Genome Resources for Seven Fungal Isolates That Cause Dollar Spot Disease in Turfgrass, Including Clarireedia jacksonii and C. monteithiana.

Fungi in the genus Clarireedia are widespread and destructive pathogens of grasses worldwide, and are best known as the causal agents of dollar spot disease in turfgrass. Here, we report genome assemblies of seven Clarireedia isolates, including ex-types of the two most widespread species, Clarireedia jacksonii and C. monteithiana. These datasets provide a valuable resource for ongoing studies of the dollar spot pathogens that include population diversity, host-pathogen interactions, marker development, and disease control.

Clarireedia: A new fungal genus comprising four pathogenic species responsible for dollar spot disease of turfgrass.

Dollar spot is one of the most destructive and economically important fungal diseases of amenity turfgrasses. The causal agent was first described in 1937 as the ascomycete Sclerotinia homoeocarpa. However, the genus-level taxonomic placement of this fungus has been the subject of an ongoing debate for over 75 y. Existing morphological and rDNA sequence evidence indicates that this organism is more appropriately placed in the family Rutstroemiaceae rather than the Sclerotiniaceae. Here we use DNA sequence data from samples of the dollar spot fungus and other members of the Rutstroemiaceae (e.g. Rutstroemia, Lanzia, Lambertella) collected throughout the world to determine the generic identity of the turfgrass dollar spot pathogen. Phylogenetic evidence from three nucleotide sequence markers (CaM, ITS and Mcm7; 1810-bp) confirmed that S. homoeocarpa is not a species of Sclerotinia; nor is it a member of any known genus in the Rutstroemiaceae. These data support the establishment of a new genus, which we describe here as Clarireedia gen. nov. The type species for the genus, Clarireedia homoeocarpa comb. nov., is described to accommodate the dollar spot fungus, and a neotype is designated. Three new species in this clade, Clarireedia bennettii sp. nov., Clarireedia jacksonii sp. nov., and Clarireedia monteithiana sp. nov. that also cause dollar spot disease are described. Clarireedia homoeocarpa and C. bennettii occur primarily on Festuca rubra (C3 grass) hosts and appear to be restricted to the United Kingdom. Clarireedia jacksonii and C. monteithiana occur on a variety of C3 and C4 grass hosts, respectively, and appear to be globally distributed. This resolved taxonomy puts to rest a major controversy amongst plant pathologists and provides a foundation for better understanding the nature and biology of these destructive pathogens.

RNA-Seq analysis of the Sclerotinia homoeocarpa--creeping bentgrass pathosystem.

Sclerotinia homoeocarpa causes dollar spot disease, the predominate disease on highly-maintained turfgrass. Currently, there are major gaps in our understanding of the molecular interactions between S. homoeocarpa and creeping bentgrass. In this study, 454 sequencing technology was used in the de novo assembly of S. homoeocarpa and creeping bentgrass transcriptomes. Transcript sequence data obtained using Illumina's first generation sequencing-by-synthesis (SBS) were mapped to the transcriptome assemblies to estimate transcript representation in different SBS libraries. SBS libraries included a S. homoeocarpa culture control, a creeping bentgrass uninoculated control, and a library for creeping bentgrass inoculated with S. homoeocarpa and incubated for 96 h. A Fisher's exact test was performed to determine transcripts that were significantly different during creeping bentgrass infection with S. homoeocarpa. Fungal transcripts of interest included glycosyl hydrolases, proteases, and ABC transporters. Of particular interest were the large number of glycosyl hydrolase transcripts that target a wide range of plant cell wall compounds, corroborating the suggested wide host range and saprophytic abilities of S. homoeocarpa. Several of the multidrug resistance ABC transporters may be important for resistance to both fungicides and plant defense compounds. Creeping bentgrass transcripts of interest included germins, ubiquitin transcripts involved in proteasome degradation, and cinnamoyl reductase, which is involved in lignin production. This analysis provides an extensive overview of the S. homoeocarpa-turfgrass pathosystem and provides a starting point for the characterization of potential virulence factors and host defense responses. In particular, determination of important host defense responses may assist in the development of highly resistant creeping bentgrass varieties.

Large scale identification of genes involved in plant-fungal interactions using Illumina's sequencing-by-synthesis technology.

Deep transcriptome profiling of pathogen-infected tissues enhances the understanding of molecular mechanisms underlying host-pathogen interactions. Illumina's next generation sequencing technology sequencing-by-synthesis (SBS) is a powerful tool to rapidly sequence genomes and transcriptomes at an affordable rate. We modified the procedure for SBS library construction to significantly increase the efficiency of library construction. Using our improved method, two Sclerotinia homoeocarpa libraries were constructed from mycelia grown in potato dextrose broth (PDB) or potato dextrose agar (PDA) for 96 h, respectively, and two creeping bentgrass libraries were constructed from leaves 96 h after inoculation with S. homoeocarpa or water sprayed, respectively. About 4-7 million mRNA signatures were sequenced from each library. Sequence analysis using BLAST was performed against sequenced fungal genomes and rice genomic sequence to identify the expressed genes in both S. homoeocarpa mycelia and creeping bentgrass. Bioinformatic analysis identified many expressed genes in the pathogen and host. A public database to access the sequence data was developed at http://www.dstidb.org . Our results demonstrate how SBS technology can unravel transcriptome complexity during the creeping bentgrass-S. homoeocarpa interaction.

Expression Analysis of Rice Defense-Related Genes in Turfgrass in Response to Magnaporthe grisea.

ABSTRACT Magnaporthe grisea (anamorph = Pyricularia grisea) causes blast on rice (Oryza sativa) and gray leaf spot on turfgrass. Gray leaf spot is a serious disease on St. Augustinegrass (Stenotaphrum secundatum), perennial ryegrass (Lolium perenne), and tall fescue (Festuca arundinacea). Virulence assays performed in this study revealed that M. grisea collected from rice could also cause disease on St. Augustinegrass and tall fescue. One rice isolate, Che86061, caused similar disease reactions on susceptible cultivars of rice and St. Augustinegrass and an incompatible interaction on resistant cultivars of both species. To explore whether similar defense-related genes are expressed in rice and St. Augustinegrass, a rice cDNA library was screened using pooled cDNAs derived from M. grisea-infected St. Augustinegrass. Thirty rice EST (expressed sequence tag) clones showing differential expression in St. Augustinegrass following M. grisea inoculation were identified and classified into six putative functional groups. Northern blot analyses of seven EST clones that collectively represented each putative functional group confirmed that the expression of five out of seven EST clones was similar in both rice and St. Augustinegrass. This study represents one of the first attempts to use a broad-scale genomic approach and resources of a model monocot system to study defense gene expression in St. Augustinegrass following M. grisea infection.

Fungicide Sensitivity of Sclerotinia homoeocarpa from Golf Courses in Ohio.

Managing dollar spot, the most common and chronic disease on intensively cultivated turfgrass, relies on the judicious use of fungicides. The heavy use of fungicides has led to the development of isolates of Sclerotinia homoeocarpa insensitive to several classes of fungicides, including benzimidazoles, demethylation-inhibitors, and dicarboximides. In vitro fungicide sensitivity assays using single discriminatory concentrations of thiophanate-methyl, propiconazole, and iprodione were developed in this study for evaluating field efficacy of these fungicides and the prevalence of fungicide insensitivity within S. homoeocarpa isolated from golf courses throughout Ohio. Discriminatory concentrations for these fungicides were determined to be: thiophanate-methyl = 1,000 µg a.i. ml-1, propiconazole = 0.1 µg a.i. ml-1, and iprodione = 1.0 µg a.i. ml-1. Effective concentration that produces 50% inhibition (EC50) was estimated based on relative mycelial growth of S. homoeocarpa on potato dextrose agar (PDA) versus PDA amended with the discriminatory concentration of each fungicide. Field trials conducted at 3 locations in 2002 and 10 locations in 2003 revealed that the in vitro assays accurately predicted field efficacy for thiophanate-methyl. When used to screen 192 S. homoeocarpa isolates collected previously from 55 golf courses throughout Ohio, the in vitro assays revealed that 34 of the golf courses sampled had S. homoeocarpa resistant to thiophanate-methyl. S. homoeocarpa with reduced in vitro sensitivities was isolated from 18 and 1 golf courses for propiconazole and iprodione, respectively.

Carbon-to-Nitrogen Ratio and Carbon Loading of Production Media Influence Freeze-Drying Survival and Biocontrol Efficacy of Cryptococcus nodaensis OH 182.9.

ABSTRACT Fusarium head blight (FHB), caused by Gibberella zeae, is a devastating disease of wheat worldwide. Cryptococcus nodaensis OH 182.9 is an effective biocontrol agent for this disease. Development of a dried product of OH 182.9 would have potential advantages of ease of handling, favorable economics, and acceptance by end users. Isolate OH 182.9 was grown for 48 and 72 h in semi-defined complete liquid (SDCL) medium with carbon-to-nitrogen (C/N) ratios of 6.5:1, 9:1, 11:1, 15:1, and 30:1, and in SDCL C/N 30:1 media with varied carbon loadings of 7, 14, 21, and 28 g/liter. Total biomass production and cell survival at 15 days after freeze-drying were evaluated. Biomass production of OH 182.9 (CFU per milliliter) was not different for all cultivation time by medium C/N or carbon loading combinations. In general, cells harvested at 48 h survived freeze-drying better than those harvested at 72 h. Survival of freeze-dried cells was greatest for cells grown for 48 h in C/N30:1 medium. Cells produced in C/N 6.5:1 medium generally exhibited the poorest survival. For the C/N 30:1 media, cells from 7 g/liter carbon loading medium harvested after 48 h had the best survival after freeze-drying. The difference in freeze-dried cell populations between superior and inferior treatments was typically 1 to 2 log units at 15 days after freeze-drying. The biomass of OH 182.9 produced in SDCL with varied C/N ratios and in SDCL C/N 30:1 media with differing carbon loadings was tested for biocontrol efficacy against FHB in greenhouse studies. The biomass harvested from SDCL C/N 9:1, 11:1, and 15:1 media after 48 h significantly reduced symptoms of FHB. None of the treatments with cells harvested at 72 h consistently reduced FHB severity (P

Effects of Choline, Betaine, and Wheat Floral Extracts on Growth of Fusarium graminearum.

Fusarium head blight has been more severe when infection occurs during anthesis, indicating that floral organs may be important infection courts. Choline acetate and glycinebetaine have been extracted from wheat and reported to be growth stimulants of Fusarium graminearum. They are hypothesized to enhance infection and tissue colonization. Growth of F. graminearum was examined on media amended with extracts from floral parts of nine wheat genotypes with various Fusarium head blight resistance levels. Results indicated no significant effect of anther, palea, or lemma extracts on radial growth when compared with unamended controls. Effects on spore germination and hyphal growth of F. graminearum by choline, betaine, and an equimolar mixture at concentrations ranging from 0.01 to 1,000 µM also were examined. Spore germination was not significantly (P ≤ 0.05) affected by choline, betaine, or a combination of the compounds compared with unamended controls. Radial hyphal growth also was not consistently affected (P ≤ 0.05) by choline or betaine when compared with controls. Equimolar mixtures of the two compounds showed significant slight reduction in growth rate at higher concentrations when compared with controls. The reduction in growth rate was due to higher concentrations of betaine. Results of this study indicate that endogenous compounds in floral parts may not be associated with wheat resistance to F. graminearum.

Biological control of Fusarium head blight of wheat and deoxynivalenol levels in grain via use of microbial antagonists.

Efforts to reduce mycotoxin contamination in food logically start with minimizing plant infection by mycotoxin producing pathogens. Fusarium graminearum (perfect state, Gibberella zeae) infects wheat heads at flowering, causing the disease Fusarium head blight (FHB) and losses of over 2.6 billion dollars in the U.S. during the last 10 years. The pathogen often produces deoxynivalenol (DON) resulting in grain size and quality reduction. Highly resistant wheat cultivars currently are not available for reducing FHB, and labeled fungicides are not consistently effective. The feasibility of biologically controlling FHB is currently being evaluated. Microbial isolates obtained from wheat anthers were screened for their ability to utilize tartaric acid, a compound that is poorly utilized by F. graminearum and could be utilized in formulations of biological control agents. Four strains that utilized tartaric acid and three that did not were effective in reducing FHB disease severity by up to 95% in greenhouse and 56% in field trials. Additional research programs around the globe have identified other antagonist strains with potential for biologically controlling FHB. Though a considerable body of research remains to be completed, strategies and microorganisms for biologically controlling FHB have reached an advanced stage of development and offer the promise of being an effective tool that could soon contribute to the reduction of FHB severity and DON contamination of grain in commercial agriculture.