Using the severe combined immunodeficiency (SCID) mouse model, we investigated the requirement of the immune system for the development of scrapie after peripheral inoculation. A total of 33% of SCID mice, all but one immunologically reconstituted SCID mice (93%), and all CB17 control mice developed the disease. PrPres was detectable in the brains of all diseased animals and in the spleens of reconstituted SCID and CB17 control mice but not of the diseased non-immunologically reconstituted SCID mice. The immune system appears to be a primary target in the pathogenesis of scrapie, but direct spread to the central nervous system from the peritoneum via visceral nerve fibers can probably also occur.

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.

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 Oacetyltransferase 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.

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.

Clarireedia jacksonii causes dollar spot disease of cool‐season turfgrasses in the United States and produces the phytotoxin oxalic acid. The role of oxalic acid in host–pathogen interactions of C. jacksonii is unknown and there are multiple challenges to studying these interactions in natural turfgrass hosts. Consequently, identification of model plants to study C. jacksonii–host interactions and the role of oxalic acid in pathogenesis is necessary. Controlled environment inoculation assays were used to evaluate pathogenesis of C. jacksonii in various model plants and investigate the role of oxalic acid in symptom development. Observations at microscopic and macroscopic levels demonstrated that infection progressed similarly in all monocots tested (creeping bentgrass, wheat, barley, rice, Brachypodium distachyon) but not in the dicot Arabidopsis thaliana. Plant oxalic acid content increased from near zero to around 0.2–0.4 mM following inoculation with C. jacksonii in creeping bentgrass, barley, and wheat. Conversely, oxalic acid content remained near zero in A. thaliana and was not well correlated with inoculation in rice and B. distachyon, both of which had higher endogenous oxalic acid levels than other monocots. Time‐course oxalic acid quantification experiments with creeping bentgrass and B. distachyon further supported a link between symptom development and in planta oxalic acid content and identified 48 hr postinoculation as a critical time‐point for investigating the role of oxalic acid in C. jacksonii pathogenesis. These studies demonstrate that various monocots can serve as tractable model systems for studying C. jacksonii–host interactions and that increases in oxalic acid content are associated with C. jacksonii symptom development.

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 grass cell walls. 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's 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.

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 hours post inoculation (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 ABC transporter gene family (OsABC1, OsABC9 and OsABC12) encoding pleiotropic drug resistance (PDR)-like ATP binding cassette (ABC) transporters were mapped to different sheath blight resistance QTL 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 section and functional validation of the ABC genes in sheath blight disease resistance.