Pulse rhizobia performance on acid soils

Take home messages

  • Inoculation of faba bean, lentil and field pea with rhizobia (Rhizobium leguminosarum bv. viciae) is critical on acid soils. Nodulation is improved by increased application rate of inoculation products.
  • The lower limit of pH(Ca) for reliable nodulation with the commercial strains of faba bean and field pea rhizobia is 5.0.
  • Liming to increase soil pH and increased rates of inoculation should be considered where soil pH(Ca) is below 5.0.
  • Several strains of rhizobia with improved acidity tolerance have shown promise in the field on faba bean and broad bean. They are being more widely tested to develop a case for commercial release.
  • Contact between rhizobia and incompatible pesticides should be avoided when sowing pulses on acid soils.

ɸExtra technical comment by Protech Consulting Pty Ltd

Background

Recent expansion of the pulse industry is seeing crops increasingly grown on soils below pH(Ca) 5.5. Faba beans are the pulse of choice in high rainfall acidic soil environments of south eastern Australia, while the high value of lentils is similarly seeing it sown on acidic soils in lower rainfall areas. The impact of acid soils on pulse production is also likely to increase as soils continue to acidify (Helyar et al. 1990), particularly where the sub-surface soil is acidic and difficult to ameliorate with lime.

Faba bean and lentil are recognised as being sensitive to soil acidity. A substantial part of this sensitivity is due to impacts on the symbiosis with reduced levels of nodulation and N2-fixation reported on acidic soils (Burns et al. 2017). Another signpost of the sensitivity is that the rhizobia (Rhizobium leguminosarum bv. viciae) that nodulate these pulses (and also field pea and vetch) persist at lower numbers or are often absent in acid soils (pH(Ca)<6). Inoculation is therefore recommended with a moderate to high chance of inoculation response on these soils (Drew et al. 2012a, 2012b, Denton et al. 2013).

Two inoculant strains are produced commercially. WSM-1455 (Group F) is produced mainly for faba bean and lentil, but is often also used on field pea. Sulfonylurea (SU)-303 (Group E) is produced for field pea and vetch. In our experience, these two inoculant strains are competent and reliably form nodules when used to inoculate pulses sown into soils above pH(Ca) 5.0, but are constrained below this level.

The performance of strains of rhizobia with improved acidity tolerance and other practices that can be used to improve pulse nodulation and N2-fixation on acid soils are described in this paper.

Acid tolerant strains of rhizobia

Strains identified that improved nodulation in low pH hydroponic experiments

Hydroponic experiments have been used to determine if strains of rhizobia isolated from acid soils provided any advantage over the commercial inoculant strains at low pH. Plant growth solutions were maintained at pH 4.2, the point where the nodulation of field pea by inoculant strains SU-303 and WSM-1455 had previously been shown to be severely reduced in the test system.

Eleven rhizobia strains, comprising five from the South Australian Research and Development Institute (SARDI) (SRDI strains) and six from Murdoch University (WSM strains selected for field pea, supplied by Dr Ron Yates), were tested for their ability to nodulate KaspaA field peas at low pH.

The strains of rhizobia varied in their ability to form nodules. Inoculant strain WSM-1455 performed better than SU-303. Of the new strains, SRDI-954, SRDI-969, WSM-4643, WSM-4644 and WSM-4645 all nodulated more than 70% of plants. SRDI-969 stood out because it also increased nodule numbers more than six-fold, compared with both commercial inoculant strains (Figure 1).

SRDI-969 stood out because it also increased nodule numbers more than six-fold, compared with both commercial inoculant strains

Figure 1. Effect of inoculation treatment on the percentage of KaspaA field pea seedlings forming nodules (left axis, columns) and the number of nodules per nodulated plant (NNP) (right axis, circles) at 20 days after inoculation.

Performance of rhizobia strains in the field

Rhizobia strains with putative acid tolerance were tested in the field between 2015 and 2017. Strains SRDI-954, SRDI-969, SRDI-970 and WSM-4643 performed best and provided substantial levels of improvement over the commercial inoculants at some sites, as described below.

2015 field trials

Strains SRDI-954 and SRDI-970 were initially provided as peat cultures to Maarten Ryder for testing in a GRDC Regional Cropping Solutions Network (RCSN) project examining a range of treatments to improve broad bean production on Kangaroo Island, SA.

In a small plot trial, both strains of rhizobia significantly increased the nodulation of broad bean compared to the current commercial strain — nodulation ratings were higher and more uniform. In addition, shoot nitrogen (N) and fixed N were almost doubled. In a complementary grower run trial (replicated four times), SRDI-954 again produced more nodules than WSM-1455, increased grain yield by 8% and the amount of N fixed by more than 40kg/ha. In these short term trials, the new rhizobia strains were more effective at improving nodulation than other agronomic treatments that included the addition of prilled lime (data not shown).

2016 field trials

Following the promising results in 2015, the cohort of rhizobia strains was expanded and tested at another three locations in 2016 (Kangaroo Island, SA, Wanilla, SA, and Ballyrogan, VIC). Strains were applied at approximately four times the recommended rate, a strategy that we now believe probably moderated the extent of differences between the commercial inoculant and new strains of rhizobia (discussed later in section on inoculation rate).

The field sites were below pH(Ca) 5.0 (4.8, 4.9 and 4.6) and responsive to inoculation, due to the absence of naturalised rhizobia. Mean nodulation across the three sites was increased five-fold by the commercial inoculant strain (Table 1). Again, strain SRDI-954 significantly increased faba bean nodulation (+64%) on Kangaroo Island and averaged 124% across the three sites. Some strains did less well (e.g. WSM-4645).

N2-fixation was significantly improved by inoculation, but was not further improved by the new strains of rhizobia (strain SRDI-969 ranked highest at 107%). On these acid soils, the best nodulated beans fixed approx. 150kg N/ha (not including roots).

Mean (three sites) grain yield with the commercial inoculant was 3.74t/ha and 3.93t/ha (105%) for strains SRDI-969 and WSM-4643, but the values were not significantly different (5% LSD). The grain yield result for WSM-4643 was largely driven by its good performance at one site.

Table 1. Mean data for nodulation, N2-fixation and grain yield across three sites expressed a percentage of the commercial E or F inoculant strain.

 

Nodulation
% commercial inoculant

N2-fixation
% commercial inoculant

Grain yield
% commercial inoculant

No rhizobia

20

47

50

Control (E or F inoculant)

100

100

100

SRDI-954

124

100

102

SRDI-969

113

107

105

SRDI-970

111

102

103

WSM-4643

99

93

105

WSM-4644

83

74

91

WSM-4645

53

74

80

Over the three measures (nodulation, grain yield and N2-fixation), strains SRDI-954 and SRDI-969 were calculated to be 108% compared to the E/F inoculant. Strain SRDI-969 delivered the most consistent benefit (113%, 107% and 105%). Strain WSM-4645 was 69% of the E/F inoculant.

Two plant bioassays assessed the persistence of rhizobial strains in the soil. Soils were collected in the summer (2017) following the trials and used to inoculate plants growing in rhizobia-free media in the greenhouse. None of the rhizobial strains had persisted in the soil at a level substantially above the control treatments, meaning re-inoculation will be necessary even if the acid tolerant strains are used. The result also indicates there is still an opportunity for improvement beyond what is offered by the strains currently being evaluated.

Further evaluation of the strains was undertaken in 2017 and included a comparison of strain performance at a standard inoculation rate.

2017 field trials

Three trials were sown in 2017, comprising two faba beans and one lentil trial.

With faba bean at Wanilla (Eyre Peninsula, SA), rhizobia strains SRDI-954 and SRDI-969 outperformed WSM-1455 for both nodulation and grain yield, when applied to seed as a peat slurry at the standard rate of inoculation (Fig. 2). This site remained dry for four weeks after sowing, adding an additional stress on the rhizobia.

With faba bean at Wanilla (Eyre Peninsula, SA), rhizobia strains SRDI-954 and SRDI-969 outperformed WSM-1455 for both nodulation and grain yield, when applied to seed as a peat slurry at the standard rate of inoculation.

Figure 2. Effect of rhizobia strain on nodule weight (left axis, columns) and grain yield (right axis, circles) of PBA SamiraA faba bean at Wanilla, Eyre Peninsula, SA in 2017. Site pH(Ca) = 4.3, sown into dry soil 28 April. Standard rate of inoculation. Standard error of means shown as bars above columns and circles.

Nodulation results from a second faba bean trial sown at Chatsworth in VIC and a lentil trial near Griffith in southern NSW are shown in Table 2. It is the first time the new strains have been examined on lentil and demonstrates they competently nodulate that species. Growing conditions (waterlogging at Chatsworth, severe frost and below average rainfall at Griffith) were more limiting to grain yield than N2-fixation at both sites. There were no significant differences in grain yield.

Table 2. Effect of strain of rhizobia on the nodulation of faba bean and lentil.

 

Chatsworth, VIC
PBA ZahraA faba bean
pH(Ca) 4.7

Griffith, NSW
PBA AceA lentils
pH(Ca) 4.9

 

Nodulation
Score (0 to 5)

Nodulation
nodules/plant

No rhizobia

0.50

1

WSM-1455 Gp F @ std rate

0.83

21

WSM-1455 Gp F @ double rate

1.15

32

SRDI-954

1.48

40*

SRDI-969

1.42

39*

SRDI-970

2.28*

Not tested

WSM-4643

2.15*

44*

Least significant difference (5%)

0.84

15

* Significantly different from WSM-1455 applied at standard rate

Overall field performance

The field results highlight the importance of good nodulation to establishing viable faba bean, lentil and field pea crops on very acid soils. Strain SRDI-954 improved nodulation over WSM-1455 at five sites and was equal at three sites where it has been tested. Strains SRDI-969, SRDI-970 and WSM-4643 improved nodulation at about a third of the sites where they have been tested. Further evaluation of the strains is planned for 2018, with increased emphasis on lentil.

The WSM strains are primarily being developed for field pea on acid soils (Ron Yates, DAFWA). Based on our assessment of those strains, WSM-4643 is preferred for the pea inoculant because it was by far the most effective of the WSM strains on faba bean.

A new strain for faba bean (and possibly lentil) could be commercially available in 2022, subject to further work being completed to satisfy the criteria required for the replacement of a major inoculant strain.

Inoculation rate

Increasing the rate of inoculation has been shown to improve the nodulation and grain yield of faba bean in an acidic soil. Doubling the rate of inoculant applied as a peat slurry increased nodulation by 52% and grain yield by 41%, despite it being limited by seasonal conditions (Fig. 3). WSM-1455 only produced an acceptable level of nodulation at double the standard rate (data not shown).

Increasing the rate of inoculation improves the nodulation and grain yield of faba bean in an acidic soil. Doubling the rate of inoculant applied as a peat slurry increased nodulation by 52% and grain yield by 41%, despite it being limited by seasonal conditions.

Figure 3. Effect of inoculation rate on nodule weight (left axis, columns) and grain yield (right axis, circles) of PBA SamiraA faba beans at Wanilla, Eyre Peninsula, SA, in 2017. Site pH(Ca) = 4.3, sown into dry soil 28 April. Values are the mean of three rhizobia strains (WSM-1455, SRDI-954 and SRDI-969). No-rhizobia treatment excluded from statistical analysis. Standard error of means shown as bars above columns and circles.

Better nodulation in response to increased inoculation rate is commonly reported (Denton et al. 2013, Roughley et al. 1993) and provides a practical way of improving nodulation where pulses are sown for the first time, especially on hostile soils. However, a note of caution; growers have provided feedback that seeder blockages have resulted when they have increased the inoculation rate, so testing a small test batch of seed first to avoid such problems is suggested.

Pesticides

Particular care needs to be taken where rhizobia are applied with pesticides on seed, especially where it is to be sown into acidic soils. Rhizobia are best applied last and as close as possible to sowing. Within six hours is commonly recommended by inoculant manufacturers. The impacts of seed applied pesticides on rhizobia is often masked where there are naturalised rhizobia present in the soil, but are more likely to be seen on acid soils where there are no rhizobia. An example of such an impact is shown in Figure 4. The treatment of faba bean seed with Apron®ɸ (metalaxyl) or P-Pickle T (PPT) (thiram and thiabendazole) fungicide prior to the application of rhizobia (as a peat slurry to the seed) caused significant reductions in both the amount of N fixed and grain yield. These reductions were the result of fewer rhizobia surviving on the seed and reduced nodulation (data not shown).

ɸ Apron® is not currently registered on faba bean. This product on faba bean is used for research purposes only. Commercial application of this product must adhere to label requirements.

Where pesticide application is necessary, granular rhizobial inoculant may provide a better option, reducing direct exposure of the rhizobia to the pesticide.

The impacts of seed applied pesticides on rhizobia is often masked where there are naturalised rhizobia present in the soil, but are more likely to be seen on acid soils where there are no rhizobia.

Figure 4. Effect of pesticide application to seed on nodule weight (left axis, columns) and grain yield (right axis, circles) of PBA SamiraA faba beans inoculated with Group F rhizobia (WSM-1455) at Ballyrogan VIC, 2016. Site pH (Ca) = 4.6. Standard error of means shown as bars above columns and circles.

Inoculant formulation

Peat inoculant applied as a slurry to seed is the most common method used by growers and is reported to provide consistent and high levels of nodulation across a broad range of environments (Denton et al. 2009, 2017). This method provided satisfactory nodulation in our studies when used to deliver the acid tolerant strains of rhizobia, although granules on occasion have provided additional benefit. Specifically, nodulation by WSM-1455 was improved on two occasions where Novozymes ‘TagTeam®’ granules were used (Table 3).

Table 3. Effect of inoculant formulation and inoculant strain on the nodulation of PBA KareemaA broad bean on Kangaroo Island, SA (sown after break) and PBA SamiraA faba beans at Wanilla, SA (sown dry). Within a site, values followed by the same letter are not significantly different.

Site

Peat slurry on seed
with strain WSM-1455

TagTeam® Granule
with strain WSM-1455

Peat slurry on seed
with strain SRDI-954

Kangaroo Island, SA
(nodule score, 0 to 5)

1.5 a

2.7 ab

3.3 bc

Wanilla, SA
(mg nodule dry weight/6 plants)

273 a

1758 b

2190 b

At the dry sown Wanilla site (2017), where the performance of various inoculant formulations containing WSM-1455 was assessed, nodulation was positively correlated with the number of cells delivered by the product (the combination of the rhizobia number in the product and application rate) (Fig. 5).

At the dry sown Wanilla site (2017), where the performance of various inoculant formulations containing WSM-1455 was assessed, nodulation was positively correlated with the number of cells delivered by the product (the combination of the rhizobia number in the product and application rate)

Figure 5. Relationship between number of rhizobia delivered at sowing by different inoculant formulations of rhizobia and the nodulation of PBA SamiraA faba beans sown at Wanilla, Eyre Peninsula, SA in 2017.

The result demonstrates that granules can work in an acidic soil, but in step with the efficacy of inoculants more generally, their performance is likely to be dependent upon the number of rhizobia they deliver. Granules provide the possibility of being able to separate the rhizobia from seed applied pesticides and fertilisers which is desirable, and so the delivery of the improved rhizobia strains in a ‘high count’ granule may provide opportunity for further improvement.

Liming

The development of new rhizobia strains should not be seen as a replacement for liming. Even with good inoculation practice on acid soils, nodulation can remain below potential and rhizobial colonisation of the soil is limited, so the addition of lime is still needed. Liming to raise soil pH above pH(Ca) 5.0 also corrects nutritional deficiencies and toxicities that more broadly limit crop performance.

Further, since nitrate leaching after pulse growth is a significant contributor to soil acidification, liming is important to counter this and prevent further acidification.

Improved rhizobia will still be of benefit where soils are limed, especially where there are acidic sub-surface soil layers that are difficult to remediate due to the slow movement of lime down the profile.

Discussion

There are reasonable prospects that a strain of rhizobia with improved acid tolerance can be selected for faba beans which are being grown on some very acid soils. An improved strain would also have the potential to be used on lentils which are in the same inoculation group. Improved acid tolerance of the rhizobia strains for faba beans and lentils may provide the potential to expand these crops into new environments and improve their performance in existing acid soil areas.

Where a rhizobia strain with improved acidity tolerance is combined with good inoculation practice, it should be possible to remove symbiotic constraints to faba bean production between pH(Ca) 4.5 and 5.0. The lower pH limit for lentils needs to be clarified, but they are generally regarded as more sensitive than faba beans. None of the rhizobia strains tested thus far appear to be able to persist in soil below pH(Ca) 5.0, therefore re-inoculation will be essential each time the crop is grown.

Until a new strain is available, growers should consider increasing their inoculation rate and avoid exposing the rhizobia to pesticides, where it is practical to do so.

Improved rhizobia should be seen as an accompaniment, not a replacement for liming. Liming remains important to prevent further acidification and is therefore critical to the longer term sustainability of the farming system. Surface soil (0-10cm) should be limed to at least pH(Ca) 5.0, noting that a higher target may be needed to achieve adequate amelioration where acidity is prevalent below the soil surface.

Further testing is needed and planned to satisfy the criteria for a rhizobia strain replacement, with a view to replacing WSM-1455 in 2022.

Useful resources

Inoculating legumes: A practical guide

Soil acidity

References

Burns H, Norton M, Tyndall P. (2017). Reduced nodulation of faba bean on acidic soils; the role of topsoil pH stratification. In: Proceedings of the 2017 Agronomy Australia Conference, 24 – 28 September 2017, Ballarat, Australia. 1-4.

Denton MD, Pearce DJ, Ballard RA, Hannah MC, Mutch LA, Norng S, Slattery JF. (2009). A multi-site field evaluation of granular inoculants for legume nodulation. Soil Biology and Biochemistry. 41, 2508-16.

Denton MD, Pearce DJ, Peoples MB. (2013). Nitrogen contributions from faba bean (Vicia faba L.) reliant on soil rhizobia or inoculation. Plant and Soil. 365, 363-74.

Denton MD, Phillips LA, Peoples MB, Pearce DJ, Swan AD, Mele PM, Brockwell J. (2017). Legume inoculant application methods: effects on nodulation patterns, nitrogen fixation, crop growth and yield in narrow-leaf lupin and faba bean. Plant and Soil. 419, 25-39.

Drew EA, Denton MD, Sadras VO, Ballard RA. (2012a). Agronomic and environmental drivers of population size and symbiotic performance of Rhizobium leguminosarum bv. viciae in Mediterranean-type environments. Crop and Pasture Science. 63, 467-77.

Drew E, Herridge DF, Ballard RA, O’Hara GW, Deaker R, Denton MD, Yates RJ, Gemell G, Hartley E, Phillips L, Seymour N. (2012b). Inoculating legumes: a practical guide. Grains Research and Development Corporation.

Helyar KR, Cregan PD Godyn DL. (1990). Soil acidity in New-South-Wales - Current pH values and estimates of acidification rates. Soil Research 28, 523-537.

Roughley RJ, Gemell LG, Thompson JA, Brockwell J. (1993). The number of Bradyrhizobium sp. (Lupinus) applied to seed and its effect on rhizosphere colonisation, nodulation and yield of lupin. Soil Biology and Biochemistry. 25, 1453-8.

Acknowledgements

The research reported in this paper is made possible by significant contributions of growers through both trial cooperation and the support of GRDC and the South Australian Grains Industry Trust (SAGIT). Trials were sown and managed by the New Variety Agronomy Group (SARDI, Clare and Port Lincoln), Lyne Dohle and Jenny Stanton (Kangaroo Island), the Southern Farming Systems Group (VIC), and Ag Grow Agronomy and Research (NSW).

WSM strains were provided by Dr Ron Yates, Department of Primary Industries and Regional Development.

Contact details

Ross Ballard
SARDI Soil Biology and Diagnostics
GPO Box 397, Adelaide SA, 5001
8303 9388
ross.ballard@sa.gov.au

GRDC Project code: DAS00128, UA00138