MANAGEMENT OF THE EMERGING TOMATO YELLOW LEAF CURL AND POTYVIRAL DISEASES OF TOMATO IN HAWAII

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: SPECIAL GRANT
• Reporting Frequency: Annual
• Accession No.: 0222999
• Grant No.: 2010-34135-21378
• Proposal No.: 2010-03383
• Project Start Date: Sep 1, 2010
• Project End Date: Aug 31, 2012
• Grant Year: 2010
• Program Code: [AH]- Tropical & Subtropical Research/T STAR

Recipient Organization
UNIV OF HAWAII
3190 MAILE WAY
HONOLULU,HI 96822

Performing Department
Plant & Environmental Protection Sciences

Non Technical Summary
Tomato yellow leaf curl, perhaps the most destructive disease of tomato, and a disease of tomato associated with potyvirus infection, have recently been discovered in Hawaii. Given the importance of tomato in Hawaiian agriculture, it is imperative to develop a management strategy to help protect Hawaii's commercial and residential tomato growers. In this proposal, we present a research plan that will aid in the short- and long-term management of these diseases in Hawaii. This project addresses TSTAR Program Goals #1 (Provide research that maintains and enhances production of established tropical and subtropical agricultural products) and #7 (Develop appropriate strategies and tactics to stem the influx of exotic diseases, insects and weeds and to control and/or eliminate extant non-indigenous species and disease). The goal of the proposed research is to use holistic approaches to develop environmentally-friendly and sustainable tomato virus disease management practices to ultimately solve the tomato virus problems.

Animal Health Component

50%

Research Effort Categories

Basic

(N/A)

Applied

50%

Developmental

50%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
212	1499	1101	100%

Knowledge Area
212 - Pathogens and Nematodes Affecting Plants;

Subject Of Investigation
1499 - Vegetables, general/other;

Field Of Science
1101 - Virology;

Keywords

tomato

viruses

Goals / Objectives
The tomato (Solanum lycopersicum L.) is one of the most widely cultivated crops in the world (Moussa et al., 2000). In Hawaii, the $10 million annual tomato crop is under a serious threat posed by two emerging viral diseases recently discovered in Hawaii's commercial tomato farms: tomato yellow leaf curl, caused by Tomato yellow leaf curl virus (TYLCV), perhaps the most destructive disease of tomato, and a previously unknown viral disease associated with the potyviruses Potato virus Y (PVY) and Tobacco etch virus (TEV). It is imperative to develop a holistic management strategy to help protect Hawaii's commercial and residential tomato growers. In this proposal, we present a research plan that will aid in the short- and long-term management of these diseases in Hawaii: 1. Sequence the entire genome of TYLCV, PVY and TEV strains from different locations 2. Conduct statewide surveys of TYLCV, PVY and TEV and county workshops 3. Determine the host range of TYLCV, PVY and TEV in Hawaii 4. Transform local tomato varieties for resistance to TYLCV and potyviruses

Project Methods
1.Sequencing the entire genomes of TYLCV and Potyvirus strains is a relatively straightforward procedure. We will design oligonucleotide primers to amplify the remaining genomic sequence (~1.2 kb) of different TYLCV strains using PCR. For potyviruses, primers containing highly conserved sequence motifs found in all published sequences of the family Potyviridae will be utilized using RT-PCR. These amplicons will be cloned and sequenced using standard protocols. Sequence comparisons will then be made, and phylogenetic relationships will be inferred using sequence analysis software. 2. Tomatoes in Hawaii will be surveyed for the presence and prevalence of viruses. We will work with county Extension Agents to visit commercial tomato farms and interested residential growers in each county. We will collect tomato fruit and leaf samples from as many locations as possible, as well as other potential hosts of the viruses. Samples will be brought back to the laboratory and frozen and processed using a non-radioactive squash-blot (tomato samples) or dot-blot hybridization assay. We will also develop and distribute a squash blot kit to Extension Agents and growers so they can perform the squash blot in the field and mail the kit to our laboratory for processing. This will obviate the need to send plant samples through the mail, which is more time consuming, expensive, and prone to sample degradation. While visiting each county for the statewide survey, we will conduct a workshop at a CES office. This workshop will educate the participants about the diseases and provide an opportunity to ask us or Extension Agents any questions they may have. We will also distribute the squash blot kits to growers at these workshops. 3. It appears that Poamoho has very high disease pressure, as almost 200 tomato plants became simultaneously infected with TYLCV. A rapid spread of Potyvirus-infected tomatoes was observed on the windward coast of Hawaii. We will conduct extensive sampling of other crops and weeds growing in these areas to determine whether they are serving as a virus reservoir. Our preliminary work has already indicated that Apple of Peru (Nicandra physalodes) and Comb hyptis (Hyptis pectinata), two common weeds in Hawaii, serve as TYLCV reservoirs. We will collect seeds from all crops and weeds and grow them in the greenhouse where they will serve as negative controls for our detections assays. 4. We will use TYLCV and Potyvirus sequences to transform locally-bred varieties of tomato such as Kewalo, Healani, and Komohana to develop transgenic tomato lines using a segmented construct comprised of multiple gene segments in order to confer multiple virus resistance. We have routinely transformed tomato for other projects and will use these established transformation protocols for this study. Putatively transgenic plants will be analyzed using molecular methods to confirm integration of the desired sequences. These plants, each representing a unique line, will be vegetatively propagated and assayed for resistance to TYLCV and potyviruses through the use of viruliferous whiteflies and alate aphids.

Progress 09/01/10 to 08/31/12

Outputs
OUTPUTS: Objective 1. The entire genome of TYLCV isolates from Hawaii (Kailua-Kona), Maui (Wailuku), and Oahu (Poamoho) were sequenced and the data deposited into GenBank (Accessions HM988987, GU322424, GU322423, respectively). These genomic sequenced indicated the TYLCV strains present in Hawaii are from the IL-clade, and likely originated from North America and/or Asia. The perceived threat posed by PVY and TEV to tomato production did not develop in 2010/2011, and additional sequence information on these two viruses was not pursued. Two additional viruses, however, were identified in Hawaii's tomato crop and partially sequenced. Southern tomato virus (STV) and Pepper mottle virus (PepMoV) were identified for the first time in Hawaii. The threat of these viruses to tomato production in Hawaii is unclear. Objective 2. Hundreds of tomato samples were collected by us, CTAHR Extension Agents, and HDOA personnel, as well as concerned growers via the ADSC were processed for the presence of TYLCV. Based on this survey, TYLCV is currently widespread on Oahu (except the windward coast) and Maui, and is limited to the Kona coast on the Big Island. We found no positive samples from Kauai, Molokai, or the Hilo side of the Big Island, although that does not necessarily mean TYLCV is absent at these locations. PVY and TEV did not become at large problem in the 2010/2011 season, so we did not survey for these viruses, however, we suspect they are widespread in Hawaii as they have been present for many years and have a wide host range. PepMoV was first identified in a single tomato field on Oahu in September 2011, where it was present in almost 20% of the ~300 samples tested. We will consider performing a statewide survey for this virus, as it has never been previously reported in Hawaii. 3. We screened 28 common agricultural weed and crop species to determine if they were alternative hosts for TYLCV, and thereby served as reservoirs for the virus. Of these species, 10 were putatively identified as hosts of the virus. It is unknown whether the virus can be naturally transmitted from any of these suspected alternative hosts to tomato. 4. With the help of other CTAHR personnel, including Jari Sugano, Theodore Radovich, Steve Fukuda, Susan Migita and others, we completed three field trials evaluating conventionally-bred virus-resistant tomato varieties. In the first trial, most varieties had good resistance to TYLCV, but were susceptible to Tomato spotted wilt virus (TSWV), another hindrance to tomato production in Hawaii. In the second and third tirials, we evaluated varieties that have resistance to both TYLCV and TSWV. Approximately 30 tomato varieties have been evaluated in the field and we have held multiple field days which allow growers to ask questions about these virus diseases and see how these varieties perform in the field. In collaboration with scientists at USDA-ARS-PBARC, we conducted a field trial evaluating three transgenic tomato lines for resistance to TSWV. All three lines performed better than non-transgenic control lines, with one of the lines appearing to be immune to TSWV infection. PARTICIPANTS: Researchers at CATAR, UHM (including Wayne Borth, Michael Melzer, Jari Sugano, Theodore Radovich, Steve Fukuda, and Susan Migita) and at USDA-ARS-PBARC (including Dennis Gonsalves, Tracie Matsumoto) were involved in the research. TARGET AUDIENCES: Plant pathologists, plant virologists, plant breeders, extension agents, growers, etc. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Sequenced the genomes of three strains of TYLCV from different Hawaiian islands Determined the current distribution of TYLCV in Hawaii Identified two additional viruses in Hawaii's tomato industry: Southern tomato virus and Pepper mottle virus Evaluated thirty four conventionally-bred tomato varieties with virus resistance Evaluated three transgenic tomato lines with virus resistance Held three educational field days for Hawaii's tomato growers on virus diseases and the varieties they can grow to combat these diseases

Publications

• Sugano et al. 2011. Field Evaluations of Tomato Yellow Leaf Curl Virus Resistant Varieties for Commercial Production. College of Tropical Agriculture and Human Resources Publication PD-78
• Melzer et al. 2012. Tomato spotted wilt. College of Tropical Agriculture and Human Resources Publication PD-81
• Melzer et al. 2012. First report of Pepper mottle virus in Hawaii. Plant Disease 96:917

Progress 09/01/10 to 08/31/11

Outputs
OUTPUTS: Objective 1. Sequence virus genome The entire genome of TYLCV isolates from Hawaii (Kailua-Kona), Maui (Wailuku), and Oahu (Poamoho) were sequenced and the data deposited into GenBank (Accessions HM988987, GU322424, GU322423, respectively). These genomic sequenced indicated the TYLCV strains present in Hawaii are from the IL-clade, and likely originated from North America and/or Asia. The perceived threat posed by PVY and TEV to tomato production did not develop in 2010/2011, and additional sequence information on these two viruses was not pursued. Two additional viruses, however, were identified in Hawaii's tomato crop and partially sequenced. Southern tomato virus (STV) and Pepper mottle virus (PepMoV) were identified for the first time in Hawaii. The threat of these viruses to tomato production in Hawaii is unclear. Objective 2. Conduct statewide surveys Hundreds of tomato samples were collected by us, CTAHR Extension Agents, and HDOA personnel, as well as concerned growers via the ADSC were processed for the presence of TYLCV. Based on this survey, TYLCV is currently widespread on Oahu (except the windward coast) and Maui, and is limited to the Kona coast on the Big Island. We found no positive samples from Kauai, Molokai, or the Hilo side of the Big Island, although that does not necessarily mean TYLCV is absent at these locations. PVY and TEV did not become at large problem in the 2010/2011 season, so we did not survey for these viruses, however, we suspect they are widespread in Hawaii as they have been present for many years and have a wide host range. PepMoV was first identified in a single tomato field on Oahu in September 2011, where it was present in almost 20% of the ~300 samples tested. We will consider performing a statewide survey for this virus, as it has never been previously reported in Hawaii. Objective 3. Determine the host range We screened 28 common agricultural weed and crop species to determine if they were alternative hosts for TYLCV, and thereby served as reservoirs for the virus. Of these species, 10 were putatively identified as hosts of the virus. It is unknown whether the virus can be naturally transmitted from any of these suspected alternative hosts to tomato. Objective 4. Local tomato varieties With the help of other CTAHR personnel, including Jari Sugano, Theodore Radovich, Steve Fukuda, Susan Migita and others, we are about to complete our third field trial evaluating conventionally-bred virus-resistant tomato varieties. In the first trial, most varieties had good resistance to TYLCV, but were susceptible to Tomato spotted wilt virus (TSWV), another hindrance to tomato production in Hawaii. In the second and third tirials, we are evaluating varieties that have resistance to both TYLCV and TSWV. Approximately 30 tomato varieties have been evaluated in the field and we have held field days which allow growers to ask questions about these virus diseases and see how these varieties perform in the field. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
Hawaii's tomato industry has expanded dramatically in the last 15 years and currently has annual sales of approximately $10 million. Tomato spotted wilt virus (TSWV) has long been a major hindrance to local tomato production. Two new and emerging viral diseases of tomato further threaten Hawaii's tomato industry. Tomato yellow leaf curl, caused by Tomato yellow leaf curl virus (TYLCV), is perhaps the most destructive disease of tomato, and a viral disease associated with the potyviruses Potato virus Y (PVY) and Tobacco etch virus (TEV) have recently been discovered in Hawaii. It is imperative to develop a holistic management strategy to help protect Hawaii's commercial and residential tomato growers.

Publications

• Sugano et al. 2011. Evaluations of Tomato Yellow Leaf Curl Virus Resistant Varieties for Commercial Production. Hānai'Ai/The Food Provider, June-July-August Issue