Geunhwa Jung

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.

Mapping and Comparative Analysis of QTL for Crown Rust Resistance in an Italian × Perennial Ryegrass Population

ABSTRACT Crown rust (Puccinia coronata f. sp. lolli) is a serious fungal foliar disease of perennial ryegrass (Lolium perenne L.) and Italian ryegrass (L. multiflorum Lam.), which are important forage and turf species. A number of quantitative trait loci (QTL) for crown rust resistance previously were identified in perennial ryegrass under growth chamber or greenhouse conditions. In this study, we conducted a QTL mapping for crown rust resistance in a three-generation Italian x perennial ryegrass interspecific population under natural field conditions at two locations over 2 years. Through a comparative mapping analysis, we also investigated the syntenic relationships of previously known crown rust resistance genes in other ryegrass germplasms and oat, and genetic linkage between crown rust resistance QTL and three lignin genes: LpOMT1, LpCAD2, and LpCCR1. The interspecific mapping population of 156 progeny was developed from a cross between two Italian x perennial ryegrass hybrids, MFA and MFB. Because highly susceptible reactions to crown rust were observed from all perennial ryegrass clones, including two grandparental clones and eight clones from different pedigrees tested in this study, two grandparent clones from Italian ryegrass cv. Floregon appeared to be a source of the resistance. Two QTL on linkage groups (LGs) 2 and 7 in the resistant parent MFA map were detected consistently regardless of year and location. The others, specific to year and location, were located on LGs 3 and 6 in the susceptible parent MFB map. The QTL on LG2 was likely to correspond to those previously reported in three unrelated perennial ryegrass mapping populations; however, the other QTL on LGs 3, 6, and 7 were not. The QTL on LG7 was closely located in the syntenic genomic region where genes Pca cluster, Pcq2, Pc38, and Prq1b resistant to crown rust (P. coronata f. sp. avenae) in oat (Avena sativa L.) were previously identified. Similarly, the QTL on LG3 was found in a syntenic region with oat genes resistant to crown rust isolates PC54 and PC59. This indicates that the ortholoci for resistance genes to different formae speciales of crown rust might be present between two distantly related grass species, ryegrass and oat. In addition, we mapped four restriction fragment length polymorphism loci for three key ryegrass lignin genes encoding caffeic acid-O-methyltransferase, cinnamyl alcohol dehydrogenase, and cinnamoyl CoA-reductase on LG7. These loci were within a range of 8 to 17 centimorgans from the QTL on LG7, suggesting no tight linkage between them. The putative ortholoci for those lignin biosynthesis genes were identified on segments of rice (Oryza sativa L.) chromosomes 6 and 8, which are the counterparts of ryegrass LG7. Results from the current study facilitate understanding of crown rust resistance and its relationship with lignin biosynthesis, and also will benefit ryegrass breeders for improving crown rust resistance through marker-assisted selection.

Overexpression of ShCYP51B and ShatrD in Sclerotinia homoeocarpa Isolates Exhibiting Practical Field Resistance to a Demethylation Inhibitor Fungicide

ABSTRACT 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.

Rapid Development of Fungicide Resistance by Sclerotinia homoeocarpa on Turfgrass

Dollar spot, caused by Sclerotinia homoeocarpa, is the most prevalent and economically important turfgrass disease in North America. Increasing levels of fungicide resistance, coupled with tightening environmental scrutiny of existing fungicides, has left fewer options for managing dollar spot. More knowledge about S. homoeocarpa populations is needed to improve dollar spot management strategies, especially with respect to minimizing the development of fungicide resistance. Population diversity of S. homoeocarpa was examined using inter-simple sequence repeat markers and vegetative compatibility assays. Two subgroups were found in S. homoeocarpa field populations on both fairway and putting green turfgrass at a research field in Wisconsin. These subgroups were genetically different, vegetatively incompatible, and had different fungicide sensitivities. The frequency of the two genetic subgroups differed significantly between the fairway and putting green, but was uniform within the fairway or within the green. Population dynamics of S. homoeocarpa in response to two systemic fungicides (thiophanate-methyl and propiconazole) were assessed based on in vitro fungicide sensitivity. Dynamics of S. homoeocarpa populations depended on the presence of fungicide-resistant isolates in the initial populations before fungicide applications and changed rapidly after fungicide applications. Shifting of the population toward propiconazole resistance was gradual, whereas thiophanate-methyl resistance developed rapidly in the population. In conclusion, field populations of S. homoeocarpa containing genetically distinct, vegetatively incompatible groups were different on turfgrass that was managed differently, and they were changed rapidly after exposure to fungicides.

Thiophanate-Methyl and Propiconazole Sensitivity in Sclerotinia homoeocarpa Populations from Golf Courses in Wisconsin and Massachusetts

Management of dollar spot, caused by the fungus Sclerotinia homoeocarpa, is dependent upon repeated fungicide applications in intensively managed turfgrass such as golf course putting greens and fairways. Repeated fungicide applications could potentially select for fungicide-resistant isolates and result in a reduction of disease control. The objectives of this study were to determine the degree of S. homoeocarpa in vitro sensitivity to the fungicides thiophanate-methyl and propiconazole using isolates collected from golf course putting greens, fairways, and roughs; and to determine the relationships of golf course age and fungicide history to the frequency of fungicide-insensitive isolates within the population. More than 1,400 S. homoeocarpa isolates were collected from putting greens, fairways, and roughs at six Wisconsin golf courses and one Massachusetts golf course and subjected to in vitro fungicide sensitivity assays with single discriminatory concentrations of thiophanate-methyl and propiconazole. Five of seven pathogen populations from rough areas were not significantly different from one another in propiconazole sensitivity. These populations were collectively the most sensitive to both fungicides and therefore, served as baseline populations for comparison with fungicide-exposed populations from putting greens and fairways. Greater propiconazole insensitivity was observed in populations collected from fairways and putting greens that received more frequent applications of the fungicide than those isolated from the roughs. In nearly all the golf courses, the frequency of thiophanate-methyl insensitivity was higher among isolates of S. homoeocarpa collected from fairways than from roughs regardless of the age of the golf course or history of benzimidazole use. Thus, while the development of resistance to propiconazole can be predicted in part by the relative frequency of demethylation inhibitor fungicide applications, the occurrence of populations resistant to thiophanate-methyl appears to be unrelated to recent use of the benzimidazole class of fungicides.

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.

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.

Reassessment of Vegetative Compatibility of Sclerotinia homoeocarpa Using Nitrate-Nonutilizing Mutants

Nitrate-nonutilizing (nit) mutants were recovered for the first time from 21 isolates of Sclerotinia homoeocarpa collected in the United States. Mutants were selected from shredded mycelium of each isolate when cultured on water agar medium amended with 4% (wt/vol) potassium chlorate. The mutants could be classified into three phenotypes: nit1, nit3, and NitM, based on their growth on minimal medium (Czapek solution agar) supplemented with NaNO(2) or hypoxanthine. Complementary heterokaryons were observed in pairings between different phenotypes of nit mutants derived from compatible isolates, but not in self-fusions or pairings between incompatible isolates. The vigor of prototrophic growth varied with isolates and mutant phenotypes. Strong and continuous heterokaryons, as well as weak and spontaneous ones, formed depending on pairings of nit mutants. Stable heterokaryons between compatible isolates, but apoptotic reactions between incompatible isolates, were observed immediately after hyphal fusion under the epifluorescence microscope. The 21 isolates used in this study, which were previously assigned into 11 different vegetative compatibility groups (VCGs) based on the formation of a barrage zone at the contact site of paired isolates on complete medium (potato dextrose agar), were regrouped into five VCGs based on heterokaryon formation between nit mutants on minimal medium.

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.

Aggressiveness of Typhula ishikariensis Isolates to Cultivars of Bentgrass Species (Agrostis spp.) Under Controlled Environment Conditions

Speckled snow mold, caused by Typhula ishikariensis, is one of the most important Typhula snow molds in subarctic zones of the Northern Hemisphere. Nine isolates of three T. ishikariensis varieties (var. ishikariensis, var. canadensis, and var. idahoensis) isolated from infected turfgrasses on golf course fairways throughout Wisconsin were evaluated for their aggressiveness toward nine cultivars of three bentgrass species (three creeping, three colonial, and three velvet cultivars) under controlled environmental conditions. Speckled snow mold severity increased as inoculum concentration of T. ishikariensis was increased. In general, bentgrass susceptibility increased between 9 and 11 weeks after seeding but gradually decreased thereafter, suggesting expression of age-related resistance as plants matured. Significant differences in aggressiveness were detected within and among T. ishikariensis varieties. Significant interactions between T. ishikariensis varieties or isolates and bentgrass species were detected, but there was no interaction between pathogen isolates and bentgrass cultivars. Disease severity evaluations showed significant differences among bentgrass cultivars and species in their response to T. ishikariensis. Since bentgrass species exhibit differential responses to T. ishikariensis varieties, representative isolates of each variety should be employed for screening of bentgrass germplasm for resistance to speckled snow mold.