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Sheath blight is a serious rice disease worldwide, and genes involved in resistance remain unclear. In the present study, a virulent field isolate of Rhizoctonia solani was used to inoculate detached leaves of a sheath blight-resistant rice cultivar, Jasmine 85; a suppression subtractive cDNA library was constructed using RNA isolated 16 h postinoculation (hpi); and differentially expressed genes were identified from the cDNA library. A total of 159 uniquely expressed sequence tags were identified, including 105 from rice with enrichment in categories related to cellular response, molecular signaling, and host defense. Coupled with gene expression studies by DNA microarray, 27 highly induced genes involved in signal transduction and defense responses were identified within 16 hpi. Three members of the ATP-binding cassette (ABC) transporter gene family ( OsABC1, OsABC9, and OsABC12) encoding pleiotropic drug resistance-like ABC transporters were mapped to different sheath blight resistance quantitative trait loci (QTLs), and their differential expressions were validated. Three high-resolution melting (HRM) markers were developed from these ABC gene family members to distinguish alleles between sheath blight-susceptible cultivar Lemont and resistant cultivar Jasmine 85. Association of sheath blight resistance to these HRM markers was examined in 77 recombinant inbred lines derived from the cross between Jasmine 85 and Lemont. The OsABC9 gene located in a major sheath blight resistance QTL qShB9-2 showed a major contribution to sheath blight resistance. These results are useful for marker-assisted selection and functional validation of the ABC genes in sheath blight disease resistance. [Formula: see text] The author(s) have dedicated the work to the public domain under the Creative Commons CC0 “No Rights Reserved” license by waiving all of his or her rights to the work worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law, 2022.

Aim The newly defined species Pectobacterium parmentieri has emerged as an aggressive pathogen that causes soft rot and blackleg diseases on potato and has been widely disseminated across the globe, jeopardizing the productivity and potato food safety. The implementation of a fast and accurate detection tool is imperative to control, monitor and prevent further spread of these pathogens. The objective of this work was to develop a specific and sensitive multiplex TaqMan qPCR to detect P. parmentieri and distinguish it from all known Pectobacterium species. A universal internal control was included to enhance the reliability of the assay. Methods and Results A comparative genomics approach was used to identify O-acetyltransferase and the XRE family transcriptional regulator as specific targets for primers/probe design for the detection of the Pectobacterium genus and P. parmentieri, respectively. Specificity was assessed with 35 and 25 strains included in the inclusivity and exclusivity panels, respectively, isolated from different geographical locations and sources. The assay specifically detected all 35 strains of Pectobacterium sp. and all 15 P. parmentieri strains. No cross-reactivity was detected during assay validation. Our assay detected up to 10 fg genomic DNA and 1 CFU ml−1 bacterial culture. No change in the detection threshold (1 CFU ml−1) was observed in spiked assays after adding host tissue to the reactions. The assay was validated with naturally and artificially infected host tissues and soil rhizosphere samples. All infected plant samples containing the target pathogens were accurately amplified. Conclusion The presented multiplex TaqMan qPCR diagnostic assay is highly specific, sensitive, reliable for the detection of Pectobacterium species and P. parmentieri with no false positives or false negatives. Significance and Impact of the Study The developed assay can be adopted for multiple purposes such as seed certification programmes, surveillance, biosecurity, microbial forensics, quarantine, border protection, inspections and epidemiology.

Dollar spot is the most economically important disease of amenity turfgrass, and more fungicides are applied targeting dollar spot than any other turfgrass disease. Dollar spot symptoms are small (3 to 5 cm), circular patches that develop in a highly variable manner within plot scale even under seemingly uniform conditions., Dollar spot, caused by the fungal pathogen Clarireedia spp., is an economically important foliar disease of amenity turfgrass in temperate climates worldwide. This disease often occurs in a highly variable manner, even on a local scale with relatively uniform environmental conditions. The objective of this study was to investigate mechanisms behind this local variation, focusing on contributions of the soil and rhizosphere microbiome. Turfgrass, rhizosphere, and bulk soil samples were collected from within a 256-m2 area of healthy turfgrass, transported to a controlled environment chamber, and inoculated with Clarireedia jacksonii. Bacterial communities were profiled by targeting the 16S rRNA gene, and 16 different soil chemical properties were assessed. Despite their initial uniform appearance, the samples differentiated into highly susceptible and moderately susceptible groups following inoculation in the controlled environment chamber. The highly susceptible samples harbored a unique rhizosphere microbiome with suggestively lower relative abundance of putative antibiotic-producing bacterial taxa and higher predicted abundance of genes associated with xenobiotic biodegradation pathways. In addition, stepwise regression revealed that bulk soil iron content was the only significant soil characteristic that positively regressed with decreased dollar spot susceptibility during the peak disease development stage. These findings suggest that localized variation in soil iron induces the plant to select for a particular rhizosphere microbiome that alters the disease outcome. More broadly, further research in this area may indicate how plot-scale variability in soil properties can drive variable plant disease development through alterations in the rhizosphere microbiome. IMPORTANCE Dollar spot is the most economically important disease of amenity turfgrass, and more fungicides are applied targeting dollar spot than any other turfgrass disease. Dollar spot symptoms are small (3 to 5 cm), circular patches that develop in a highly variable manner within plot scale even under seemingly uniform conditions. The mechanism behind this variable development is unknown. This study observed that differences in dollar spot development over a 256-m2 area were associated with differences in bulk soil iron concentration and correlated with a particular rhizosphere microbiome. These findings provide interesting avenues for future research to further characterize the mechanisms behind the highly variable development of dollar spot, which may inform innovative control strategies. Additionally, these results suggest that small changes in soil properties can alter plant activity and hence the plant-associated microbial community, which has important implications for a broad array of agricultural and horticultural plant pathosystems.

Dollar spot, caused by the fungal pathogen Clarireedia spp., is an economically important disease of amenity turfgrass in temperate climates worldwide. This disease often occurs in a highly variable manner, even on a local scale with relatively uniform environmental conditions. The objective of this study was to investigate mechanisms behind this local variation, focusing on contributions of the soil and rhizosphere microbiome. Turfgrass, rhizosphere, and bulk soil samples were taken from within a 256 m2 area of healthy turfgrass, transported to a controlled environment chamber, and inoculated with C. jacksonii. Bacterial communities were profiled targeting the 16s rRNA gene, and 16 different soil chemical properties were assessed. Despite their initial uniform appearance, the samples differentiated into highly susceptible and moderately susceptible groups following inoculation in the controlled environment chamber. The highly susceptible samples harbored a unique rhizosphere microbiome with lower relative abundance of antibiotic-producing bacterial taxa and higher predicted abundance of genes associated with xenobiotic biodegradation pathways. In addition, stepwise regression revealed that bulk soil iron content was the only significant soil characteristic that positively regressed with decreased dollar spot susceptibility during the peak disease development stage. These findings suggest that localized variation in soil iron induces the plant to select for a particular rhizosphere microbiome that alters the disease outcome. More broadly, further research in this area may indicate how plot-scale variability in soil properties can drive variable plant disease development through alterations in the rhizosphere microbiome.IMPORTANCEDollar spot is the most economically important disease of amenity turfgrass, and more fungicides are applied targeting dollar spot than any other turfgrass disease. Dollar spot symptoms are small (3-5 cm), circular patches that develop in a highly variable manner within plot-scale even under seemingly uniform conditions. The mechanism behind this variable development is unknown. This study observed that differences in dollar spot development over a 256 m2 area were associated with differences in bulk soil iron concentration and correlated with a particular rhizosphere microbiome. These findings provide important clues for understanding the mechanisms behind the highly variable development of dollar spot, which may offer important clues for innovative control strategies. Additionally, these results also suggest that small changes in soil properties can alter plant activity and hence the plant-associated microbial community which has important implications for a broad array of important agricultural and horticultural plant pathosystems.

Dollar spot is caused by the fungus Clarireedia jacksonii and is the most common disease of golf course turfgrass in temperate climates. Oxalic acid (OA) is an important pathogenicity factor in other fungal plant pathogens, such as the dicot pathogen Sclerotinia sclerotiorum, but its role in C. jacksonii pathogenicity on monocot hosts remains unclear. Herein, we assess fungal growth, OA concentration, and pH change in potato dextrose broth (PDB) following incubation of C. jacksonii. In addition, OA production by C. jacksonii and S. sclerotiorum was compared in PDB amended with creeping bentgrass or common plant cell wall components (cellulose, lignin, pectin, or xylan). Our results show that OA production is highly dependent on the environmental pH, with twice as much OA produced at pH 7 than pH 4 and a corresponding decrease in PDB pH from 7 to 5 following 96 h of C. jacksonii incubation. In contrast, no OA was produced or changes in pH observed when C. jacksonii was incubated in PDB at a pH of 4. Interestingly, C. jacksonii increased OA production in response to PDB amended with creeping bentgrass tissue and the cell wall component xylan, a major component of all walls of grasses. S. sclerotiorum produced large amounts of OA relative to C. jacksonii regardless of treatment and no treatment increased OA production by this fungus, though pectin suppressed S. sclerotiorum OA production. These results suggest that OA production by C. jacksonii is reliant on host specific components within the infection court, as well as the ambient pH of the foliar environment during its pathogenic development.

Brachypodium distachyon is a C3 grass that is an attractive model host system for studying pathogenicity of major turfgrass pathogens due to its genetic similarity to many cool-season turfgrasses. Infection assays with two or more isolates of the casual agents of dollar spot, brown patch, and Microdochium patch resulted in compatible interactions with B. distachyon inbred line Bd21-3. The symptoms produced by these pathogens on Bd21-3 closely resembled those observed on the natural turfgrass host (creeping bentgrass), demonstrating that B. distachyon is susceptible to the fungal pathogens that cause dollar spot, brown patch, and Microdochium patch on turfgrasses. The interaction between Sclerotinia homoeocarpa isolates and Brachypodium ecotypes was also investigated. Interestingly, differential responses of these ecotypes to S. homoeocarpa isolates was found, particularly when comparing B. distachyon to B. hybridum ecotypes. Taken together, these findings demonstrate that B. distachyon can be used as a model host system for these turfgrass diseases and leveraged for studies of molecular mechanisms contributing to host resistance.

Clarireediasp. (formerly calledSclerotinia homoeocarpa), the fungal pathogen that causes dollar spot of turfgrasses, produces oxalic acid but the role of this toxin inClarireediasp. pathogenesis is unknown. In the current study, whole plant inoculation assays were used to evaluate pathogenesis ofClarireediasp. in various model hosts and investigate the role of oxalic acid in dollar spot disease. These assays revealed that both host endogenous oxalate content and pathogen-produced oxalic acid influence the timing and magnitude of symptom development. In time-course expression analysis, oxalate oxidase and related defense-associated germin-like protein genes in creeping bentgrass showed strong up-regulation starting at 48-72 hpi, indicating that germin-like protein genes are most likely involved in defense following initial contact with the pathogen and demonstrating the importance of oxalic acid inClarireediasp. pathogenesis. Overall, the results of these studies suggest that oxalic acid and host endogenous oxalate content are important for pathogenesis byClarireediasp. and may be associated with the transition from biotrophy to necrotrophy during host infection.

Dollar spot, caused by Sclerotinia homoeocarpa, is one of the most economically devastating diseases of amenity turfgrasses worldwide. The pathogen is readily isolated from active lesions, but detection from seed, dormant host tissue and other plant debris that may serve as a source of primary inoculum is difficult. A semi-selective medium was developed to enhance isolation of S. homoeocarpa. Various fungicides used on turfgrass, the pH indicator dye bromophenol blue, and two pH levels, were assessed for their effects on, and in the case of bromophenol blue, response to growth of S. homoeocarpa and contaminant fungi frequently isolated from field and seed samples. Amendment of the medium to pH 4 promoted growth of S. homoeocarpa in the absence of fungicides and enhanced pathogen growth relative to contaminant isolates on medium amended with 0.1 μg mL−1 triticonazole or 5 μg mL−1 azoxystrobin. The growth rate of S. homoeocarpa on these three media, as determined by in vitro radial growth assays, wa...

Rhizoctonia solani, teleomorph Thanatephorus cucumeris, is a polyphagous necrotrophic plant pathogen of the Basidiomycete order that is split into 14 different anastomosis groups (AGs) based on hyphal interactions and host range. In this investigation, quantitative real-time PCR (qRT-PCR) techniques were used to determine potential pathogenicity factors of R. solani in the AG1-IA/rice and AG3/potato pathosystems. These factors were identified by mining for sequences of pathogen origin in a library of rice tissue infected with R. solani AG1-IA and comparing these sequences against the recently released R. solani AG3 genome. Ten genes common to both AGs and two specific to AG1-IA were selected for expression analysis by qRT-PCR. Results indicate that a number of genes are similarly expressed by AG1 and AG3 during the early stages of pathogenesis. Grouping of these pathogenicity factors based on relatedness of expression profiles suggests three key events are involved in R. solani pathogenesis: early host contact and infiltration, adjustment to the host environment, and pathogen proliferation through necrotic tissue. Further studies of the pathogenesis-associated genes identified in this project will enable more precise elucidation of the molecular mechanisms that allow for the widespread success of R. solani as a phytopathogen and allow for more targeted, effective methods of management.

Dollar spot is the most economically important disease of amenity turfgrasses in the United States, yet little is known about the source of primary inoculum for this disease. With the exception of a few isolates from the United Kingdom, Sclerotinia homoeocarpa, the causal agent of dollar spot, does not produce spores. Consequently, it was assumed that overwintering of this organism in soil, thatch, and plant debris provides primary inoculum for dollar spot epidemics. Overwintering of S. homoeocarpa in roots and shoots of symptomatic and asymptomatic creeping bentgrass turfgrass was quantified over the course of a three-year field experiment. Roots did not consistently harbor S. homoeocarpa, whereas S. homoeocarpa was isolated from 30% of symptomatic shoots and 10% of asymptomatic shoots in the spring of two out of three years. The presence of stroma-like pathogen material on leaf blades was associated with an increase in S. homoeocarpa isolation and colony diameter at 48 hpi. Commercial seed has also been hypothesized to be a potential source of initial inoculum for S. homoeocarpa. Two or more commercial seed lots of six creeping bentgrass cultivars were tested for contamination with S. homoeocarpa using culture-based and molecular detection methods. A viable, pathogenic isolate of S. homoeocarpa was isolated from one commercial seed lot and contamination of this lot was confirmed with nested PCR using S. homoeocarpa specific primers. A sensitive nested PCR assay detected S. homoeocarpa contamination in eight of twelve (75%) commercial seed lots. Seed source, but not cultivar or resistance to dollar spot, influenced contamination by S. homoeocarpa. Overall, this research suggests that seeds are a potential source of initial inoculum for dollar spot epidemics and presents the need for further research in this area.