EFFECTS OF THE BACTERIAL WILT PATHOGEN RALSTONIA SOLANACEARUM ON TOMATO ROOT ARCHITECTURE

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: TERMINATED
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 0233593
• Project No.: IND011293
• Project Start Date: Apr 1, 2013
• Project End Date: Sep 30, 2017

Project Director
Iyer-Pascuzzi, AN.

Recipient Organization
PURDUE UNIVERSITY
(N/A)
WEST LAFAYETTE,IN 47907

Performing Department
Botany & Plant Pathology

Non Technical Summary
The soil-borne bacterium Ralstonia solanacearum causes bacterial wilt, one of the most destructive diseases of tomato worldwide. Host plant resistance is the most effective measure of disease control, but is poorly understood. Since Ralstonia first infects root systems, understanding how root architecture is affected by this pathogen is an important step in understanding bacterial wilt resistance in tomato.

Animal Health Component

(N/A)

Research Effort Categories

Basic

(N/A)

Applied

(N/A)

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
102	1460	1160	25%
102	4010	1160	25%
212	1460	1160	25%
212	4010	1160	25%

Knowledge Area
212 - Pathogens and Nematodes Affecting Plants; 102 - Soil, Plant, Water, Nutrient Relationships;

Subject Of Investigation
1460 - Tomato; 4010 - Bacteria;

Field Of Science
1160 - Pathology;

Keywords

tomato bacterial wilt

plant disease resistance

root architecture

soil-borne pathogen

ralstonia solanacearum

temperature

rhizotron

Goals / Objectives
Ralstonia solanacearum, the causal agent of bacterial wilt, is a devastating soil-borne pathogen of tomato. Despite this, host plant resistance to Ralstonia remains poorly understood. The long-term goal of our research is to identify mechanisms underlying resistance to bacterial wilt in tomato. Since Ralstonia first infects the root, understanding how root systems are affected by the pathogen is an important first step towards this goal. Thus the specific objectives of this proposal are: 1. Development of a tomato-tron to non-destructively observe infected and uninfected tomato roots growing in soil over time. 2. Investigate how root architecture is differentially altered by Ralstonia solanacearum in wild and domestic tomato varieties with different levels of resistance and susceptibility. 3. Examine the effects of temperature on tomato root systems infected with Ralstonia. The work presented here will lay the foundation for future research dissecting the molecular and genetic mechanisms of bacterial wilt resistance in tomato roots.

Project Methods
Objective 1: Development of a tomato-tron to non-destructively observe infected and uninfected tomato roots growing in soil over time. A long-term goal of our work is to understand how the bacterial wilt pathogen Ralstonia solanacearum affects root development, particularly root system architecture (RSA, Lynch 1995). One way to investigate this is to examine root architectural changes in resistant and susceptible tomato varieties infected with Ralstonia. Examination of root architecture after pathogen infection requires the ability to non-destructively view soil-grown root systems over time. We will develop the tomato-tron, a soil-based root growth and observation rhizotron for seedlings. The tomato-tron will be composed of two glass plates spaced approximately 1 - 3 mm apart and filled with soil. Seeds will be placed in soil at the top of the plates and root architecture viewed through the glass plates as root systems grow. For Ralstonia infection, plates will be immersed in a solution containing the bacteria. Additional plants will be mock-inoculated with water. Plates can be scanned to examine root architecture at any point pre- or post-infection over a multiple-day period. In the future we will use this system as a platform to explore the molecular and genetic basis of wilt. Objective 2: Investigate how root architecture is differentially altered by the tomato pathogen Ralstonia solanacearum in wild and domesticated tomato varieties with different levels of resistance. Since Ralstonia invades the plant through the root systems, and root systems can adapt to explore resources in changing environments, we hypothesize 1) that the development of root system architecture will be altered by bacterial infection, and 2) that resistant and susceptible plants will be differentially affected by infection. We will examine the root system architecture of several resistant and susceptible tomato varieties. Plants will be grown in the tomato-trons and rhizotron plates will be scanned at specific time-points after Ralstonia or mock infection. RSA traits will be measured from scanned images using ImageJ software. Objective 3: Examine the effects of temperature on tomato root systems infected with Ralstonia Bacterial wilt infection is strongly influenced by environmental factors, including temperature (Hayward et al 1991; Scott et al 2005). Root development is also strongly impacted by changing environmental conditions (Lynch 1995). We hypothesize that changes in root architecture under different temperature regimes may contribute to the altered disease pressure observed at different temperatures. We will use accessions from objective 2 that showed altered root architecture after infection with Ralstonia. Root architecture changes at three different temperature regimes with three different Ralstonia strains will be investigated using the same methods in objective 2. In addition, we will test whether bacterial movement within the root is altered under each temperature regime, and whether root architecture changes and/or bacterial movement correlates with aerial wilt symptoms.

Progress 04/01/13 to 09/30/17

Outputs
Target Audience:Plant scientists - faculty, postdocs, graduate students, undergraduates - at acadmic and research insitutions and in industry, both in the US and globally. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project has trained one postdoc, graduate student and several undergraduates in plant pathology, plant genetics, molecular biology, and root biology. They have all learned microscopy techniques and bioinformatics skills. As a result, they are better trained for the workforce. How have the results been disseminated to communities of interest?Results have been disseminated in the form of publications, national conferences, and seminars at other institutions. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? We have identified QTL for resistance to R. solanacearum in a population of recombinant inbred lines of tomato. We are currently adding additional molecular markers to the map so that we can bettter localize the QTL. Using a reverse genetics approach, we identified a gene that contribuest to tomato resistance to R. solanacearum. This work has just been accepted for publication in Mol. Plant Microbe Interactions. We performed a screen of wild species of tomato with one strain of R. solananacearum.

Publications

• Type: Journal Articles Status: Published Year Published: 2017 Citation: Kim H, Kim BS, Shim JE, Hwang S, Yang S, Kim E, Iyer-Pascuzzi AS, and Lee I. 2017. Network-based unveiling of complex traits in tomato, a model crop for fleshy fruits. Molecular Plant, 10: 652-655.
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Caldwell D, Kim BS, Iyer-Pascuzzi AS. 2017. Ralstonia solanacearum differentially colonizes roots of resistant and susceptible tomato plants. Phytopathology, 107: 528-536.
• Type: Journal Articles Status: Accepted Year Published: 2017 Citation: French E, Kim BS, Rivera-Zuluaga K, Iyer-Pascuzzi AS. 2017. Whole root transcriptomic analysis suggests a role for auxin pathways in resistance to Ralstonia solanacearum in tomato. MPMI, accepted

Progress 10/01/15 to 09/30/16

Outputs
Target Audience:During this reporting period, Dr. Iyer-Pascuzzi reached three types of target audiences: 1) Scientists: scientists were reached throughthree publications: two about bacterial wilt in tomato and onein Arabidopsis root development,2) High school students and community citizens: Dr. Iyer-Pascuzzi reached out to minority high-school students and community citizens with two-fourlectures in a summer workshop about microbes and plants held at Purdue. 3) Undergraduate students: Dr. Iyer-Pascuzzi reached undergraduate students at Purdue through teaching a Spring semester class about Plant growth and development, and by hosting undergraduates in her lab. Changes/Problems:No major changes or problems. What opportunities for training and professional development has the project provided?One post-doc, twograduate students, and one undergraduate student have been receiving training in plant biology and plant pathology with this project. How have the results been disseminated to communities of interest?Dr. Iyer-Pascuzzi published two papers describing bacterial wilt disease in 2016. What do you plan to do during the next reporting period to accomplish the goals?Dr. Iyer-Pascuzzi will continue to publish additional manuscripts and will give scientific talks at meetings in 2017.

Impacts
What was accomplished under these goals? We have been continuing to focuson Aim 2: Investigate how root architecture is differentially altered by Ralstonia solanacearum in wild and domestic tomato varieties with different levels of resistance and susceptibility. We have found significant differences between resistant and susceptible lines in terms of lateral root production and our recent work suggests that this is due to the plant hormone auxin.

Publications

• Type: Journal Articles Status: Published Year Published: 2016 Citation: Kim, BS., French, E., Caldwell, D., Harrington, EJ., Iyer-Pascuzzi, AS., Bacterial wilt disease: Host resistance and pathogen virulence mechanisms Physiological and Molecular Plant Pathology 95, 37-43
• Type: Journal Articles Status: Published Year Published: 2016 Citation: French, E., Kim, BS., Iyer-Pascuzzi AS., Mechanisms of quantitative disease resistance in plants Seminars in cell & developmental biology

Progress 10/01/14 to 09/30/15

Outputs
Target Audience:During this reporting period, Dr. Iyer-Pascuzzi reached three types of target audiences: 1) Scientists: scientists were reached with the publication of a review on genomics in Arabidopsis root development, and by two international talks (London and Japan) about the role of roots in bacterial wilt disease. 2) High school students: Dr. Iyer-Pascuzzi reached out to minority high-school students with two lectures in a summer workshop about microbes and plants held at Purdue. 3) Undergraduate students: Dr. Iyer-Pascuzzi reached undergraduate students at Purdue through teaching a Spring semester class about Plant growth and development, and by hosting undergraduates in her lab. Changes/Problems:We focused this year on Aim 2, and no major changes or problems occurred. What opportunities for training and professional development has the project provided?One post-doc, one graduate student, and one undergraduate student have been receiving training in plant biology and plant pathology with this project. How have the results been disseminated to communities of interest?Dr. Iyer-Pascuzzi gave two international talks in 2015 describing results and ongoing work on this project. What do you plan to do during the next reporting period to accomplish the goals?Dr. Iyer-Pascuzzi plans to publish several papers on this project next year.

Impacts
What was accomplished under these goals? We have been focusing on Aim 2: Investigate how root architecture is differentially altered by Ralstonia solanacearum in wild and domestic tomato varieties with different levels of resistance and susceptibility. We have found significant differences between resistant and susceptible lines in terms of lateral root production and are currently investigating the molecular basis of this.

Publications

• Type: Journal Articles Status: Published Year Published: 2015 Citation: Karve R and Iyer-Pascuzzi AS. Digging deeper: high-resolution genome-scale data yields new insights into root biology. Curr Opin Plant Biol. 2015 doi: 10.1016/j.pbi.2015.01.007. PMID: 25636037

Progress 10/01/13 to 09/30/14

Outputs
Target Audience: Dr. Iyer-Pascuzzi reached scientists with the publication of a review on Arabidopsis root development, and reached middle and elementary school students through her participation in 'zipTrips'. zipTrips is a Purdue-based outreach that brings virtual electronic field trips with Purdue Scientists to middle and elementary school science classes in video form. Dr. Iyer-Pascuzzi participated in the Sept 2014 ZipTrip entitled 'Plant Science: The Green Machine'. Changes/Problems: We tried using the 'tomato-tron' but could not get plants to become infected with the pathogen, and could not see the roots well. Now, we grow plants in cones and remove them to view the root systems. We wash dirt from the root systems, scan and measure them for the desired traits. What opportunities for training and professional development has the project provided? One postdoctoral student and one graduate student are currently being trained in this project. The postdoc attended the Mid-Western ASPB meeting and the North-Central APS meeting and displayed a poster of his results at both. How have the results been disseminated to communities of interest? Public understanding of this work was enhanced through Dr. Iyer-Pascuzzi's participation in Purdue's zipTrips, electronic field trips for middle and elementary age students. Dr. Iyer-Pascuzzi participated in the Plant Science: Green Machine zipTrip, held in Sept 2014. She explained the concepts of disease resistance and QTL analysis, and discussed the pathogen with which she works. What do you plan to do during the next reporting period to accomplish the goals? We will finish the QTL analysis and write the first paper describing these results, and we will examine the effect of temperature upon infection. We will also participate in additional outreach.

Impacts
What was accomplished under these goals? We have investigated the effects of Ralstonia solanacearum, the casual agent of bacterial wilt, on root growth of inoculated resistant (R) and susceptible (S) tomato plants. We do not use the tomato-trons because we were not able to infect the tomatoes with the pathogen, and we could not see the roots well. Thus we now use a destructive method in which we grow plants in pots, remove them, wash the roots, scan them, and then take our measurements. This works quite well and we are able to get high-quality data. For the second objective, we have found that S plants have a more significantly reduced root biomass, weight and length after inoculation compared to R plants. The changes in these traits correlate with susceptibility across a number of different varieites. Further, the length of the initial primary root is more significantly reduced in S plants. We are currently conducting a QTL analysis for BW resistance using wilt scores, root traits and shoot traits to determine the genetic basis for resistance. We are the first to show that root traits correlate with resistance, and the first, to my knowledge to use such traits in a QTL analysis. For the third objective, we have found that differences in heat alter the root systems of tomato plants, and we will be examining how temperature alters infection in the future.

Publications

Progress 04/01/13 to 09/30/13

Outputs
Target Audience: Plant pathologists and tomato breeders. Changes/Problems: We did not receive our permit to use Ralstonia until October 2013, and thus are delayed in achieving the project goals. What opportunities for training and professional development has the project provided? A technician was mentored in plant biology by the professor. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? During the reporting period, we developed a rhizotron soil-based system to view tomato root architecture (goal 1). However, since a permit from APHIS is required to use this pathogen and I did not receive the permit until mid-October 2013, the rest of the goals have not yet been accomplished. We have just recently begun inoculations and examinations of root architecture after Ralstonia infection, but this does not fall within the reporting period.

Publications

• Type: Journal Articles Status: Awaiting Publication Year Published: 2014 Citation: Anjali Iyer-Pascuzzi and Rosangela Sozzani, Postembryonic Control of Root Meristem Growth and Development. 2014. Current Opinion in Plant Biology 17: 7-12, in press