Page 278 - Plant Canada 2024 Proceeding

PLANT CANADA 2024

symptom induction, and interference of cellular components. Based on these results, vegetative
propagation of hop may be limited in its success of reducing HLVd spread with prior detection, suggesting
a need for further preventative action.

[P148] OPTICAL DENSITY ASSAY TO ASSESS THE SENSITIVITY OF PYTHIUM AND
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GLOBISPORANGIUM ISOLATES TO MEFENOXAM. Umbrin Ilyas , Lindsey J. du Toit , and Mary Ruth
McDonald . Department of Plant Agriculture, University of Guelph, Guelph, Ontario, Canada, N1G 2W1;
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and Department of Plant Pathology, Washington State University, Mount Vernon, WA, USA, 98273
Correspondence to: uilyas@uoguelph.ca

Cavity spot of carrot is an economically important disease caused by several species of Pythium and
Globisporangium. Disease management in conventional production primarily relies on applications of the
fungicide mefenoxam. Frequent reports in Ontario of severe disease even with mefenoxam applications
suggest prevalence of mefenoxam resistance in populations of the pathogen. The objectives of the study
were to (1) develop a fast and accurate optical density (OD) assay to assess mefenoxam sensitivity (2)
evaluate the sensitivity of Pythium and Globisporangium isolates to mefenoxam (3) determine if
mefenoxam sensitivity varies among species. OD assays are used routinely to screen for antibiotic
resistance in bacteria, but there are challenges with filamentous oomycetes. To account for variability in
growth using an OD assay, the growth was measured at nine positions per well in a 96-well plate at 600
nm, and the readings averaged. Z-scores (>0.4) and growth curves were used to confirm the isolate was
growing actively as isolates of different species grow at different rates. Validity of the method to assess
mefenoxam sensitivity was confirmed by comparing growth inhibition and half maximal effective
concentration (EC50) values from the OD assay with those from the standard assay of measuring colony
growth on mefenoxam-amended agar medium. A total of 188 isolates collected from 13 carrot fields in the
Holland Marsh, Ontario, in 2020-23 belonged to seven species: P. sulcatum, G. violae, G. rostratifingens,
G. intermedium, G. sylvaticum, G. irregulare, and G. ultimum. A 2-mm-diameter mycelial plug of each
isolate was transferred to mefenoxam-amended V8 broth with six concentrations (0.1 – 100 μg/mL). The
positive control treatment included five isolates of G. ultimum resistant to mefenoxam and one isolate of
this species sensitive at 10 μg/mL. The optimum time for determining mefenoxam sensitivity for slow-
growing species (<20 mm/day) was 72 hours, and for fast-growing (>20 mm/day) was 48 hours based on
the z-score and growth curves with the OD assay. There was a significant positive correlation (r= 0.94
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and 0.74) and linear relationship (R adj= 0.97 and 0.97) between EC50 and growth inhibition values
calculated for the OD and agar medium assay. All isolates tested were sensitive to mefenoxam at 10
μg/mL. The percentage inhibition at 1 μg/mL varied significantly among species, with isolates of G.
irregulare most sensitive, and isolates of P. sulcatum least sensitive. This optimized OD assay is an
efficient and accurate method to monitor mefenoxam resistance in filamentous oomycetes.

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