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ARS Home » Pacific West Area » Salinas, California » Crop Improvement and Protection Research » Research » Research Project #441277

Research Project: Understanding the Molecular Mechanisms of Resistance to Tipburn and INSV+Pythium wilt

Location: Crop Improvement and Protection Research

Project Number: 2038-21530-003-005-T
Project Type: Trust Fund Cooperative Agreement

Start Date: Apr 1, 2022
End Date: Mar 31, 2024

Monterey County, California harvests approximately 100,000 acres of lettuce (Lactuca sativa L.) valued at $1.2 billion per year. Local issues threatening this industry include, but are not limited to, the increased prevalence of Impatiens Necrotic Spot Virus (INSV) along with the co-occurrence of soil-borne pathogens such as Pythium wilt, and physiological disorders such as tipburn. INSV and the co-occurrence of Pythium wilt are relatively new problems in Monterey county since 2019, and considerable basic research needs to be done before solutions can be found. Resistance to tipburn is a continuing priority of the lettuce board, but losses due to INSV and other diseases in the field have prevented meaningful results from tipburn trials. Alternative greenhouse trials for tipburn resistant germplasm are necessary to continue to make progress in this area. Developing lettuce germplasm with genetic resistance to INSV and tipburn continues to be the most economical and environmentally safe method of control. Breeders are developing resistant lines, but continually need new information about the genetics and mechanisms by which the plant resists these threats in order to develop new, completely resistant varieties. Our objectives are: 1.) to develop a greenhouse protocol for screening breeding lines for tipburn; 2.) to screen existing breeding lines for markers associated with tipburn resistance; 3.) to develop a greenhouse protocol for inoculating lettuce with INSV and Pythium to observe and understand the basic immune response of the plant to these diseases separately and in conjunction.

In order to develop a greenhouse protocol to screen breeding lines for tipburn, we will grow known susceptible cultivars in growth chambers under variable temperatures and humidity until tipburn is achieved. We will then replicate these conditions in greenhouses and score for tipburn using collaborators’ scoring protocols. Scores in greenhouses will be compared to field data collected in previous years. Developing markers associated with tipburn resistance will be approached by developing a PCR assay to screen for these markers in the parental lines from the published manuscript and the parental lines of the collaborator’s populations. These sequence data will be analyzed to confirm sequence similarity to published markers. Additional markers will then be developed, with emphasis on markers that can be assayed using restriction enzymes, or markers that amplify one variant and not the other. This will allow screening of populations to be based on a simple and inexpensive PCR and agarose gel assay, rather than requiring expensive sequencing. Plants from two tipburn-resistant populations developed by a collaborator will be grown in the greenhouse and screened for these markers. Plants with no tipburn under greenhouse conditions and the appropriate resistant markers will be developed further in future years when field trials can resume. Finally, to develop a greenhouse protocol for inoculating lettuce with INSV and Pythium, we will consult with collaborators with experience inoculating with INSV and Pythium. Pythium will be isolated from the field and developed on millet seed. Plants will be grown in the greenhouse and infected millet seed will be placed in the soil. INSV will be introduced via infected thrips. qPCR assays will be developed to quantify Pythium and ELISA assays will be used to quantify virus titers. We will collect data for regression analysis based on disease score x Pythium concentration, disease score x thrip pressure, disease score x INSV titers, disease score x Pythium concentration x INSV titers, disease score x days after inoculation with Pythium, disease score x days after inoculation with INSV, and we will conduct experiments in growth chambers on the effects of elevated temperature on disease progression.