Project Number: 8030-12210-001-05-S
Project Type: Non-Assistance Cooperative Agreement
Start Date: Jul 1, 2018
End Date: Jun 30, 2019
1) Collect H. solani isolates from representative potato production areas. 2) Determine in vitro sensitivity of H. solani isolates to fungicides and compare the accuracy and precision of the spiral gradient method relative to traditional dilution plating. 3) Examine fungicide target genes in H. solani for specific mutations associated with reduced sensitivity to benzimidazole, QoI, SDHI and triazole fungicides in other fungi.
The H. solani isolates will be isolated from tubers with silver scurf from Maine, Michigan and Oregon and received from additional cooperators in the states of Idaho, New York, North Dakota, Washington and Wisconsin. This isolate collection will cover the geographical location of major potato production in the US. Monoconidial isolates from individual tubers will be obtained by isolating and sub-culturing a single conidium of H. solani onto clarified V8 (CV8) media amended with CaCO3. Subsequently, hyphal tipping will be used to obtain pure cultures of H. solani. Fungicides in sensitivity testing will include; Azoxystrobin, difenoconazole, fludioxonil, sedaxane, and solatenol. For dilution plating, fungicide stock solutions of 0.00, 0.01, 0.1, 1,10 and 100 mg/liter will be used to amend water agar. Isolate sensitivities will be expressed as relative germination or radial growth. Isolate sensitivity will be expressed in mg/liter and determined by the effective concentration in inhibiting spore germination or relative growth by 50% (EC50). For gradient plating, stock solutions of 1000 mg/liter of each fungicide will be prepared by dissolving commercial-grade fungicides in a sterile solvent, H2O or dimethyl sulfoxide (DMSO). The stock solution will be added to water agar across a gradient from 0 to 1000 ppm using a spiral gradient plater (Spiral Plate Biotechnology Inc.). Conidial suspensions (10 mL) will be spread across the fungicide gradient plate radially from edge to center. The point coordinates at which the colony growth starts and ends will be used to calculate the effective concentration for 50% growth inhibition (EC50) for each isolate for each of the fungicides. DNA sequencing of sensitive and insensitive isolates will be used to identify genetic markers responsible for fungicide resistance in H. solani. Subsequently, PCR based diagnostics will be used to detect mutations in target genes responsible for fungicide resistance in individual isolates. Additionally, PCR diagnostics will be used to detect mutations from H. solani isolates and directly from silver scurf lesions recovered on tuber tissue. Mutants resistant to the selected fungicides will be generated by plating conidial suspension on fungicide-amended agar. EC50 will be determined for each mutant. Risk assessment of fungicide resistance will be examined by measuring their frequency of mutation and fitness, including sporulation, mycelial growth under various temperatures, morphology, virulence of infection. Cross resistance will also be examined by the sensitivities of the mutants to fungicides that have same mode of action with the chemical used for mutant generation. The potential for alternative management practices will be assessed in greenhouse and growth chamber trials on plants (for effects on subsequent tubers) and tubers under storage conditions, as needed.