The role of ice nucleating bacteria in frost sensitivity of cereals in Western Australia

Overall Objective

The aim of the project was to determine the occurrence and impact of ice nucleating-active bacteria (INB) on severity of frost damage in cereal crops grown in frost prone areas of Western Australia.

Project Synopsis

Frost related damage to Australian cereal crops is estimated between $360 and $700 million each year. Apart from the economic loss, it takes a heavy toll on emotional well-being of the growers. In the WA grain production system, frost damage could be relatively minor at -2^oC. However, severe frost damage has been observed when light showers in the afternoon or evening precedes a frost event, even though the temperatures are not substantially colder. One possibility that remained unexplored in the WA production system, was whether small rain events on the plant canopy played a role in exacerbating frost severity in cereal crops. The project aimed to determine the occurrence and impact of ice nucleation-active bacteria (INB) on severity of frost damage in cereal crops grown in frost prone areas of Western Australia.

To determine the occurrence and impact of ice nucleation bacteria population, a series of field and lab-based experiments were conducted using microbiological and molecular techniques, thermal imagery and measured agronomic traits including grain yield. Pseudomonas syringae strains containing the ice nucleating gene were isolated and identified. The ice nucleating activity test evaluated the temperature at which a given sample initiates ice formation as measured by droplet freezing assay.  A qPCR system was developed in a separate internally funded DPIRD project to allow in-field quantification of ice nucleating gene contained in plant tissue samples

A total of ten bacterial strains in genus Pseudomonas were isolated and identified from different crop residues of cereal and legumes crops as well as from rainwater collected across the WA wheatbelt. Through DNA sequencing, these strains were found to contain the ice nucleating gene, which produces the ice nucleation protein required to initiate freezing of plants at warmer sub-zero temperatures around -2^oC

The test on samples collected during three growing seasons (2019–2021) have consistently confirmed that stubble and older senescing/senesced leaves of wheat plants, showed higher ice nucleation activity consistent with the presence of high number of INB. Ice nucleating bacteria were found in much lower numbers on the green, healthy upper canopy leaves. Unfortunately, peak INB activity of older leaves and stubble overlaps with the most frost susceptible stage of wheat (heading and flowering) which also coincides with a wetter and colder time of the growing season when spring frost occurs.

In addition, high nucleation activity was found in some of the spring rainfall events (<10 mm). Initially, it was hypothesised that such rainfall events that occurred prior to a frost event (temperature less than 2°C) on a crop in stubble retained system, led to increased frost damage due to greater biological ice nucleation activity earlier in the night resulting in more terminal damage. Consequently, a field experiment to understand how late afternoon or evening rainfall, as well as stubble retention, exacerbated frost damage in wheat was conducted at Dale, WA. Simulated rainfall events containing high ice nucleation activity, were applied using a commercial ice nucleating protein to wheat plots at flowering stage prior to a predicted frost event. In-field thermography in these plots indicated freezing of multiple heads and flag leaves almost at the same time. The observation sheds light on a likely mechanism by which a late afternoon shower with high ice nucleation activity increases flower sterility in wheat. Field trials from two seasons (2020–2021) using a dose response of the ice nucleation activity samples, confirmed that the floret sterility in these plots showed a significant increase in frost damage, compared to the sterile water by itself.

Further thermal imagery taken during a frost event, from a flowering wheat crop grown under a stubble retention system allowed visualisation of ice initiation and movement along the wheat canopy. Ice nucleation started first on the crop residue in the inter-row, before moving to the older senesced leaves and up to the plant leaf sheath, stems and reproductive tissue. This observation was consistent with the higher ice nucleation activity measured in the stubble and older leaves of wheat crops reported in this project.

In conclusion, uncovering the major role of INB in increasing the severity of frost damage in cereal crops in WA has brought a step change in the way in which frost research is approached. These findings and findings in the last 15 years of natural frost have laid the foundation for further research work that will focus on the management of ice nucleating bacteria in the cereal cropping system through agronomic and/or genetic solutions. Such efforts can potentially benefit growers by reducing frost damage which has been estimated to cost Australian growers between $360 and $700 million in direct and indirect yield losses every year.