Development of a PCR-Based Assay for Identifying Members of the Pseudomonas syringae Species Complex from Environmental Samples

View the project final report

Commodities

• Vegetables: tomatoes

Practices

• Pest Management: integrated pest management, prevention

There is a growing appreciation for the diversity of bacteria within the Pseudomonas syringae species complex (Pssc) that are capable of causing disease on tomato. Further, non-pathogenic lineages of the Pssc that are present in environmental reservoirs have been shown to be capable of adapting to agricultural settings and emerging as highly virulent pathogens. The goal of the proposed research is to develop an assay for screening various types of environmental samples that is both highly specific to the Pssc and offers a high degree of discriminatory ability within the Pssc. This assay involves the amplification of a genomic region partially encoding an R-type tailocin ubiquitous in the Pssc. Using primers that have been developed and tested both in-silico and in-planta, I will assess the ability of the assay to specifically amplify Pssc members from plant material, soil, and rain - all of which are important reservoirs for the Pssc. I will also determine the effectiveness and lower detection limit on commercial tomato seed, a common means of transmission for Pssc tomato pathogens. In addition to validating the molecular biology, I will also develop a software pipeline for accurately and reproducibly analyzing data obtained from the assay. The results of this study will provide extension staff and researchers with a valuable tool for studying and managing Pssc-mediated disease on tomato. Results will be communicated in publications and presentations geared toward extension staff, farmers, and agricultural researchers.

In this project, I propose to develop an assay for researchers to detect and monitor a broad range of Pssc lineages associated with disease in tomato.

The specific objectives of my proposal are:

1. Assess the specificity and discriminatory ability of a Pssc-tailocin primer set compared to culture-based method of detection within natural microbial communities. Expected outcome: this objective will describe the ability of PCR assay to exclusively detect the Pssc while differentiating between different clades within Pssc communities.
2. Determine the lower limits of detection in seed and water, both common sources of pathogen transmission. Expected outcome: This objective will determine how abundant different lineages need to be for the assay to detect them in common sources of pathogens. This has important implications for source tracking during epidemics and in the case of seed, screening contaminated seed that might have very low but still infectious numbers of pathogenic strains.
3. Develop a software pipeline to predict identities of lineages detected through PCR-based assay. Expected outcome: This objective will provide extension faculty with a simple, accurate approach for analyzing the data obtained from PCR-amplification with the Pssc-tailocin primers. The output of the pipeline will include most probable identities of unique lineages detected as well as calculated measures of diversity.

Overall, the expected outcome for all three objectives together is to provide a fully-developed assay for extension faculty and plant epidemiologists to efficiently and accurately survey natural sources of Pssc members, identify known tomato pathogens throughout the complex, and better detect environmental lineages that might represent emerging tomato pathogens.