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.

Resistance of Sclerotinia homoeocarpa Field Isolates to Succinate Dehydrogenase Inhibitor Fungicides.

Sclerotinia homoeocarpa isolates were collected from golf courses in Japan and the United States (2016-2017). Japan isolates were collected during a monitoring study and the U.S. isolates were collected due to field failure. Five succinate dehydrogenase inhibitor (SDHI) active ingredients (boscalid, fluopyram, fluxapyroxad, isofetamid, and penthiopyrad) were examined using in vitro sensitivity assays to determine cross-resistance. Sequence analysis revealed a point mutation leading to an amino acid substitution (H267Y) and a silent mutation (CTT to CTC) at codon 181 in the SdhB subunit gene. Isolates with the B-H267Y (n = 10) mutation were resistant to boscalid and penthiopyrad and had increased sensitivity to fluopyram. SdhB silent mutation 181C>T isolates (n = 2) were resistant to boscalid, isofetamid, and penthiopyrad. Sequence analysis revealed 3 mutations leading to an amino acid substitution (G91R, n = 5; G150R, n = 1; G159W, n = 1) in the SdhC subunit gene. Isolates harboring the SdhC (G91R or G150R) mutations were resistant to boscalid, fluxapyroxad, isofetamid, and penthiopyrad. A genetic transformation system was used to generate mutants from a SDHI sensitive isolate to confirm the B-H267Y and C-G91R mutations were direct determinants of SDHI resistance and associated with in vitro sensitivity assay results.

Antifungal mechanisms of ZnO and Ag nanoparticles to Sclerotinia homoeocarpa.

Fungicides have extensively been used to effectively combat fungal diseases on a range of plant species, but resistance to multiple active ingredients has developed in pathogens such as Sclerotinia homoeocarpa, the causal agent of dollar spot on cool-season turfgrasses. Recently, ZnO and Ag nanoparticles (NPs) have received increased attention due to their antimicrobial activities. In this study, the NPs' toxicity and mechanisms of action were investigated as alternative antifungal agents against S. homoeocarpa isolates that varied in their resistance to demethylation inhibitor (DMI) fungicides. S. homoeocarpa isolates were treated with ZnO NPs and ZnCl2 (25-400 µg ml-1) and Ag NPs and AgNO3 (5-100 µg ml-1) to test antifungal activity of the NPs and ions. The mycelial growth of S. homoeocarpa isolates regardless of their DMI sensitivity was significantly inhibited on ZnO NPs (≥200 µg ml-1), Ag NPs (≥25 µg ml-1), Zn2+ ions (≥200 µg ml-1), and Ag+ ions (≥10 µg ml-1) amended media. Expression of stress response genes, glutathione S-transferase (Shgst1) and superoxide dismutase 2 (ShSOD2), was significantly induced in the isolates by exposure to the NPs and ions. In addition, a significant increase in the nucleic acid contents of fungal hyphae, which may be due to stress response, was observed upon treatment with Ag NPs using Raman spectroscopy. We further observed that a zinc transporter (Shzrt1) might play an important role in accumulating ZnO and Ag NPs into the cells of S. homoeocarpa due to overexpression of Shzrt1 significantly induced by ZnO or Ag NPs within 3 h of exposure. Yeast mutants complemented with Shzrt1 became more sensitive to ZnO and Ag NPs as well as Zn2+ and Ag+ ions than the control strain and resulted in increased Zn or Ag content after exposure. This is the first report of involvement of the zinc transporter in the accumulation of Zn and Ag from NP exposure in filamentous plant pathogenic fungi. Understanding the molecular mechanisms of NPs' antifungal activities will be useful in developing effective management strategies to control important pathogenic fungal diseases.

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.

Overexpression of ShCYP51B and ShatrD in Sclerotinia homoeocarpa isolates exhibiting practical field resistance to a demethylation inhibitor fungicide.

We investigated genetic factors that govern the reduced propiconazole sensitivity of Sclerotinia homoeocarpa field isolates collected during a 2-year field efficacy study on dollar spot disease of turf in five New England sites. These isolates displayed a >50-fold range of in vitro sensitivity to a sterol demethylation inhibitor (DMI) fungicide, propiconazole, making them ideal for investigations of genetic mechanisms of reduced DMI sensitivity. The CYP51 gene homolog in S. homoeocarpa (ShCYP51B), encoding the enzyme target of DMIs, is likely a minor genetic factor for reduced propiconazole sensitivity, since there were no differences in constitutive relative expression (RE) values and only 2-fold-higher induced RE values for insensitive than for sensitive isolate groups. Next, we mined RNA-Seq transcriptome data for additional genetic factors and found evidence for the overexpression of a homolog of Botrytis cinerea atrD (BcatrD), ShatrD, a known efflux transporter of DMI fungicides. The ShatrD gene showed much higher constitutive and induced RE values for insensitive isolates. Several polymorphisms were found upstream of ShatrD but were not definitively linked to overexpression. The screening of constitutive RE values of ShCYP51B and ShatrD in isolates from two golf courses that exhibited practical field resistance to propiconazole uncovered evidence for significant population-specific overexpression of both genes. However, linear regression demonstrated that the RE of ShatrD displays a more significant relationship with propiconazole sensitivity than that of ShCYP51B. In summary, our results suggest that efflux is a major determinant of the reduced DMI sensitivity of S. homoeocarpa genotypes in New England, which may have implications for the emergence of practical field resistance in this important turfgrass pathogen.

The Association Between In Vitro Propiconazole Sensitivity and Field Efficacy of Five New England Sclerotinia homoeocarpa Populations.

Dollar spot (Sclerotinia homoeocarpa) is a major turfgrass disease requiring fungicide application to maintain acceptable conditions for golf. A 2-year field experiment was conducted to determine the association between field efficacy of propiconazole and in vitro fungicide sensitivity of isolates from five S. homoeocarpa populations. Four golf courses with prior propiconazole exposure (Hartford Golf Club, Hickory Ridge Country Club, Shuttle Meadow Country Club, and Wintonbury Hills Golf Club), and a baseline site with no prior propiconazole exposure (Joseph Troll Turf Research Facility) were chosen as field sites. Experimental plots at each site received the following treatments at 21-day intervals: untreated, propiconazole (0.44, 0.88, 1.32, and 1.76 kg a.i. ha-1), and chlorothalonil (8.18 kg a.i. ha-1). S. homoeocarpa isolates were sampled at three time points during 2009 and 2010: initial (directly before fungicide treatment), 7 days after treatment (DAT), and 21 days after the last treatment. Isolates sampled from dollar spot infection centers at 7 DAT (2009 and 2010) were considered to exhibit "practical field resistance". In parallel, S. homoeocarpa isolates from each site were assayed for in vitro sensitivity to propiconazole by determining relative mycelium growth percentages (RMG%) on potato dextrose agar amended with propiconazole at a discriminatory concentration of 0.1 µg a.i. ml-1. S. homoeocarpa isolates from the four exposed populations displayed significantly higher RMG% values than the baseline population. In general, field efficacy at all propiconazole rates tested was lower at the four locations with prior propiconazole exposure when compared with the baseline population. Increased RMG% values on the propiconazole discriminatory concentration 0.1 µg a.i. ml-1 were associated with decreased relative control values for all propiconazole rates in 2009 and 2010. Results suggest RMG values above 50% at the propiconazole discriminatory concentration of 0.1 µg a.i. ml-1 may be a suitable threshold for detection of S. homoeocarpa isolates that cause practical DMI field resistance.

In vitro Assessment of Sclerotinia homoeocarpa Resistance to Fungicides and Plant Growth Regulators.

Dollar spot (caused by Sclerotinia homoeocarpa) is the most economically important turfgrass disease in North America. This disease is primarily controlled by fungicide applications on golf courses; however, fungicide resistance has been confirmed in three of the four systemic fungicide classes commonly used to control dollar spot. The main objective of this study was to evaluate S. homoeocarpa sensitivity to multiple chemical classes and cross-resistance among active ingredients within the same class; in particular, the association between the fungistatic effect of demethylation inhibitors (DMIs) and plant growth regulators (PGRs). Fifty-eight isolates were selected arbitrarily from four locations in the United States and assayed for in vitro sensitivity to six DMI, two dicarboximide, one carboximide, and one benzimidazole fungicide as well as three type II PGRs. A series of concentrations for each active ingredient was used to determine the mean 50% effective concentration (EC50) values and correlation coefficients were calculated for all active ingredients. The EC50 values of all active ingredients from the DMI class were highly correlated (P < 0.0001) to each other as well as to the one dicarboximide (iprodione) and two PGRs (flurprimidol and paclobutrazol). Isolates resistant to thiophanatemethyl had significantly higher EC50 values than sensitive isolates for all active ingredients assayed except for boscalid. Findings showed that multiple and cross-resistance has developed in S. homoeocarpa and that the two PGRs have a fungistatic effect on this pathogen similar to that of DMI fungicides. The high correlation of in vitro sensitivities among PGRs and DMI fungicides further suggests that PGRs may contribute to the selection of DMI-resistant isolates or facilitate decreased sensitivity to DMI fungicides in the field.

A pleiotropic drug resistance transporter is involved in reduced sensitivity to multiple fungicide classes in Sclerotinia homoeocarpa (F.T. Bennett).

Dollar spot, caused by Sclerotinia homoeocarpa, is a prevalent turfgrass disease, and the fungus exhibits widespread fungicide resistance in North America. In a previous study, an ABC-G transporter, ShatrD, was associated with practical field resistance to demethylation inhibitor (DMI) fungicides. Mining of ABC-G transporters, also known as pleiotropic drug resistance (PDR) transporters, from RNA-Seq data gave an assortment of transcripts, several with high sequence similarity to functionally characterized transporters from Botrytis cinerea, and others with closest blastx hits from Aspergillus and Monilinia. In addition to ShatrD, another PDR transporter showed significant over-expression in replicated RNA-Seq data, and in a collection of field-resistant isolates, as measured by quantitative polymerase chain reaction. These isolates also showed reduced sensitivity to unrelated fungicide classes. Using a yeast complementation system, we sought to test the hypothesis that this PDR transporter effluxes DMI as well as chemically unrelated fungicides. The transporter (ShPDR1) was cloned into the Gal1 expression vector and transformed into a yeast PDR transporter deletion mutant, AD12345678. Complementation assays indicated that ShPDR1 complemented the mutant in the presence of propiconazole (DMI), iprodione (dicarboximide) and boscalid (SDHI, succinate dehydrogenase inhibitor). Our results indicate that the over-expression of ShPDR1 is correlated with practical field resistance to DMI fungicides and reduced sensitivity to dicarboximide and SDHI fungicides. These findings highlight the potential for the eventual development of a multidrug resistance phenotype in this pathogen. In addition, this study presents a pipeline for the discovery and validation of fungicide resistance genes using de novo next-generation sequencing and molecular biology techniques in an unsequenced plant pathogenic fungus.