Field Assessment of Six Point-Mutations in SDH Subunit Genes Conferring Varying Resistance Levels to SDHIs in Clarireedia spp.

Dollar spot, caused by Clarireedia spp. (formerly Sclerotinia homoeocarpa F.T. Bennett), is the most economically important turfgrass disease causing considerable damage on golf courses. While cultural practices are available for reducing dollar spot infection, chemical fungicide use is often necessary for maintaining optimal turf quality. Since the release of boscalid in 2003, the succinate dehydrogenase inhibitor (SDHI) class has become an invaluable tool for managing dollar spot. However, resistance to this class has recently been reported in Clarireedia spp. and many other plant pathogenic fungi. After SDHI field failure on four golf courses and one university research plot, a total of six unique SDH mutations conferring differential in vitro sensitivities to SDHIs have been identified in Clarireedia spp. In 2018 and 2019, turf research plots were inoculated with sensitive, non-mutated isolates of Clarireedia spp., as well as resistant isolates harboring each unique identified mutation. Fungicide efficacy trials were conducted on inoculated plots to assess differential sensitivity to five SDHI active ingredients (boscalid, fluxapyroxad, isofetamid, fluopyram, and pydiflumetofen) across mutations under field conditions. Results indicate unique mutations are associated with distinct SDHI field efficacy profiles as shown in in-vitro sensitivity assays. Isolate populations with B subunit mutations (H267Y/R) were more sensitive to fluopyram, whereas isolate populations with C subunit mutations (C-G91R, C-G150R) showed resistance to all SDHIs tested. Mutation-associated differential sensitivity observed under field conditions indicates a need for a nation-wide survey and frequent monitoring of SDHI sensitivity of dollar spot populations on golf courses in the USA. Further, the information gained from this study will be useful in providing sustainable management recommendations for controlling site-specific resistant populations of Clarireedia spp.

A Method for the Examination of SDHI Fungicide Resistance Mechanisms in Phytopathogenic Fungi Using a Heterologous Expression System in Sclerotinia sclerotiorum.

Succinate dehydrogenase inhibitors (SDHIs) are a class of broad-spectrum fungicides used for management of diseases caused by phytopathogenic fungi. In many cases, reduced sensitivity to SDHI fungicides has been correlated with point mutations in the SdhB and SdhC target genes that encode components of the succinate dehydrogenase complex. However, the genetic basis of SDHI fungicide resistance mechanisms has been functionally characterized in very few fungi. Sclerotinia sclerotiorum is a fast-growing and SDHI fungicide-sensitive phytopathogenic fungus that can be conveniently transformed. Given the high amino acid sequence similarity and putative structural similarity of SDHI protein target sites between S. sclerotiorum and other common phytopathogenic ascomycete fungi, we developed an in vitro heterologous expression system that used S. sclerotiorum as a reporter strain. With this system, we were able to demonstrate the function of mutant SdhB or SdhC alleles from several ascomycete fungi in conferring resistance to multiple SDHI fungicides. In total, we successfully validated the function of Sdh alleles that had been previously identified in field isolates of Botrytis cinerea, Blumeriella jaapii, and Clarireedia jacksonii (formerly S. homoeocarpa) in conferring resistance to boscalid, fluopyram, or fluxapyroxad and used site-directed mutagenesis to construct and phenotype a mutant allele that is not yet known to exist in Monilinia fructicola populations. We also examined the functions of these alleles in conferring cross-resistance to more recently introduced SDHIs including inpyrfluxam, pydiflumetofen, and pyraziflumid. The approach developed in this study can be widely applied to interrogate SDHI fungicide resistance mechanisms in other phytopathogenic ascomycetes.

A Rapid Molecular Detection System for SdhB and SdhC Point Mutations Conferring Differential Succinate Dehydrogenase Inhibitor Resistance in Clarireedia Populations.

Dollar spot, caused by the ascomycete fungus Clarireedia (formerly Sclerotinia), is one of the most resource-demanding diseases on amenity turfgrasses in North America. Differential resistance to the succinate dehydrogenase inhibitor (SDHI) fungicide class, conferred by singular point mutations on the SdhB, SdhC, and SdhD subunits of the succinate dehydrogenase enzyme (SDH), has been reported in dollar spot as well as many other plant-pathogenic fungal diseases. Four unique mutations were previously reported from Clarireedia field isolates collected from two different cool-season golf courses in Japan and Rhode Island: an amino acid substitution H267Y and a silent mutation (CTT to CTC) at codon 181 on the SdhB subunit gene, and amino acid substitutions G91R and G150R on the SdhC subunit gene. To properly diagnose and monitor SDHI resistance in the field, a rapid detection system for known mutations is crucial. As part of this study, additional SDHI-resistant Clarireedia isolates were collected from Rutgers University research plots and in vitro sensitivity to four SDHI active ingredients was assessed. SdhB, SdhC, and SdhD subunits of these isolates were sequenced to reveal an additional mutation on the SdhB subunit gene, H267R, not previously observed in Clarireedia. Cleaved amplified polymorphic sequence (CAPS) and derived CAPS molecular markers were developed to detect five mutations conferring SDHI resistance in Clarireedia isolates and validated using samples from two additional golf courses in Connecticut and Wisconsin experiencing SDHI field failure. This PCR-based molecular detection system will be useful for continued monitoring, assessment, and delay of SDHI resistance in the field.

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.

Chlorothalonil biotransformation by cytochrome P450 monooxygenases in Sclerotinia homoeocarpa.

Cytochrome P450s have been shown to play a vital role in the xenobiotic detoxification system of Sclerotinia homoeocarpa, the causal agent of the turfgrass disease dollar spot. A previous study indicated that three CYP450s were validated to play a functional role in resistance against different fungicide classes including propiconazole and plant growth regulator, flurprimidol. In this study, we present these CYP450s possess the capability to modify the multi-site mode of action fungicide chlorothalonil. Chlorothalonil is an extensively used contact fungicide and has been shown to persist in soils. High Performance Liquid Chromatography (HPLC) indicated faster rates of chlorothalonil biotransformation by CYP561 and CYP65 overexpression strains when compared to the wild-type and CYP68 overexpression strain. Our GC-MS results show that the primary transformation intermediate found in soils, 4-hydroxy-2,5,6 trichloro-isophthalonitrile is produced by CYP450s' metabolism. These findings suggest fungal CYP450s can biotransform chlorothalonil for biodegradation or detoxification.

A Xenobiotic Detoxification Pathway through Transcriptional Regulation in Filamentous Fungi.

Fungi are known to utilize transcriptional regulation of genes that encode efflux transporters to detoxify xenobiotics; however, to date it is unknown how fungi transcriptionally regulate and coordinate different phases of detoxification system (phase I, modification; phase II, conjugation; and phase III, secretion). Here we present evidence of an evolutionary convergence between the fungal and mammalian lineages, whereby xenobiotic detoxification genes (phase I coding for cytochrome P450 monooxygenases [CYP450s] and phase III coding for ATP-binding cassette [ABC] efflux transporters) are transcriptionally regulated by structurally unrelated proteins. Following next-generation RNA sequencing (RNA-seq) analyses of a filamentous fungus, Sclerotinia homoeocarpa, the causal agent of dollar spot on turfgrasses, a multidrug resistant (MDR) field strain was found to overexpress phase I and III genes, coding for CYP450s and ABC transporters for xenobiotic detoxification. Furthermore, there was confirmation of a gain-of-function mutation of the fungus-specific transcription factor S. homoeocarpa XDR1 (ShXDR1), which is responsible for constitutive and induced overexpression of the phase I and III genes, resulting in resistance to multiple classes of fungicidal chemicals. This fungal pathogen detoxifies xenobiotics through coordinated transcriptional control of CYP450s, biotransforming xenobiotics with different substrate specificities and ABC transporters, excreting a broad spectrum of xenobiotics or biotransformed metabolites. A Botrytis cinerea strain harboring the mutated ShXDR1 showed increased expression of phase I (BcCYP65) and III (BcatrD) genes, resulting in resistance to fungicides. This indicates the regulatory system is conserved in filamentous fungi. This molecular genetic mechanism for xenobiotic detoxification in fungi holds potential for facilitating discovery of new antifungal drugs and further studies of convergent and divergent evolution of xenobiotic detoxification in eukaryote lineages.IMPORTANCE Emerging multidrug resistance (MDR) in pathogenic filamentous fungi is a significant threat to human health and agricultural production. Understanding mechanisms of MDR is essential to combating fungal pathogens; however, there is still limited information on MDR mechanisms conferred by xenobiotic detoxification. Here, we report for the first time that overexpression of phase I drug-metabolizing monooxygenases (cytochrome P450s) and phase III ATP-binding cassette efflux transporters is regulated by a gain-of-function mutation in the fungus-specific transcription factor in the MDR strains of the filamentous plant-pathogenic fungus Sclerotinia homoeocarpa This study establishes a novel molecular mechanism of MDR through the xenobiotic detoxification pathway in filamentous fungi, which may facilitate the discovery of new antifungal drugs to control pathogenic fungi.

Nucleic adaptability of heterokaryons to fungicides in a multinucleate fungus, Sclerotinia homoeocarpa.

Sclerotinia homoeocarpa is the causal organism of dollar spot in turfgrasses and is a multinucleate fungus with a history of resistance to multiple fungicide classes. Heterokaryosis gives rise to the coexistence of genetically distinct nuclei within a cell, which contributes to genotypic and phenotypic plasticity in multinucleate fungi. We demonstrate that field isolates, resistant to either a demethylation inhibitor or methyl benzimidazole carbamate fungicide, can form heterokaryons with resistance to each fungicide and adaptability to serial combinations of different fungicide concentrations. Field isolates and putative heterokaryons were assayed on fungicide-amended media for in vitro sensitivity. Shifts in fungicide sensitivity and microsatellite genotypes indicated that heterokaryons could adapt to changes in fungicide pressure. Presence of both nuclei in heterokaryons was confirmed by detection of a single nucleotide polymorphism in the ß-tubulin gene, the presence of microsatellite alleles of both field isolates, and the live-cell imaging of two different fluorescently tagged nuclei using laser scanning confocal microscopy. Nucleic adaptability of heterokaryons to fungicides was strongly supported by the visualization of changes in fluorescently labeled nuclei to fungicide pressure. Results from this study suggest that heterokaryosis is a mechanism by which the pathogen adapts to multiple fungicide pressures in the field.

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.

Draft Genome Sequences of the Turfgrass Pathogen Sclerotinia homoeocarpa.

Sclerotinia homoeocarpa (F. T. Bennett) is one of the most economically important pathogens on high-amenity cool-season turfgrasses, where it causes dollar spot. To understand the genetic mechanisms of fungicide resistance, which has become highly prevalent, the whole genomes of two isolates with varied resistance levels to fungicides were sequenced.

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.

Evidence for Genetic Similarity of Vegetative Compatibility Groupings in Sclerotinia homoeocarpa.

Vegetative compatibility groups (VCGs) are determined for many fungi to test for the ability of fungal isolates to undergo heterokaryon formation. In several fungal plant pathogens, isolates belonging to a VCG have been shown to share significantly higher genetic similarity than those of different VCGs. In this study we sought to examine the relationship between VCG and genetic similarity of an important cool season turfgrass pathogen, Sclerotinia homoeocarpa. Twenty-two S. homoeocarpa isolates from the Midwest and Eastern US, which were previously characterized in several studies, were all evaluated for VCG using an improved nit mutant assay. These isolates were also genotyped using 19 microsatellites developed from partial genome sequence of S. homoeocarpa. Additionally, partial sequences of mitochondrial genes cytochrome oxidase II and mitochondrial small subunit (mtSSU) rRNA, and the atp6-rns intergenic spacer, were generated for isolates from each nit mutant VCG to determine if mitochondrial haplotypes differed among VCGs. Of the 22 isolates screened, 15 were amenable to the nit mutant VCG assay and were grouped into six VCGs. The 19 microsatellites gave 57 alleles for this set. Unweighted pair group methods with arithmetic mean (UPGMA) tree of binary microsatellite data were used to produce a dendrogram of the isolate genotypes based on microsatellite alleles, which showed high genetic similarity of nit mutant VCGs. Analysis of molecular variance of microsatellite data demonstrates that the current nit mutant VCGs explain the microsatellite genotypic variation among isolates better than the previous nit mutant VCGs or the conventionally determined VCGs. Mitochondrial sequences were identical among all isolates, suggesting that this marker type may not be informative for US populations of S. homoeocarpa.

Comparative genome analysis between Agrostis stolonifera and members of the Pooideae subfamily, including Brachypodium distachyon.

Creeping bentgrass (Agrostis stolonifera, allotetraploid 2n = 4x = 28) is one of the major cool-season turfgrasses. It is widely used on golf courses due to its tolerance to low mowing and aggressive growth habit. In this study, we investigated genome relationships of creeping bentgrass relative to the Triticeae (a consensus map of Triticum aestivum, T. tauschii, Hordeum vulgare, and H. spontaneum), oat, rice, and ryegrass maps using a common set of 229 EST-RFLP markers. The genome comparisons based on the RFLP markers revealed large-scale chromosomal rearrangements on different numbers of linkage groups (LGs) of creeping bentgrass relative to the Triticeae (3 LGs), oat (4 LGs), and rice (8 LGs). However, we detected no chromosomal rearrangement between creeping bentgrass and ryegrass, suggesting that these recently domesticated species might be closely related, despite their memberships to different Pooideae tribes. In addition, the genome of creeping bentgrass was compared with the complete genome sequence of Brachypodium distachyon in Pooideae subfamily using both sequences of the above-mentioned mapped EST-RFLP markers and sequences of 8,470 publicly available A. stolonifera ESTs (AgEST). We discovered large-scale chromosomal rearrangements on six LGs of creeping bentgrass relative to B. distachyon. Also, a total of 24 syntenic blocks based on 678 orthologus loci were identified between these two grass species. The EST orthologs can be utilized in further comparative mapping of Pooideae species. These results will be useful for genetic improvement of Agrostis species and will provide a better understanding of evolution within Pooideae species.

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.

Geographic Distribution of Fungicide-Insensitive Sclerotinia homoeocarpa Isolates from Golf Courses in the Northeastern United States.

Chemical management of dollar spot in turf may lead to the development of Sclerotinia homoeocarpa populations with reduced fungicide sensitivity. The objective of this study was to determine the scope of S. homoeocarpa insensitivity to fungicides commonly used to control dollar spot on golf courses in the northeastern United States. A total of 965 and 387 isolates of S. homoeocarpa from intensively or individually sampled sites, respectively, were evaluated for in vitro sensitivity to iprodione, propiconazole, and thiophanate-methyl. Mean baseline sensitivities to iprodione and propiconazole were 0.2763 and 0.0016 µg a.i. ml-1, respectively, and all baseline isolates were sensitive to thiophanate-methyl at 1,000 µg a.i. ml-1. When compared with the baseline population, 14 and 18 of 20 total populations were less sensitive to iprodione and propiconazole, respectively. Individually sampled isolates obtained from fairways, putting greens, or tees were less sensitive to iprodione and propiconazole when compared with the baseline. For thiophanate-methyl, five populations were sensitive, six were resistant, and the remaining nine populations contained various proportions (2 to 92%) of resistant isolates. Individually sampled isolates obtained from fairways and putting greens were evaluated for associations in sensitivity among the three fungicides. A weak but positive correlation in sensitivity to iprodione and propiconazole was observed for isolates resistant to thiophanate-methyl but correlations for sensitive isolates were not significant. Furthermore, isolates with highly reduced sensitivity to iprodione clustered in a narrow range of propiconazole sensitivity. These data suggest the possible existence of resistance mechanisms common to diverse fungicide classes. Overall, results indicate that insensitivity of S. homoeocarpa to iprodione, propiconazole, and thiophanate-methyl exists in varying degrees on golf courses in the northeastern United States.

Transferability of cereal EST-SSR markers to ryegrass.

A large number of expressed sequence tags (ESTs) in public databases have provided an opportunity for the systematic development of simple sequence repeat (SSR) markers. EST-SSRs derived from conserved coding sequences show considerable cross-species transferability in related species. In the present study, we assessed the utility of cereal EST-SSRs in ryegrass (Lolium spp.). A total of 165 cereal EST-SSRs were tested; a high rate of transferability (57%) and polymorphism (67% of functional EST-SSRs) was demonstrated between cereals and ryegrass. A total of 46 segregating loci derived from 37 EST-SSRs were mapped on an existing ryegrass genetic map. The mapped loci were uniformly distributed across all seven linkage groups without significant clustering at the distal regions of linkage groups. Sequences of ryegrass amplicons generated by randomly selected 16 EST-SSRs were aligned with reference sequences of cereal EST-SSRs. The SSR motifs and repeat lengths of the cereal EST-SSR markers were different from the majority of ryegrass amplicons. Furthermore, a majority of EST-SSRs amplified different flanking sequences of SSRs in ryegrass than the original cereal sequences. Our results suggest that the high degree of cereal EST-SSR transferability to ryegrass can be a useful enhancement to the molecular database of PCR-based markers but sequence analysis is essential before transferring genetic information using comparative mapping.

Antifungal Activity of Silver Ions and Nanoparticles on Phytopathogenic Fungi.

Silver in ionic or nanoparticle forms has a high antimicrobial activity and is therefore widely used for various sterilization purposes including materials of medical devices and water sanitization. There have been relatively few studies on the applicability of silver to control plant diseases. Various forms of silver ions and nanoparticles were tested in the current study to examine the antifungal activity on two plant-pathogenic fungi, Bipolaris sorokiniana and Magnaporthe grisea. In vitro petri dish assays indicated that silver ions and nanoparticles had a significant effect on the colony formation of these two pathogens. Effective concentrations of the silver compounds inhibiting colony formation by 50% (EC50) were higher for B. sorokiniana than for M. grisea. The inhibitory effect on colony formation significantly diminished after silver cations were neutralized with chloride ions. Growth chamber inoculation assays further confirmed that both ionic and nanoparticle silver significantly reduced these two fungal diseases on perennial ryegrass (Lolium perenne). Particularly, silver ions and nanoparticles effectively reduced disease severity with an application at 3 h before spore inoculation, but their efficacy significantly diminished when applied at 24 h after inoculation. The in vitro and in planta evaluations of silver indicated that both silver ions and nanoparticles influence colony formation of spores and disease progress of plant-pathogenic fungi. In planta efficacy of silver ions and nanoparticles is much greater with preventative application, which may promote the direct contact of silver with spores and germ tubes, and inhibit their viability.