Transformation of Epichloë typhina by electroporation of conidia.

BACKGROUND: Choke, caused by the endophytic fungus Epichloë typhina, is an important disease affecting orchardgrass (Dactylis glomerata L.) seed production in the Willamette Valley. Little is known concerning the conditions necessary for successful infection of orchardgrass by E. typhina. Detection of E. typhina in plants early in the disease cycle can be difficult due to the sparse distribution of hyphae in the plant. Therefore, a sensitive method to detect fungal infection in plants would provide an invaluable tool for elucidating the conditions for establishment of infection in orchardgrass. Utilization of a marker gene, such as the green fluorescent protein (GFP), transformed into Epichloë will facilitate characterization of the initial stages of infection and establishment of the fungus in plants. FINDINGS: We have developed a rapid, efficient, and reproducible transformation method using electroporation of germinating Epichloë conidia isolated from infected plants. CONCLUSIONS: The GFP labelled E. typhina provides a valuable molecular tool to researchers studying conditions and mechanisms involved in the establishment of choke disease in orchardgrass.

Tilletia vankyi, a new species of reticulate-spored bunt fungus with non-conjugating basidiospores infecting species of Festuca and Lolium.

A bunt fungus, exhibiting a spore germination pattern unique to known reticulate-spored species of Tilletia was found infecting plants in seed production fields of Festuca rubra ssp. rubra (red fescue) and F. rubra ssp. fallax (Chewing's fescue) in Oregon, and in seed lots of Lolium perenne (perennial ryegrass) from Australia and Germany. Teliospores germinated to form 20-40 uninucleate, non-conjugating basidiospores, and colonies derived from single basidiospores produced teliospores in culture. In inoculation studies using single basidiospore colonies, perennial ryegrass and L. perenne ssp. multiflorum (Italian or annual ryegrass) were infected. A phylogenetic analysis, based on ITS region rDNA, eukaryotic translation elongation factor 1 alpha, and the second largest subunit of RNA polymerase II demonstrated that the fescue and ryegrass bunts are conspecific, and distinct from known species of Tilletia.

Quantitative Assessment of Anguina sp. and Rathayibacter rathayi in Dactylis glomerata Seed Production Fields in Oregon and Estimates of Yield Loss.

Anguina sp. is a nematode that infests the inflorescence of orchardgrass and forms galls that replace the seed. Anguina sp. is also a vector of the bacterial pathogen Rathayibacter rathayi, which causes galls or gummosis in orchardgrass (Dactylis glomerata) panicles. The percentage of orchardgrass panicles infected or percentage of seed loss from Anguina sp. or R. rathayi in five commercial orchardgrass seed-production fields in Oregon during 2003 and 2004 was determined. The percentage of panicles with Anguina sp. ranged from 9 to 24%, although the percentage of seed replaced by Anguina sp. was less than 0.2%. The number of galls per panicle ranged from 1 to 29. However, more than 50% of Anguina-infested panicles contained only a single Anguina gall and few panicles had eight or more galls. The percentage of panicles with R. rathayi ranged from 3 to 27%. Percentage of seed loss from R. rathayi ranged from 0.1 to 7.3%. Seed loss in orchardgrass seed-production fields assessed for both Anguina sp. and R. rathayi was found to be as great as 8%. The number of Anguinagalls remaining in fields following harvest ranged from 0 to 40 per square meter.

Evaluation of Resistance to Ergot, Caused by Claviceps purpurea, in Kentucky Bluegrass, Based on Incidence and Severity Estimates.

Ergot, caused by Claviceps purpurea, is an important disease of Kentucky bluegrass grown for seed. Resistance is a preferred means of disease control, although approaches to evaluating ergot resistance in Kentucky bluegrass are not well established. A large scale disease assessment trial was established to compare within-year and year-to-year variability among measures of ergot incidence or severity in Kentucky bluegrass and to determine the most efficient approaches for cultivar evaluation. The susceptibility of 104 Kentucky bluegrass cultivars (78 commercial and 26 experimental) to ergot was assessed, based on percentage of panicles with sclerotia (incidence), sclerotia per panicle (severity), percentage of seeds replaced by sclerotia, or percentage of sclerotia among seed by weight. Cultivar ranking for susceptibility within years was similar for all methods of ergot assessment. Yearly mean incidence of ergot among cultivars ranged from 1.0 to 97.5% for susceptible cv. HV102 to 0.0 to 2.5% for resistant cv. Huntsville, with remaining cultivars distributed between the extremes. Yearly mean values of ergot incidence and severity differed among years. Much of this variability was due to days with rain during the period from flower initiation to maturity (period of susceptibility). Disease incidence was correlated (P ≤ 0.05) with days with rain in 22 of the cultivars. Duration of flowering was correlated (P ≤ 0.05) with ergot incidence in 11 cultivars. Results from this study suggest that variability in host and environment would likely offset any gain in precision of severity data. In large trials in particular, collection of incidence data (percentage of panicles with ergot) would provide the most timely and efficient means for ergot assessment.

Use of a Seed Scarifier for Detection and Enumeration of Galls of Anguina and Rathayibacter Species in Orchard Grass Seed.

Seed galls, caused by Anguina spp., are normally detected visually in cereals such as wheat and barley. However, in grasses such as orchard grass, the presence of galls induced by Anguina or Rathayibacter spp. are difficult to detect visually due to their infrequent occurrence and masking by lemmas and paleas. To develop improved seed assays for the presence of the nematode and bacterial galls, a small scarifier was used to remove lemmas and paleas without causing major damage to seeds or galls. Following scarification, the galls were visually identified and manually counted under a dissecting microscope. Using the scarifier, several orchard grass seed lots were screened for Anguina and Rathayibacter spp. The percentage of samples of orchard grass seed harvested in the Willamette Valley of Oregon during 1996, 1997, and 2000 containing galls of Anguina sp. were 37, 46, and 48, respectively. The percentage of samples containing bacterial galls with Rathayibacter sp. was 27, 31, and 40, respectively. Total galls with Anguina sp. per 25 g of orchard grass sample ranged from 1 to 24. The mean of Anguina sp. galls per sample in 1996, 1997, and 2000 were 4, 5, and 5, respectively. Total galls with bacteria per 25 g of sample ranged from 27 to 40; mean number of galls per sample in 1996, 1997, and 2000 were, 6, 5, and 11, respectively. This is the first report confirming the presence of Rathayibacter sp. galls in orchard grass in Oregon.