Key messages

• A method has been developed for field-evaluation of sclerotinia stem rot (SSR) resistance in Brassica napus.
• Using this technique, SSR-resistant Australian B. napus breeding lines were found to be more resistant than the canola cultivar ATR Bonito, suggesting they may be useful for breeding.
• ATR Bonito was found to be more resistant than the cultivar HyTTec® Trophy, suggesting a range of susceptibilities in current cultivars.
• Develop a scalable technique for in-field evaluation of SSR resistance of B. napus varieties.
• Use it to assess cultivars and candidate SSR-resistant breeding lines in the field.

Aims

• Develop a scalable technique for in-field evaluation of SSR resistance of B. napus varieties.
• Use it to assess cultivars and candidate SSR-resistant breeding lines in the field.

Introduction

SSR is a canola disease caused by the fungus Sclerotinia sclerotiorum. It is challenging to manage using rotation because of the wide host-range of S. sclerotiorum. Since there are no Australian canola cultivars with SSR resistance, SSR is usually managed with preventative fungicide sprays at flowering. However, fungicides can be costly and, because SSR is a sporadic disease, their return on investment is difficult to predict.

Canola cultivars partially resistant to SSR would improve its management. A few such cultivars are available in Canada and the UK, but not Australia. Resistant material and scalable methods for evaluating plants in the field are required to breed SSR-resistant cultivars.

We previously presented a novel method for inoculating B. napus with S. sclerotiorum in the field. In published research, we have also identified three Australian B. napus breeding lines with stem resistance to SSR in glasshouse and controlled environments. However, these lines have not been evaluated using a whole plant field inoculation method, and our current method requires refinement.

Method

Based on previous research, we used ground red rice as a carrier for S. sclerotiorum mycelium. This method was selected for its low cost, reliability and ease of inoculum application. Briefly, 1 kg of red rice, 500ml of potato dextrose broth (PDB) and 500ml of distilled water were added to an autoclave bag and left to soak for four hours at 4°C. Excess liquid was drained, then the bag was sealed and autoclaved twice for 20 minutes at 121°C. Two 8mm potato dextrose agar (PDA) plugs of each of four genotypically distinct S. sclerotiorum isolates from WA were added to the bag and incubated for eight days at 22°C. Resulting inoculum was dried on sterilised trays at 32°C for six days, with daily stirring. Inoculum was then processed to a finer consistency with a Nutri-bullet 900 watt blender for 10 seconds and stored at 4°C until required.

Two field trials were conducted near Northam, WA. Field trial 1 (FT1) included the cultivars ATR Bonito and HyTTec® Trophy. Field trial 2 (FT2) included ATR Bonito, the Australian B. napus breeding lines Chisaya, Chikuzen and Norin 28, and the historical Australian cultivar Mystic (released in 1999). Chisaya, Chikuzen and Norin 28 were previously shown to develop smaller lesions than ATR Bonito when their stems were inoculated with PDA plugs carrying S. sclerotiorum in a glasshouse or controlled environment. Using the same technique, Mystic was found to be the most resistant historical Australian cultivar.

Three replicate plots per treatment and variety were sown for both trials. Plants were inoculated at 30% flowering by spreading the red rice inoculum over plots at a rate of 15 g/m2. A single inoculation was applied to FT1, while FT2 had two inoculations two weeks apart. While FT2 was uniformly irrigated to maintain high humidity, FT1 plots had either no irrigation or misting, or both irrigation and misting. Disease severity was assessed using a 0–5 rating scale: 0 = no symptoms; 1 = leaf and/or pod symptoms; 2 = small superficial stem lesion, <10% branches with lesions; 3 = large stem lesion; 4 = ~50% main stem girding, leaves wilted; 5 = main stem girdled, plant collapse and/or lodging. A cumulative link mixed model determined statistical differences using the “ordinal” package in R.

Results

In FT1 there was a 0–7% incidence of natural SSR in uninoculated plots. Irrigation and misting did not significantly impact disease incidence or severity, so were ignored in statistical models. Plots inoculated with red rice carrier had significantly higher SSR incidence than uninoculated plots (P<0.001). HyTTec® Trophy had a higher SSR incidence than ATR Bonito (P<0.001) and a greater increase in SSR incidence compared to uninoculated plots when inoculated with the red rice carrier (P<0.01). Though disease incidence increased from uninoculated to inoculated plots, average severity declined from 3.9 in uninoculated to 3.5 in inoculated plants. ATR Bonito plants had an average disease severity of 3.4, which was lower the average of 3.8 for HyTTec® Trophy.

These data show the reliability of the red rice carrier technique for producing consistent disease pressure in the field and highlight a potential difference in SSR susceptibility between two current Australian cultivars. This warrants further exploration of the range of SSR susceptibilities in canola.

In FT2, Chisaya, Chikuzen and Norin 28 flowered earlier than Mystic. The SSR severity of these varieties was assessed relative to the first planting of ATR Bonito. The SSR severity of Mystic was assessed relative to the second planting of ATR Bonito in separate statistical models. These models did not detect any significant differences in SSR severity between Mystic and ATR Bonito. Here, we present the full results relative to ATR Bonito for Chikuzen, Chisaya, and Norin 28. All results are based on scores assessed 28 days post-inoculation (DPI).

Based on the full dataset, there was no significant difference in average SSR severity between plants inoculated once or twice (P=0.85). Chikuzen and Chisaya, but not Norin 28, had significantly lower average SSR severity than ATR Bonito (P<0.0001). Average SSR severity in FT2 was 3.05, 2.49, 2.63 and 2.91 for ATR Bonito, Chikuzen, Chisaya and Norin 28, respectively.

A single statistical block in FT2 had excessively high SSR scores for plots containing Chisaya and Norin 28 after two inoculum applications. These plots were outliers compared with others, which had much lower SSR severity. Since these plots were on the edge of the trial in an exposed area, they underwent severe lodging in the windy conditions in the two weeks between inoculations. This likely artificially inflated SSR severity scores.

Excluding this block showed a significant decrease in average SSR severity in Chikuzen, Chisaya and Norin 28 relative to ATR Bonito (P<0.0001). Average SSR severity across plants was 3.12, 2.46, 2.50 and 2.66 for ATR Bonito, Chikuzen, Chisaya and Norin 28, respectively. This suggests that these varieties may be useful for SSR resistance breeding. Further testing for Norin 28 may be warranted given scoring inconsistencies.

Conclusion

Our field inoculation method has been assessed in two years and multiple locations, demonstrating its scalability and consistency. We have shown potential differences in SSR susceptibility among current canola cultivars and useful levels of SSR resistance in Australian B. napus breeding lines. Since resistant lines were identified in previous screens using agar plug stem inoculations in glasshouse and controlled environment experiments, this study suggests some consistency between controlled stem infections and whole plant field inoculations.

Acknowledgments

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.

Contact details

Mark Derbyshire
Centre for Crop and Disease Management
Curtin University, Kent Street, Bentley, WA 6102
08 9266 0365
mark.derbyshire@curtin.edu.au