Location: Vegetable Research2013 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:
The research in this subordinate project relates to inhouse project Objective 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 these diseases. Timely and accurate disease diagnosis through sensitive virus identification is an important component of integrated viral disease management. In this study, we developed several molecular-based methods for sensitive virus detection and strain differentiation, including the recently developed deep sequencing of small RNA and assembly technology, real-time RT-PCR, and loop-mediated isothermal amplification. Using simple sample preparation through limited dilution of crude tissue extract, we were able to develop these technologies for routine disease diagnosis for known viruses (Pepino mosaic virus), an emerging virus (Tomato necrotic stunt virus- ToNStV) and viroids (Potato spindle tuber viroid, Mexican papita viroid and Tomato chlorotic stunt viroid). In a host range study, the new virus, ToNStV, systemically infected many species in the family Solanaceae and locally infected some species in the family Amaranthaceae, Brassicaceae, and Cucurbitaceae. The genomic organization and phylogenetic analysis revealed a new species of potyvirus, which shared less than 60% amino acid sequence identity to other known potyviruses, especially those infecting solanaceous plants. Mexican papita viroid (MPVd), in the genus Pospiviroid and family Pospiviroidae, is another emerging viroid in greenhouse tomatoes. MPVd was first isolated from wild papita (Solanum cardiophyllum Lindl) plants in Mexico in 1996. Since 2009, several disease outbreaks on greenhouse tomatoes in Canada and Mexico were linked to MPVd infection. However, Koch’s Postulates for MPVd have not been established. In the current study, infectious cDNA clones expressing the full genome of MPVd in two different MPVd isolates (MX and Mex8) were developed and their infectivity was confirmed through mechanical inoculation on ‘Moneymaker’ tomato plants. The developed infectious cDNA clones will provide us a powerful tool in identification of RNA sequences, structure motif analysis or gene domains responsible for symptom expression. Increasing global trade and seed exchange significantly affects viral disease incidence on tomato. In greenhouse tomatoes, we detected a dramatic shift in the prevalent genotype of PepMV in North America. Our studies indicated that commercial tomato seed lots are the likely sources of initial PepMV inocula that resulted in the genotype shift as observed in North America. The development of a rapid and sensitive genotype-specific RT-LAMP system will facilitate timely identification of the PepMV genotypes for accurate assessment of virus population and epidemiology. An efficient management strategy for greenhouse viruses is to use chemical disinfectant to prevent virus or viroid spread. However, disinfectant effectiveness against these viruses and viroids on greenhouse tomatoes are largely unknown. In the current study, we designed an experimental protocol and evaluated 16 commercial disinfectants for their effectiveness to deactivate virus infectivity on cutting devises against several tomato viruses and viroids. Several chemicals (including 10% Chlorox bleach, 2% Vircon-S, nonfat dried milk) were shown to demonstrate broad reactivity against four test tomato viruses and viroids.