Jeremiah K. S. Dung

Spatial Patterns of Ergot and Quantification of Sclerotia in Perennial Ryegrass Seed Fields in Eastern Oregon

Ergot, caused by Claviceps purpurea, is a major disease of perennial ryegrass grown for seed in eastern Oregon. The objective of this research was to quantify and describe the spatial patterns of ergot severity in each of three 50-ha commercial fields of perennial ryegrass grown for seed in 2012 and 2013. In total, 1,433 and 1,405 quadrats were sampled among the three fields in 2012 and 2013, respectively, and the percentage of quadrats with ergot ranged from 59 to 90%. The mean incidence of infected seed heads in each quadrat ranged between 13 and 29%, while mean severity in each quadrat ranged from 0.2 to 0.5 sclerotia per seed head. Significant autocorrelation and clustering were observed in all three fields in both years, as indicated by Morans I and spatial analysis by distance indices of aggregation. The mean number of ergot sclerotia collected from each field after harvest ranged between 4 and 15 sclerotia m-2 in 2012 and 18 and 119 sclerotia m-2 in 2013. Sclerotia left in perennial fields after harvest are a significant source of inoculum that should be targeted for control. This is the first study to quantify spatial patterns of ergot in perennial ryegrass and provides insights into possible mechanisms that contribute to ergot etiology and epidemiology.

A rapid, sensitive and field-deployable isothermal assay for the detection of Verticillium alfalfae

Abstract The genus Verticillium contains 10 plant pathogenic species that are responsible for billions of dollars of crop losses annually on more than 400 plant species worldwide. On alfalfa, Verticillium alfalfae causes Verticillium wilt, a disease with the potential to reduce yields of susceptible cultivars by the second harvest year and to limit the productive stand life to less than 3 years. Susceptible alfalfa cultivars exhibit disease symptoms that include chlorotic V-shaped lesions at the leaf tip that cause entire leaflets to become bleached and twisted, leading to defoliation. Stems often remain green after all leaflets have desiccated, and discolouration, stunting and wilting of shoots is visible. Internally, the taproot can show yellow to brown vascular discolouration. International quarantines sometimes prohibit the entry of alfalfa samples contaminated with the Verticillium pathogen. In this investigation, we developed a recombinase polymerase amplification (RPA) assay coupled with a lateral flow device for the specific and sensitive detection of V. alfalfae. The primers targeted the translation elongation factor 1α (TEF-1α) gene and were designed with mismatches to increase the specificity of the assay. This assay detected as little as 800 fg of DNA from all tested isolates of V. alfalfae. Moreover, non-specific amplification of closely related V. nonalfalfae or other Verticillium species was not observed. Detection of V. alfalfae in inoculated alfalfa cultivars with high or low resistance further demonstrated the specificity and sensitivity of the diagnostic assay. Additionally, RPA detected V. alfalfae from inoculated plants using a crude DNA extraction method. The assay is easy to use and will allow growers, diagnostic labs and regulatory agencies to determine if V. alfalfae is present in alfalfa products.

Late summer disease symptoms in western Washington red raspberry fields associated with co-occurrence of Phytophthora rubi, Verticillium dahliae, and Pratylenchus penetrans, but not Raspberry bushy dwarf virus.

Sixty percent of the

Simple sequence repeat markers that identify Claviceps species and strains

109 million processed red raspberry industry of the United States occurs in northern Washington State. In 2012, late-summer symptoms of vascular wilt and root disease were observed in many raspberry plantings. These symptoms were initially attributed to Verticillium dahliae. However, diagnostic tests for the pathogen were often contradictory and other soilborne pathogens (Phytophthora rubi and Pratylenchus penetrans) or Raspberry bushy dwarf virus (RBDV) might also have been involved. Therefore, a survey was conducted in 2013 and 2014 to (i) establish the incidence and soil population levels of V. dahliae in red raspberry production fields, (ii) compare among diagnostic methods and laboratories for detecting and quantifying V. dahliae from raspberry field soil, and (iii) assess which pathogens are associated with late-summer disease symptoms of raspberry. Plant and soil samples were collected from 51 disease sites and 20 healthy sites located in 24 production fields. Samples were analyzed for the presence and quantity of each pathogen using traditional plating and extraction methods (V. dahliae, P. rubi, and P. penetrans), quantitative polymerase chain reaction (qPCR) (V. dahliae and P. rubi), and enzyme-linked immunosorbent assay (RBDV). Results showed that V. dahliae was present in 88% of the production fields and that detection of the pathogen differed by method and by laboratory: qPCR detected V. dahliae in the soil from approximately three times as many sites (51 of 71 total sites) as by plating on NP10 semi-selective medium (15 of 71 total sites). Soil populations of V. dahliae were slightly greater at disease sites, but the pathogen was detected with similar frequency from healthy sites and it was rarely isolated from diseased plants (4%). P. rubi, P. penetrans, and RBDV were also common in production fields (79, 91, and 53% of fields, respectively). Both P. rubi (soil and root samples) and P. penetrans (root populations only), but not RBDV, were more frequently found at disease sites than healthy sites, and the amount of P. rubi detected by qPCR was greater from disease sites than healthy sites. In addition, P. rubi was isolated from 27% of the symptomatic plants located at disease sites. Regardless of detection method, V. dahliae, P. rubi, and P. penetrans, either with or without RBDV, were more likely to co-occur at disease sites (73%) than healthy sites (35%), suggesting that a soilborne disease complex is present in raspberry production fields. Results indicate that P. rubi is the primary pathogen most strongly associated with late-summer symptoms of disease, but root populations of P. penetrans and higher soil populations of V. dahliae may also be of concern. Therefore, disease control methods should focus on all three soilborne pathogens.