South Australian scientists have developed an innovative method of quantifying rhizobia in soil using a high-throughput DNA test.

The project’s success will help southern region grain growers save significant time and money when inoculating their legume crops and will also streamline future rhizobia studies.

Research to establish an efficient DNA-based method to quantify rhizobia levels in soil has been one focus of the strategic partnership between GRDC and the South Australian Research and Development Institute (SARDI). The project is being led by Dr Alan McKay, who pioneered the PREDICTA® B platform for assessing soil-borne pathogen loads in soil prior to seeding.

Dr McKay says there was a clear need to improve methods for measuring rhizobia levels in soil. “The existing methods using plant-based bio-assays were laborious and costly, and were a major bottleneck in research. They were completely unsuitable as the basis for a grower service,” he says.

“We had been considering developing tests for rhizobia for some time, but the available sequence data provided no useful leads. Many discussions were had with Ross Ballard, sub-program leader for beneficial microbes at SARDI, and he indicated the species names attributed to sequence data in the public databases may not be correct.”

Local re-sequencing leads to successful test

With support from the GRDC and SARDI strategic partnership, the researchers decided to re-sequence isolates from Rhizobia Group E/F (which nodulate lentils, faba beans, field peas and vetch), along with closely related non-target species that had been characterised by Mr Ballard’s group.

This re-sequencing proved critical and, while the differences were small, eventually enabled the team to identify a test design that worked.

“Using the DNA method, we can now process thousands of soil samples with relative ease, whereas processing 100 samples was a major task using the bio-assay,” Dr McKay says.

Growers could sample by soil type to create a rhizobia map for the paddock, then use precision ag equipment for targeted variable-rate inoculant applications.

Since then, the team has been ground-truthing the new method using field soils, with encouraging results.

Mr Ballard says they are now verifying the test using soil samples collected from paddocks rather than laboratory cultures and can reliably quantify rhizobia in soil with a high degree of specificity.

“Based on soil samples from 40 different paddocks in South Australia and Victoria, we have achieved an 80 per cent correlation with the plant-based bio-assay method, which is quite good for this type of test,” he says.

Practical benefits for growers

GRDC has now invested in an extension of the project to evaluate the test nationally. This will allow the researchers to calibrate the assay for cropping soils in New South Wales and Western Australia.

The researchers have also looked at the best method for sampling whole paddocks. They have found rhizobia levels are marginally higher in the crop row than the inter-row at the end of a season where a paddock was sown to wheat.

Initial studies have also shown rhizobia numbers are reasonably uniform within a soil type in a paddock. “This means growers could sample by soil type to create a rhizobia map for the paddock, then use precision ag equipment for targeted variable-rate inoculant applications,” Mr Ballard says.

“It will save them input costs in areas where rhizobia levels are high and the likelihood of inoculation response is low – and where rhizobia are absent or present in low numbers, inoculant rates can be increased to improve nodulation.”

Early prediction of rhizobia levels

Mr Ballard says the team is also investigating how early soil samples can be collected to predict rhizobia levels at legume seeding. “This recognises that growers plan their cropping programs up to a year in advance of sowing and prefer to order inoculants well before the legume is sown,” he says.

“We will investigate if rhizobia levels at harvest, early spring and even at seeding in the season before the legume crop is sown are useful predictors of the need to inoculate in the next season. Rhizobia populations can vary from nearly one million per gram of soil when crop biomass and soil moisture is high to as few as 100 individuals per gram at the end of summer, so we need to get it right.

“An added complication is that levels of rhizobia can decline rapidly in acidic soils. It is vital to validate our results before we start offering a service to growers. The cost of saying they won’t need to inoculate when they do, for example, could be very significant.”

Having established a successful DNA screening methodology for Rhizobia Group E/F, the team is looking forward to developing tests for the rhizobia that nodulate with other important legumes, including chickpeas, lupins and mungbeans.

“We will also explore whether the test can be used to test inoculant stored on the farm, in order to confirm its viability before use,” Mr Ballard says.

Further benefits from improved rhizobia research

The research team is equally enthusiastic about the new test’s potential to assist and accelerate rhizobia research. The method is currently being used to assist in the selection of new acid-tolerant rhizobia strains by measuring their colonisation of soils.

“Having a more efficient way of identifying and counting rhizobia will make it easier to measure the impact of agronomic practices on rhizobia populations and develop new strains,” Mr Ballard says. “In the end, the value of this test for research will probably provide even greater benefit to growers, through the faster development of rhizobia strains and improved inoculation practices.”

The outcomes of this project are being delivered via the strategic research partnership between GRDC and SARDI. The GRDC-SARDI partnership has facilitated a range of projects which provide innovative research outcomes relevant to South Australia’s cropping zones.

More information: PIRSA Media, pirsa.media@sa.gov.au