2012 Annual Report 1a.Objectives (from AD-416): 1. To improve plant disease diagnostic technologies for accurate, rapid and cost-effective identification of causal agents in tomato plants at the earliest possible time relative to manifestation of disease symptoms. 2. To elucidate factors affecting the epidemiology of the viroid disease, to evaluate the potential synergistic interaction with Pepino mosaic virus on tomato and to identify vulnerable areas (e.g., seed or alternative host) that could potentially provide innovative and effective disease management opportunities. 3. To develop and evaluate innovative technologies including the use of disinfectants, seed treatment or laser knifes to determine their effectiveness in preventing the highly contagious pathogens (i.e., viroids, PepMV, CMM, and botrytis) from spreading. 1b.Approach (from AD-416): 1. Through gene mining in the GenBank databases, computer-assisted alignment of a group of viroid/virus sequences will be used to identify the conserved genomic regions that can be used for primer and probe design in the development of species-specific or genus-specific Real-time RT-PCR systems. Once the real-time RT-PCR detection system is developed and validated, it will then be used to conduct individual or a panel virus/viroid test to monitor the presence and frequency of certain virus or viroid on tomatoes. This surveillance system will serve to identify the critical control points for these pathogens. 2. To determine the etiology of the emerging tomato stunting chlorosis disorder, pathogenicity tests will be conducted. Presence of a specific viroid or virus will be evaluated through symptom expression on the indicator plants and by confirmation tests using above developed real-time RT-PCR techniques and nucleotide sequencing. To investigate the synergic effect between TCDVd and PepMV, one set of tomato plants will be inoculated with PepMV or TCDVd and another set with a mixed culture of both pathogens. The impact of pathogen infection on plant growth and fruit production will be measured over time. The factors affecting the efficiency of mechanical transmission of TCDVd will be conducted in production greenhouse, in collaboration with cooperating growers, to periodically monitor the occurrence and distribution patterns of TCDVd throughout the tomato growing season. 3. The efficacy of the selected disinfectants will be screened initially through mechanical inoculation of treated TCDVd or PepMV tissue extract on indicator plants and tomato. Any promising disinfectant will be evaluated by simulating crop work practices such as de-leafing, plant training and harvesting using a disinfected cutting knife or pruning shears. Similar efficacy test will be carried out with the newly developed no-contact cutting device(s) (laser and/or water jet knife) that will be developed by one of the project collaborators. Specialty Crops Research Initiative. 3.Progress Report: This project corresponds with in-house Objectives 1: Develop sensitive diagnostic tools for the emerging viral diseases of greenhouse tomatoes and bacterial diseases on vegetable Brassicas; and Objective 2: Characterize the etiology of emerging diseases of vegetables and identify critical factors affecting the epidemiology of those diseases. In recent years, greenhouse tomato growers have experienced a significant genotype shift in Pepino mosaic virus (PepMV), from a prevalent European genotype to an aggressive Chinese genotype, resulting in severe symptoms and increased yield loss. Growers are demanding certified PepMV-free seed from seed suppliers, as well as, genotype-specific detection of their PepMV infected tomato plants. Positive and accurate identification of the disease causal agent(s) is a prerequisite to achieve an effective disease management. Current detection technologies for PepMV, including immunostrip, Enzyme Linked Immunosorbant Assay (ELISA), real-time reverse transcriptase polymerase chain reaction (RT-PCR), could not be used to differentiate various genotypes. Sequencing and analysis of PCR products is the most definitive determination of a genotype. However, this technology requires highly skilled technical support and may not be practical for large-scale disease diagnosis. Thus, development of a genotype-specific Loop isothermal amplification (LAMP) technology would greatly enhance our capabilities to achieve a simple and sensitive detection and determination of virus genotype. In collaboration with major greenhouse growers in the U.S. and Mexico, several emerging diseases infecting greenhouse tomatoes were identified and characterized. A natural infection of greenhouse tomatoes by Potato spindle tuber viroid (PSTVd) was identified in the U.S. and Pepino mosaic virus (PepMV) was recently reported in Mexico. A potentially damaging bacterial disease (Candidatus Liberibacter solanocearum) was also identified in Mexico. A mixed infection of several pathogens in field samples was common. In some cases, a mixed infection of different pathogens could induce a synergistic effect on the infected plant, which may result in greater damage to plant health. It could also complicate the situation in decision-making for disease management. To achieve accurate and complete pathogen identification in field samples, a novel technology with next generation sequencing of small Ribonucleic Acid (RNA) was used to evaluate viruses in tomato samples collected in North America. This technology was shown to be capable of differentiating virus genotypes and to allow effective identification of viroids as well. A disease survey and pathogen identification was performed and the results were transferred to the respective growers for disease management considerations. These research results were presented at the Tomato Disease Workshop and the annual American Phytopathological Society Meeting. The technology of deep-sequencing of small RNAs in plants will potentially revolutionize virus and viroid identification and characterization.