Understanding the basis of agrochemical resistance in biotrophic grapevine pathogens

Abstract

Powdery mildew (caused by Erysiphe necator), Botrytis bunch rot (Botrytis cinerea) and downy mildew (Plasmopara viticola) are significant diseases of grapevine, which are generally managed by growers with the use of agrochemicals. However pathogen resistance to a number of fungicides has been observed widely in Australian vineyards. The development of resistance leads to reduced efficacy and field failure of agrochemicals, causing significant impacts on crop quantity and quality. This project augmented the SARDI project SAR 1701-1.2 (Improving the understanding of fungicide resistance in Australian viticulture) by applying genome sequencing approaches and metagenomics to determine the genetic basis of resistance. Ultimately the aim was to link the scientific basis of resistance (e.g. through the identification of resistance genes) in the various fungal pathogens and the incidence of field failure for the various fungicide chemistries.

Summary

The development of resistance to agrochemicals is an ever-increasing problem in agriculture, and one for which the Australian wine sector is not immune. Results of a previous SARDI/AWRI research project have shown that there is widespread prevalence of resistance to many of the commonly used agrochemicals in the main fungal grapevine pathogens Erysiphe necator (powdery mildew), Plasmopara viticola (downy mildew) and Botrytis cinerea (bunch rot). The intention of this project was to isolate and obtain genomic DNA from phenotyped fungal isolates so that whole genome sequencing and assembly may be performed, and the genetic basis of resistance determined.

Significant project outcomes are summarised below:

• Generally, powdery mildew isolates showed reduced sensitivity to the selected fungicides:
  • Group 11, Quinone outside Inhibitors (QoIs). Reduced sensitivity, but no phenotype resistance was recorded at field rate.
  • Group 3, Demethylation Inhibitors (DMIs). No phenotype resistance was recorded at field rate of DMI fungicides.
  • Group 5, Spiroxamine. Most isolates were sensitive but no resistance was recorded for this group.
  • Group 13, proquinazid and quinoxyfen. 60% of the tested isolates were resistant to quinoxyfen, with the remainder having reduced sensitivity.
  • Group 7, boscalid and pydiflumetofen. Reduced sensitivity but no resistance detected at field rate.
  • Group 50 pyriofenone. Reduced sensitivity.
  • Group U6 cyflufenamid. All isolates tested were sensitive and no reduced sensitivity or resistance was detected.
• For botrytis bunch rot, low resistance frequency was found for groups 9, 12 and 17. Some new genotypes were characterised, with mutations identified that provide targets for future pPCR assays.
• For downy mildew, resistance of P. viticola at field rate was detected for QoI fungicides and PA, mostly in NSW and Vic. Reduced sensitivity was detected for groups 40, 45, 11 and group 4 (metalaxyl). It was recommended that PA and QoI fungicides be withdrawn from a spray program where resistance to metalaxyl and pyraclostrobin has been detected.
• Relationships between phenotype, genotype and field efficacy
  • Strong relationships between phenotyping and genotyping for group 11 (QoIs). Bioassay results matched with the presence of the mutant G143A, making it an ideal candidate for high-throughput and in-field testing.
  • The relationship between phenotyping and genotyping for group 3 (DMIs) in powdery mildew was weak and unclear as other factors could contribute to the resistance, such as gene copy number or other mutations. The Y136F mutant was detected in most of the isolates but the phenotyping results showed only limited reduction in sensitivity. More research required before reliable genotyping can be performed for DMI resistance.
  • The mutation linked to SDHI resistance was not detected in any E. necator populations, however reduced sensitivity was detected. Similarly, the G1105S mutant, linked to CAA (group 40) resistance, was not detected in any isolates of P. viticola, however reduced sensitivity was detected. Continued monitoring for SDHI and CAA resistance will be important.
• Mixing sulfur with three DMI fungicides (penconazole, myclobutanil and difenoconazole) had no effect on the their efficacy in controlling powdery mildew, under greenhouse conditions. This supports label recommendations for the use of sulfur.
• Strategies to manage fungicide resistance
  • Powdery mildew: monitoring (bioassays and/or genetic analysis), rotating fungicide chemistries, applying chemicals only when necessary, applying multi-site fungicides and other alternatives such as inorganic fungicides (sulfur) and avoiding chemicals that showed resistance in the pathogen populations.
  • Downey mildew: Avoid using metalaxyl and QoIs where resistance has been observed, in addition to continuing monitoring (bioassays and/or genetic analysis), rotating chemistries and applying farm biosecurity practices, by avoiding shared equipment and practicing hygienic agriculture practices.
  • Botrytis bunch rot: Low resistance frequency was observed during the project, but some heavily sampled regions showed elevated frequencies. Sound resistance management practices are required to maintain an overall low resistance frequency status and suppress any areas with elevated frequencies.