Evaluation of a Sclerotinia homoeocarpa Population with Multiple Fungicide Resistance Phenotypes Under Differing Selection Pressures

Dollar spot, caused by Sclerotinia homoeocarpa, is one of the most significant diseases of cool-season turfgrass on golf courses. Resistance to the benzimidazole, dicarboximide, and succinate dehydrogenase inhibitor (SDHI) classes and reduced sensitivity to the sterol-demethylation inhibitor (DMI) in S. homoeocarpa populations have been widely reported in the United States. Moreover, the occurrence of S. homoeocarpa populations with multiple fungicide resistance (MFR) is a growing problem on golf courses. The present study was undertaken to evaluate the efficacy of DMI, dicarboximide, and SDHI against a S. homoeocarpa population with MFR on a Connecticut golf course fairway from 2014 to 2016. Also, because the S. homoeocarpa population consisted of four different phenotypes with differing resistance profiles to benzimidazole, dicarboximide, and DMI, in vitro sensitivity assays were used to understand the dynamics of the MFR population in the presence and absence of fungicide selection pressures. Results indicated that boscalid fungicide (SDHI) was able to provide an acceptable control of the MFR dollar spot population. Propiconazole or iprodione application selected isolates with both DMI and dicarboximide resistance (DMI-R/Dicar-R). In the absence of fungicide selection pressures, the percent frequency of DMI-R/Dicar-R or DMI and benzimidazole resistance (DMI-R/Ben-R) isolates declined in the population. Out of the four phenotypes, the percent frequency of isolates with DMI, dicarboximide, and benzimidazole resistance (DMI-R/Dicar-R/Ben-R) was the lowest in the population regardless of fungicide selection pressures. Our first report of MFR population dynamics will help develop effective strategies for managing MFR and potentially delay the emergence of future resistant populations in S. homoeocarpa.

Molecular Mechanisms Involved in Qualitative and Quantitative Resistance to the Dicarboximide Fungicide Iprodione in Sclerotinia homoeocarpa Field Isolates

The dicarboximide fungicide class is commonly used to control Sclerotinia homoeocarpa, the causal agent of dollar spot on turfgrass. Despite frequent occurrences of S. homoeocarpa field resistance to iprodione (dicarboximide active ingredient), the genetic mechanisms of iprodione resistance have not been elucidated. In this study, 15 field isolates (seven suspected dicarboximide resistant, three multidrug resistance (MDR)-like, and five dicarboximide sensitive) were used for sequence comparison of a histidine kinase gene, Shos1, of S. homoeocarpa. The suspected dicarboximide-resistant isolates displayed nonsynonymous polymorphisms in codon 366 (isoleucine to asparagine) in Shos1, while the MDR-like and sensitive isolates did not. Further elucidation of the Shos1 function, using polyethylene glycol–mediated protoplast transformation indicated that S. homoeocarpa mutants (Shos1I366N) from a sensitive isolate gained resistance to dicarboximides but not phenylpyrrole and polyols. The deletion of Shos1 resulted in higher resistance to dicarboximide and phenylpyrrole and higher sensitivity to polyols than Shos1I366N. Levels of dicarboximide sensitivity in the sensitive isolate, Shos1I366N, and Shos1 deletion mutants were negatively correlated to values of iprodione-induced expression of ShHog1, the last kinase in the high-osmolarity glycerol pathway. Increased constitutive and induced expression of the ATP-binding cassette multidrug efflux transporter ShPDR1 was observed in six of seven dicarboximide-resistant isolates. In conclusion, S. homoeocarpa field isolates gained dicarboximide resistance through the polymorphism in Shos1 and the overexpression of ShPDR1.

Spatial Distribution of Single-Nucleotide Polymorphisms Related to Fungicide Resistance and Implications for Sampling

Spatial distribution of single-nucleotide polymorphisms (SNPs) related to fungicide resistance was studied for Botrytis cinerea populations in vineyards and for B. squamosa populations in onion fields. Heterogeneity in this distribution was characterized by performing geostatistical analyses based on semivariograms and through the fitting of discrete probability distributions. Two SNPs known to be responsible for boscalid resistance (H272R and H272Y), both located on the B subunit of the succinate dehydrogenase gene, and one SNP known to be responsible for dicarboximide resistance (I365S) were chosen for B. cinerea in grape. For B. squamosa in onion, one SNP responsible for dicarboximide resistance (I365S homologous) was chosen. One onion field was sampled in 2009 and another one was sampled in 2010 for B. squamosa, and two vineyards were sampled in 2011 for B. cinerea, for a total of four sampled sites. Cluster sampling was carried on a 10-by-10 grid, each of the 100 nodes being the center of a 10-by-10-m quadrat. In each quadrat, 10 samples were collected and analyzed by restriction fragment length polymorphism polymerase chain reaction (PCR) or allele specific PCR. Mean SNP incidence varied from 16 to 68%, with an overall mean incidence of 43%. In the geostatistical analyses, omnidirectional variograms showed spatial autocorrelation characterized by ranges of 21 to 1 m. Various levels of anisotropy were detected, however, with variograms computed in four directions (at 0°, 45°, 90°, and 135° from the within-row direction used as reference), indicating that spatial autocorrelation was prevalent or characterized by a longer range in one direction. For all eight data sets, the β-binomial distribution was found to fit the data better than the binomial distribution. This indicates local aggregation of fungicide resistance among sampling units, as supported by estimates of the parameter θ of the β-binomial distribution of 0.09 to 0.23 (overall median value = 0.20). On the basis of the observed spatial distribution patterns of SNP incidence, sampling curves were computed for different levels of reliability, emphasizing the importance of sample size for the detection of mutation incidence below the risk threshold for control failure.

Resistance to dicarboximide and benzimidazole fungicides in Botrytis cinerea from greenhouse tomatoes in New Zealand

Isolates from Botrytis cinerea collected from greenhouse tomato crops throughout New Zealand were tested for resistance to the dicarboximide fungicide vinclozolin and the benzimidazole fungicide carbendazim by assessing fungal growth on agar media amended with the fungicides (100 ppm active ingredient for both fungicides) Benzimidazole resistant strains of B cinerea were found on 15 of the 18 properties examined and dicarboximideresistant strains were found on 11 properties Dicarboximide resistance was more prevalent in the Auckland region with 71 of isolates resistant compared to 5 of isolates resistant for the other three regions All of the strains that were resistant to dicarboximide were also resistant to benzimidazole The high levels of dicarboximide resistant strains in the Auckland region correspond to reported loss of fungicide efficacy and loss of disease control in some greenhouses in this region

Fitness of Botrytis cinerea Associated with Dicarboximide Resistance

Fitness costs in Botrytis cinerea associated with dicarboximide resistance were studied. Spearman rank correlation coefficients were calculated between resistance to iprodione and survival ability both outside and inside the greenhouse, measured on isolates randomly chosen from a collection done in a survey of commercial greenhouses in Southeastern Spain in 1992. Survival was measured at 47, 83, and 110 days as percentage of surviving mycelia in a sample of artificially inoculated tomato stem pieces and as percentage of viable sclerotia from a sample of sclerotia produced on potato dextrose agar. Resistance to iprodione was measured by the fungicide concentration that reduces fungal growth by 50% (EC50 values). Significant (P < 0.05) negative correlation coefficients between survival of sclerotia and resistance to iprodione were found for some samplings dates, which indicates that sclerotia of resistant isolates survive less well than sclerotia from sensitive isolates. For mycelia, no relationship between survival and resistance was found.

Sensitivity of Botrytis cinerea from Connecticut Greenhouses to Benzimidazole and Dicarboximide Fungicides

Botrytis cinerea was isolated from infected plants in six greenhouses in Connecticut. Forty-five isolates were evaluated in vitro to determine fungicide sensitivity to benzimidazole (benomyl and thiophanate-methyl) and dicarboximide fungicides (vinclozolin and iprodione). B. cinerea isolates with fungicide resistance were recovered from each greenhouse sampled. Benzimida-zole resistance was more common than dicarboximide resistance (74 to 76% versus 36 to 43%, respectively). Multiple fungicide resistance was common. Nineteen isolates were resistant to both a benzimidazole and a dicarboximide fungicide. The level (EC50) of resistance to dicer-boximides was low compared with resistance to benzimidazoles. Isolate growth rate was not correlated to fungicide sensitivity or EC50. Fungicide resistance was apparently unrelated to the patterns of fungicide use in greenhouses sampled.