1a.Objectives (from AD-416):
1) Identify pathogenic Pythium species using RFLP and sequencing based assay.
2) Provide a faster means of estimating pathogenic Pythium species populations using real time PCR.
3) Develop internal control to determine if PCR inhibitors influences the amplification of pathogenic Pythium species target sequences.
1b.Approach (from AD-416):
Plant and soil samples will be collected randomly from under the diseased calla lily plants from the Golden State Bulb Growers in Moss Landing, CA. Infected roots will be cut in 5-mm segment and placed on Pythium selective media plates and will be established in Potato Dextrose Agar after 2 days. Pathogenicity test will be performed in the greenhouse. Identification of Pythium species will be done using PCR-RFLP of the mitochondrially encoded cytochrome oxidase II (cox II) gene followed by sequencing of cox II gene. Soil DNA extraction will be performed using procedures identified previously. Quantification of pathogenic Pythium species will be accomplished using real time PCR quantification assay since it offers a rapid, sensitive, and specific method for the diagnosis of plant pathogens in soil samples. Inoculum densities will also be determined using soil plating. Evaluation of DNA extraction procedures will be done with different soil types.
This agreement supports Objective b “Determine pathogen and weed control efficacy of emerging chemicals as alternatives to methyl bromide in field trials for ornamental systems and strawberry systems” of Objective 4 “Develop effective, practical, economical and environmentally acceptable approaches to replace methyl bromide as a pre-plant soil fumigant in ornamental and strawberry production systems” of the parent project. Thirty-nine isolates of Pythium sp. were recovered from diseased roots of calla lily taken from multiple locations in production fields and tested for pathogenicity in a greenhouse test. All the pathogenic isolates were characterized using PCR amplification and restriction digestion of the Cyclooxygenase-1 & 2 genes. Thirty-five of the isolates were pathogenic and expressed the same genotype; the non-pathogenic isolates exhibited different genotypes. These data suggest the patogenic Pythium isolates from these multiple locations are identical; identification of this isolate down to species is in progress. Subsequently, pathogen DNA was extracted and successfully amplified from soil samples collected from diseased fields. The development of a quantitative Polymerase Chain Reaction (PCR) assay is in development. This work will facilitate rapid identification and quantification of a serious calla lily pathogen which impacts grower decisions of soil fumigation.