Location: Horticultural Crops Disease and Pest Management Research Unit
Project Number: 2072-22000-045-010-G
Project Type: Grant
Start Date: Sep 1, 2021
End Date: Oct 31, 2024
Objective:
1. Phenotype sensitivity of Botrytis spp. isolates to site-specific fungicides with field-level resolution.
2. Determine virulence of Botrytis sp. isolates with single and multi-fungicide resistance.
Approach:
Objective 1: We will sample at least 40 blueberry fields (conventional and organic farms) in western Washington and Oregon. Metadata pertaining to each field such as GPS location, cultivar, age of the planting, and fungicide usage will be collected from the grower. Sampling will be done twice (early and late season) and the tissue to be sampled will be either twig, flower, or fruit. Samples with Botrytis-like conidiophores will be transferred to PDA and then single spored. Asymptomatic samples will be processed as described in Amiri et al. (2018a) to enhance sporulation and pure cultures will be obtained. All Botrytis spp. will be identified to the species level by PCR using standard molecular markers (Plesken et al. 2015; Saito et al. 2016). Fungal stock cultures will be made and stored at -80' until further usage.
Technical-grade fungicides containing a single active ingredient will be added to standard media for resistance assays. Salicylhydroximic acid (SHAM) will be added to media to inhibit the pathogen’s alternative oxidase pathway when testing for resistance to FRAC 11. Previously developed discriminatory fungicide dosages will be used for conducting fungicide sensitivity assays (Weber and Hahn 2011). Freshly harvested fungal spores will be added to different fungicide-amended media in wells of 24-well plates and incubated at 22' and radial diametric colony growth will be measured over four days. The experiment will be repeated twice. Each isolate will be classified as sensitive or resistant based on percent colony growth compared to the well diameter as done by Cosseboom et al. (2019). Botrytis spp. populations will then be categorized according to the number of FRAC classes they were resistant to. ANOVA will be used to compare average nCCR from early and late sampling times.
Objective 2: Detached fruit assays will be conducted based on protocols developed by Saito et al. (2016). Briefly, organically grown disease-free blueberry fruits will be surface disinfested, and sprayed with uniform quantity of commercial-grade fungicides belonging to different FRAC classes. Fungicide label recommended dosages will be followed for these assays. Fruits treated with sterile water will serve as controls. After four hours of drying, fruits will be inoculated with a subset of Botrytis spp. isolates that have either single or multiple chemical class resistances. The fruits will be wounded using a sterile toothpick and then inoculated with a spore concentration adjusted to 1 X 105 conidia/mL. Inoculated fruits will be incubated in a humid container for 5 days at 20' in the dark. Disease severity will be estimated using a 0 to 4 severity scale and disease incidence will be calculated by dividing number of fruits exhibiting disease symptoms by the total number of fruits assessed. All experiments will be repeated twice. ANOVA will be conducted to test differences among isolates for disease incidence and severity values. A Tukey’s highly significant difference test will be used to compare means of each treatment and each isolate.
