Non Technical Summary
To mitigate plant epidemics, we need to understand the threats (plant pathogens). This project focuses on Ralstonia pathogens that cause bacterial wilt disease on crops that are major components of the American diet, including potato, tomato, banana, and peanut. These wilt pathogens are complex organisms that persist in the environment for decades. Moreover, pathogens have genetically encoded variability in their traits. Little is known about the population diversity of Ralstonia in the United States nor how they survive in the environment.

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
212	0210	1100	50%
212	2499	1100	50%

Knowledge Area
212 - Pathogens and Nematodes Affecting Plants;

Subject Of Investigation
0210 - Water resources; 2499 - Plant research, general;

Field Of Science
1100 - Bacteriology;

Goals / Objectives
Objective 1: Identify and characterize traits that contribute to Ralstonia's ability to persist in freshwater. We will use a high-throughput forward genomic screen (RBTnSeq) to identify genetic factors that allow Ralstonia to persist in water. The RB-TnSeq screens will be performed in parallel using representatives of each of the three major Ralstonia clades. We will perform forward genomic screens in freshwater sampled from an irrigation well, an agricultural drainage canal, and a creek in an ecological preserve to evaluate the effects of variations in water chemistry on Ralstonia's genetic mechanisms for persistence. To identify traits that contribute to Ralstonia's exceptional survival ability, we will quantify relative abundance and culturability of barcoded RB-TnSeq mutants using Illumina sequencing and test hypotheses through physiological bioassays. We will use an evolutionary approach to extrapolate the mechanistic findings to the breadth of Ralstonia diversity. This knowledge will support our long-term goal to develop innovative, biologically based strategies to manage bacterial phytopathogens.Objective 2: Develop a scalable framework for surveilling domestic Ralstonia populations. Although knowledge of local Ralstonia populations is necessary to achieve sustainable control of bacterial wilt, there is a major gap in knowledge about the diversity of Ralstonia populations in the U.S. Based on a few studies and our own preliminary genome sequencing efforts, we know that there are at least 11 clonal groups of Ralstonia in the Southeast U.S. and an unknown number in Hawai?i. We will fill this gap in knowledge through an integrative approach where we will use phylogenomics to design and validate a costeffective tool to identify lineages of domestic Ralstonia. To disseminate knowledge on Ralstonia diversity, we will develop an online, interactive pathogen dashboard that stakeholders can use to investigate epidemiological questions. This objective supports our long-term goal to surveille domestic Ralstonia populations in waterways and agricultural production regions by monitoring how they spread and evolve.

Project Methods
Methods will include forward genetic screens (random barcoded transposon site sequencing: RB-TnSeq), targeted mutagenesis, biochemical assays, whole genome sequencing, culturing bacteria from infected plant samples, and multiplex PCR assay development.