The data was explored for violation of assumptions (normality, heterogeneity, independence, sphericity, interactions) prior to analysis. Data was analysed by fitting response variables to linear mixed effects model with an appropriate link (guassian, binomial or poisson) with predictor (rhizobia strain) as fixed effects and replication as random effect in package lme4 (Bates et al 2014).

Results

Gibson

Uninoculated legume seed attained very low nodulation in the extremely acidic soils and this resulted in lower values for all other measurements (Figure 1). Field peas (CVs Butler PBR symbol) inoculated with WSM4643 demonstrated a significant increase in biomass N fixed (kg/ha) compared to plots inoculated with WSM1455, indicating higher tissue N concentrations. Similar results were reached for nodule score, peak biomass, seed yield and %Ndfa. Significant increases were observed for grain yield, %Ndfa and the amount of N fixed (kg/ha) by the lentil plots (CVs Bolt PBR symbol) inoculated with WSM4643 compared to inoculation with WSM1455 (Figure 1). It is important to note that lentils are usually not recommended to be grown in soils containing these chemical constraints (Table 1). Woolly pod vetch (Vicia villosa CVs RM4 PBR symbol) seeds inoculated with WSM4643 demonstrated a significant increase in nodulation and biomass N fixed (kg/ha) over seed inoculated with WSM1455 (Figure 1). It is noteworthy that seed inoculated with WSM4643 achieved significantly higher biomass N fixed (kg/ha) for all three legumes studied.

Muresk

Lentil plants inoculated with WSM4643 recorded significantly higher nodulation and seed yield (t/ha) compared to the current commercial inoculant WSM1455, and significantly greater peak biomass to the uninoculated plots (Figure 2). Similarly, field peas inoculated with WSM4643 exhibited significantly higher seed yield than the uninoculated or WSM1455 treatments. Uninoculated plots containing lentil (cv. Bolt PBR symbol) or field peas (cv. Butler PBR symbol) measured less for nodulation, peak biomass and seed yield (Figure 2).

Figure 1. Average nodule rating per plant, peak dry weight biomass (t/ha), percentage nitrogen derived from atmosphere per plot (%Ndfa), average seed yield per plot (t/ha) and amount of biomass N fixed kg/ha of lentils (cv. Bolt), field peas (cv. Butler) and vetch (cv. RM4) at Gibson, Western Australia field site (2019) when inoculated with 2 rhizobial strains (WSM1455 and WSM4643) or left as uninoculated controls. Centre points represent estimated marginal means, shading represents 95% confidence intervals and arrows indicate Tukey comparison values.

Figure 2. Average nodule rating per plant, peak dry weight biomass (t/ha) and average seed yield per plot (t/ha) of lentils (cv. Bolt) and field peas (cv. Butler) at Muresk, Western Australia field site (2021) when inoculated with 2 rhizobial strains (WSM1455 and WSM4643) or left as uninoculated controls. Centre points represent estimated marginal means, shading represents 95% confidence intervals and arrows indicate Tukey comparison values.

Conclusion

Legume seed inoculated with WSM4643 exhibited a consistent increase in nodulation, biomass, N fixation and seed yield when compared to the current commercial strain. The trial results highlighted the extreme importance of inoculation and the production gains that growers can achieve through administering superior rhizobial strains. Analysis showed WSM4643 demonstrated statistically significantly higher results in 7 of the 21 measurements compared to the current commercial strain WSM1455. Additionally, WSM4643 was equivalent in performance to WSM1455 for all of the remaining measurements. These results combined with similar field experiments trialling strain WSM4643 around southern Australia have been compiled to make a successful case to the Australian Nitrogen Fixation Committee to provisionally replace the commercial inoculant group E/F strain (WSM1455). Strain WSM4643 has now been given to the Australian inoculation companies to evaluate manufacture efficiency and genetic stability in their production process with the view that it will be commercially available for the 2023 growing season. Matching rhizobium strain to legume genotype, soil and climate, in combination with successful administration, is the key to maximising yield and profit from legumes. Legumes inoculated with strain WSM4643 can offer more flexible cropping rotations and deliver higher levels of N₂ fixation on acidic soils which subsequently can reduce the application of fertiliser N.  Importantly, growers must consider the suitability of legume to soil conditions and environment, and combine inoculant delivery optimisation with advanced liming and soil amelioration techniques.

Acknowledgments

The authors would like to thank the various support staff and project technicians from the Legume and Rhizobium Studies (LRS, Murdoch University) that have contributed to this project and the DPIRD Esperance and Northam Research Support Unit for sowing, managing and harvesting the trial. The authors would also like to thank farmer Wayne Lewis and family (Gibson) and the Muresk Institute (WA Government), and finally the GRDC supported National Fixation Project (NFP). The research undertaken as part of this project is made possible by the significant contributions of growers through both trial cooperation and the support of the GRDC, the author would like to thank them for their continued support.

PBR symbol Varieties displaying this symbol beside them are protected under the Plant Breeders Rights Act 1994.

Paper reviewed by: George Mwenda, DPIRD.

References

Atieno, M, Lesueur, D (2019) Opportunities for improved legume inoculants: Enhanced stress tolerance of rhizobia and benefits to agroecosystems. Symbiosis 77, 191-205.

Bates, D, Mächler, M, Bolker, B, Walker, S (2014) Fitting linear mixed-effects models using lme4. arXiv preprint arXiv:1406.5823

Bullard, G, Roughley, R, Pulsford, D (2005) The legume inoculant industry and inoculant quality control in Australia: 1953–2003. Australian Journal of Experimental Agriculture 45, 127-140.

Drew, E, Herridge, D, Ballard, R, O’Hara, G, Deaker, R, Denton, M, Yates, R, Gemell, G, Hartley, E, Phillips, L (2012) Inoculating legumes: a practical guide. GRDC.

Evans, J (2005) An evaluation of potential Rhizobium inoculant strains used for pulse production in acidic soils of south-east Australia. Australian Journal of Experimental Agriculture 45, 257-268.

Howieson, J, Ballard, R (2004) Optimising the legume symbiosis in stressful and competitive environments within southern Australia—some contemporary thoughts. Soil Biology and Biochemistry 36, 1261-1273.

Howieson, J, Malden, J, Yates, R, O'Hara, G (2000) Techniques for the selection and development of elite inoculant strains of Rhizobium leguminosarum in southern Australia. Symbiosis 28, 33-48.

Howieson, J, Yates, R, Foster, K, Real, D, Besier, R (2008) Prospects for the future use of legumes. In 'Nitrogen-fixing leguminous symbioses.' (Eds M Dilworth, E James, J Sprent, W Newton.) pp. 363-394. (Springer: Dordrecht)

Moore, G. A. (2001). Soil Guide: A handbook for understanding and managing agricultural soils. Perth, WA, Western Australian Department of Agriculture.

Peoples, MB, Unkovich, MJ, Herridge, DF (2009) Measuring symbiotic nitrogen fixation by legumes. Nitrogen fixation in crop production 52, 125-170.

Team, RC, 2019. R: A language and environment for statistical computing (Version 3.0. 2) [Computer software]. Vienna, Austria: R Foundation for Statistical Computing.

Unkovich, M, Herridge, D, Peoples, M, Cadisch, G, Boddey, B, Giller, K, Alves, B, Chalk, P (2008) Soil and land systems group, school of earth and environmental science. In M. Unkovich D. F. Herridge M. Peoples G. Cadisch B. Boddey K. Giller & P. Chalk. (Eds.), Measuring plant-associated nitrogen fixation in agricultural systems, (pp. 258). Canberra, Australia: ACIAR.

van Gool, D (2011) Wheat yield potential and land management constraints in the South West of Western Australia. Department of Agriculture and Food, Western Australia, Perth.

Yates, R, Abaidoo, R, Howieson, J (2016) Field experiments with rhizobia. In 'Working with rhizobia.' (Eds J Howieson, M Dilworth.) pp. 145-166. (ACIAR: Canberra, Australia)

Yates RJ, Harrison RJ, Loi A, Steel EJ, Edwards TJ, Nutt BJ, Porqueddu C, Gresta F, Howieson JG (2021a) Sourcing Rhizobium leguminosarum biovar viciae strains from Mediterranean centres of origin to optimise nitrogen fixation in forage legumes grown on acid soils. Grass and Forage Science 76, 33-43.

Yates RJ, Steel EJ, Poole CM, Harrison RJ, Edwards TJ, Hackney B, Stagg GR, Howieson JG (2021b) Optimising the growth of forage and grain legumes on low pH soils through the application of superior Rhizobium leguminosarum biovar viciae strains. Grass and Forage Science 76, 44-56.

Contact details

Ron Yates
Murdoch University / DPIRD
90 South St, Murdoch WA 6150 / 3 Baron-Hay Court, South Perth WA 6151
+61 427550125
Email: ron.yates@murdoch.edu.au or ronald.yates@dpird.wa.gov.au