Location: Temperate Tree Fruit and Vegetable Research
Project Number: 2092-21220-002-002-T
Project Type: Trust Fund Cooperative Agreement
Start Date: Apr 1, 2017
End Date: Mar 31, 2022
Objective:
Identify if one or more pathogens cause the unusual symptoms occurring mid- to late-season in potato fields in the Columbia Basin. Objective 1: Grafting diseased plant tissue. Objective 2. Molecular diagnostics of diseased plants.
Sub-objective A: Established molecular diagnostic tools will be used to determine if known fungal, bacterial, and/or viral pathogens are present in the symptomatic plant materials. Sub-objective B: Molecular diagnostic tools designed to identify unknown pathogens will be used to determine if fungal and/or bacterial pathogens are present in the symptomatic plant material.
Objective 3. Fulfill Koch’s postulate for pathogens identified in field samples.
Approach:
Symptomatic plants in commercial and research fields will be identified, symptoms recorded, and the overall disease pressure in the field of interest and nearby fields will be noted. Foliar tissue from the symptomatic plants and asymptomatic plants located nearby will be collected. Scions will be generated from terminal or axillary leaf buds of symptomatic tissue from the diseased plants, and grafted onto healthy potato plants grown in the greenhouse. Grafted plants will be maintained in the greenhouse and observed on a weekly basis for up to six weeks. Symptoms arising in the indicator plants will be noted. Symptomatic and asymptomatic plants collected as above for grafting will simultaneously be analyzed for the presence of known pathogens using molecular methods. Nucleic acids will be extracted from different tissue of symptomatic and asymptomatic plants using the dellaporta protocol. DNA extracts will be used as templates for targeted PCR analyses to determine if known fungal, bacterial, and/or viral agents are present. All reactions will be run in a methodical matter, focusing on pathogens most likely to cause the specific disease symptoms of each individual symptomatic plant to be analyzed. Standard PCR or RT-PCR procedures will be followed. Nucleic acid extractions will be used as template for PCR analyses using universal primers that target diverse species that are either fungal or bacterial in nature. Fungal Pathogens: The Internal Transcribed Spacer (ITS) regions within fungal ribosomal DNA genes will be targeted using primers designed to amplify any fungal DNA present, with limited to no amplification of plant DNA. Bacterial Pathogens: The bacterial 16S ribosomal DNA coding region will be targeted using degenerate primers that specifically amplify the bacterial DNA and not the plant DNA. Any unknown fungal or bacterial product produced by PCR analysis will be purified and subject to cloning, followed by sequence analysis to identify the specific pathogen(s) present. Note that a high number of clones will be selected for sequence analysis to ensure that pathogens will be detected despite pathogen titer in the plants. If the symptoms are confirmed to be of biological nature by grafting, and a particular pathogen(s) is detected, the pathogen(s) will be purified by known species-specific methods. Healthy potato plants grown in the greenhouse will be inoculated. An incubation time of one to two months will be used to allow the inoculated plants adequate time to develop symptoms. Any plants that develop symptoms will be collected, the pathogen(s) isolated and purification will be done according to species-specific methods. Any re-isolated pathogen will be identified by standard molecular biology methods.
