Ice nucleating bacteria (INB) can promote ice growth more effectively than any other known material but, until recently, their role in causing frost damage in grain crops has been little-researched.

A three-year co-investment by the Council of Grain Grower Organisations and the Western Australian Department of Primary Industries and Regional Development (DPIRD), supplemented with another one-year project supported through the WA Government’s Royalties for Regions program (Boosting Grains R&D) has made headway into the involvement of bacteria in frost severity.

The research, carried out under the mentorship of Dr Ben Biddulph and led by Dr Amanuel Bekuma from DPIRD, has begun to unravel the part these bacteria could play in causing frost damage to crops.

Dr Bekuma, originally from Ethiopia, brings a thorough understanding of plant-microbe interactions to the project from his AusAid-supported PhD undertaken at Murdoch University, together with experience in conducting large-scale field trials. He now leads the DPIRD frost team together with Brenton Leske, building on the work of Dr Ben Biddulph.

“Over the years of research that Dr Biddulph had conducted extensive frost field trials, he had seen patches and trails of more-severe frost damage in paddocks and was starting to consider that not only was it an abiotic stress, but something else biologically was going on,” Dr Bekuma says.

The possible role of INB in causing frost damage, particularly in horticultural crops, has been known for decades. But we now have new technology, together with extensive dedicated field facilities, at hand to examine its role in grains in more detail.

INB, such as Pseudomonas syringae, can be present in rainfall prior to frost events associated with weak frontal systems or in crop residue and dispersed by rainfall or be actively growing on the plant canopy. In the latter case, older leaves at the lower canopy have more INB than green and healthy leaves.

Compared to situations where stubble is removed, stubbles retained or applied during the growing season further exacerbate frost risk by potentially increasing the INB population that initiate freezing from the ground to canopy level.

“We know that wheat varieties differ in their susceptibility to frost, but we were interested to find out if this may be related to genetic differences in the plants’ ability to host INB, as we thought this may be a logical first stage of approach,” Dr Bekuma says.

“We have been able to demonstrate that the presence of a bacterial ice nucleating protein significantly increased frost damage in field-grown wheat at the DPIRD Dale frost nursery, so the next step was to investigate if there is a wheat variety by INB interaction.”

For the purpose of this DPIRD-supported study, seed samples of four wheat varieties – Young , Wyalkatchem, LRPB Scout and Cutlass – differing in response to frost ranging from susceptible to tolerant, respectively, were sown at the frost nursery at different times of sowing during the 2018 and 2019 field season.

Seeds were harvested from all plots at maturity, threshed, cleaned and stored in a cold room at 4°C. Detailed methodology, using the latest molecular techniques, was developed to extract, replicate and quantify the amount of INB from each time-of-sowing by year treatment.

Frosted grains harbour more INB

In both 2018 and 2019, the proportion of grains affected by frost was higher when the wheat was sown early (see Figures 1a and b). INB populations were found to be higher in seed samples from earlier times of sowing (April-sown) compared to late-sown (around mid-May) wheat in both years.

Figure 1: The percentage of frosted grains and the number of ice nucleation bacteria (INB) present on seeds of four wheat genotypes grown at Dale, WA in (a) 2018 and (b) 2019 for five times of sowing (from April to mid-May). The data is log transformed for variance stabilisation. Error bars indicate the mean ± SEM.

Source: DPIRD

“Early sowing usually increases the likelihood of overlapping frost events with the susceptible stage of wheat plants. For all varieties, the 2018 samples had a higher INB population compared to the 2019 samples, which corresponds to the greater proportion of frosted grains for these varieties,” Dr Bekuma says.

“At present, the correlation between INB population and percentage of frosted grain is not understood. For instance, it is not clear if the presence of INB on the seed caused the frost or if frost-affected seeds provide favourable conditions for opportunistic INB to flourish.”

Grains that are frosted during early grain fill have a typical pinched appearance with creases along the seed axis, and this crease may provide habitat for the bacteria. Frosted grains also have a high sugar content, potentially serving as a nutrient source for INB and other microbes. The cause and effect of the presence of INB on frosted grains needs to be investigated further.

Grain frost seems to be correlated with (or associated with higher) INB colonisation on the seed surface, but the number of INB populations varies depending on wheat genotypes and the severity of the frost damage. For instance, Wyalkatchem showed high levels of INB present on the grain when exposed to a moderate level of frost damage and there were significantly fewer INB present on Young under the same conditions.

“An increased level of sugars in frosted grains and plant tissue in response to cold exposure might hold the key to understanding the link between INB colonisation and genetic tolerance. The role of seed-borne INB in repopulating the subsequent crop and sustaining the frost cycle for the next crop remains to be investigated. In the meantime, it would be safer to avoid keeping frosted grain for seeding purposes to reduce frost risk.”

Future research

This three-year study has just begun to unravel the role that INB may have in causing frost in grain crops.

“We need to expand our dataset and verify the extent to which different wheat varieties may host INB,” Dr Bekuma says.

We need also to examine seed treatment effects on INB and the effect the type of grain storage may have on the amount of INB hosted on grain samples in an on-farm setting.

“To be able to support growers in their management decisions for frost, we need to further investigate potential management interventions for INB and their application methods.”

Dr Amanuel Bekuma, 0402 634 366, amanuel.bekuma@dpird.wa.gov.au

Read more: GRDC Update Paper - Wheat genotypes differ in seed-borne ice nucleating bacterial (INB) population.