A Comparison of Disinfectants to Prevent Spread of Potyviruses in Greenhouse Tomato Production

Potyviruses, transmitted by a diverse array of common aphid species, infect a broad range of vegetable crops, and can be problematic in greenhouse tomato production. Once introduced, these viruses are believed to be transmitted plantto-plant during pruning operations, and can infect large sections of a greenhouse, resulting in significant losses in fruit quality and yield. Several methods are used for virus management in greenhouse production, including rouging of diseased plants and treatment of tools and facilities with virucides to eradicate the virus responsible. To clarify potyvirus transmission efficiency from an infected source during pruning operations, experiments were conducted using direct and serial mechanical inoculation of Potato virus Y (PVY) using a scalpel dipped in a suspension of PVY-infected plant sap. Tests demonstrated that both serial and direct inoculation resulted in significant PVY transmission, but that transmission rates declined after the first few plants in serial transmission. Additional tests evaluated the efficiency of two virucides, a quaternary ammonium solution and sodium hypochlorite, for virus inactivation during pruning operations using a range of concentrations and time points. Results demonstrated that 0.5% sodium hypochlorite treatment for two seconds was sufficient for virus control, and superior to treatment with quaternary ammonium solutions. Introduction Viruses within the genus Potyvirus, family Potyviridae, can be a significant problem for greenhouse tomato production. Two potyviruses, Potato virus Y (PVY) and Tobacco etch virus (TEV), occur with some frequency in tomato greenhouses with adjacent or nearby production of other affected crops, particularly vegetables or potato (3,4). However, limited information is available on spread and management of potyviruses in greenhouse tomato production. Potyviruses can cause symptoms of foliar mottling, puckering, and slight distortion or rugosity (10), and can be spread from plant-to-plant not only by aphid vectors, but also by mechanical transmission. Some tomato-infecting potyviruses, such as Tobacco etch virus, can produce fruit mottling, preventing marketability (10) and significantly reducing yields. Efforts to limit losses and prevent spread of potyviruses throughout the greenhouse can result in the grower not only removing infected plants and those plants adjacent to the infected plants, but also taking significant sections of a greenhouse out of production for a period of time, further reducing profits. Removal of infected plants is often necessary to eliminate sources of infection prior to replanting in order to prevent further infections from developing. Potyviruses can enter greenhouses by a number of methods. Two common modes of accidental introduction include movement of viruliferous aphid vectors from nearby infected field crops or weeds into a greenhouse through vents, doors, or other points of entry. Alternatively potyviruses can enter on contaminated nursery material. Once infected plants are introduced, the viruses can spread from plant-to-plant through transmission by aphid vectors that may be present in the greenhouse. Growers have speculated that potyviruses may also spread down rows during pruning operations, but little information is available on how readily this actually occurs. Growers routinely attempt to 21 February 2011 Plant Health Progress control mechanically transmitted viruses by removing diseased plants and those around them in an effort to reduce the source of inoculum. Additional measures include the use of chemical or heat treated tools for trimming and/or harvesting operations. Chemicals (including those referred to as virucides that eliminate viruses) can vary from sodium hypochlorite (bleach) based solutions, to dipping tools in milk, to new compounds specifically designed to inactivate viruses and other pathogens through surface sterilization (3,7). Although these methods are often used to control mechanically transmitted viruses in greenhouse operations, limited information exists on their effectiveness for potyvirus control in greenhouses. Studies were conducted to determine the ease at which potyviruses can spread in a tomato production greenhouse as a result of pruning operations, as well as the effectiveness of two common methods for treatment of tools and equipment to prevent virus spread. The purpose of these studies was three fold: (i) to determine the ability of a potyvirus to spread within rows during pruning operations; (ii) to determine if treatment of tools with virucides can reduce or eliminate within row spread of a potyvirus during pruning operations; and (iii) to determine how length and concentration of virucide treatment influences effectiveness of potyvirus control. Virus and Inoculum Preparation The PVY isolate used for these studies was obtained from a tomato greenhouse in southern California during a potyvirus outbreak, and was maintained at the USDA-ARS in Salinas, CA. Its identity was confirmed by sequencing of RT-PCR products obtained using potyvirus group specific primers CWC6 and CWC7 (5) targeting the NIb/CP region of the virus genome and comparison of the sequences of clones derived from RT-PCR amplicons to known sequences in GenBank. Inoculum for the experiments consisted of PVYinfected Nicotiana benthamiana, inoculated 3 weeks prior to the beginning of the experiment and exhibiting strong symptoms at the time of use as inoculum. N. benthamiana was used as a propagation host for PVY inoculum due to its ability to rapidly produce high concentrations of virus. For each inoculation, 3.5 g of symptomatic N. benthamiana leaves were ground in 10 ml of 100 mM sodium phosphate buffer, pH 8.0, containing 0.1% sodium sulfite, and kept on ice for the duration of inoculation. Inoculation was performed by dipping a scalpel blade in the inoculum suspension, then using the scalpel to make a diagonal slice in the stem of a tomato plant just above a leaf attachment point (node). Plants were inoculated when approximately 30 cm tall. Tomatoes (Solanum lycopersicum) used in all experiments were the variety VFN8 (University of California, Davis), kindly provided by Seminis Vegetable Seeds (Woodland, CA). The study consisted of two experiments, each containing five to six replicates of nine treatments with 8 plants per treatment, arranged in a randomized complete block design. Experiments were conducted sequentially in a greenhouse at the USDA-ARS in Salinas, CA. Potyvirus Spread During Pruning Operations To determine the ability of potyviruses to spread down a row through pruning operations, VFN8 tomato plants were inoculated with PVY using two inoculation approaches: direct inoculation and serial inoculation. Direct inoculation involved inoculation of each tomato plant with a scalpel dipped in freshly prepared ground plant sap prepared as described above, immediately prior to inoculation. Tomato seedlings were inoculated by slicing diagonally into the stem to a depth of approximately one third of the stem diameter, to provide an illustration of PVY movement in the greenhouse as a result of mechanical transmission during pruning operations. Serial inoculation was performed similarly, with the first plant inoculated as described for direct inoculation, followed by consecutive inoculation of a series of plants without cleaning of inoculation tools or dipping in fresh inoculum. This approach was designed to illustrate how many times a potyvirus can be transmitted in consecutive cuts once the virus is acquired on tools from a single infected source plant in a row, as would occur during routine greenhouse pruning operations. For each 21 February 2011 Plant Health Progress replicate within each experiment, a negative control treatment was included consisting of an identical set of plants, sliced with a virus-free scalpel (no inoculum). Treatment of Pruning Tools for Control of Potyviruses A commercial quaternary ammonium virucide used for disinfection of greenhouses and associated pruning tools (5-10% alkyl dimethyl ethylbenzyl ammonium chloride and 5-10% alkyl dimethyl benzyl ammonium chloride, hereafter referred to as quaternary ammonium) was compared with chlorine bleach (5% sodium hypochlorite) for effectiveness in controlling potyvirus transmission in greenhouse tomato production (Table 1). Quaternary ammonium was used at two concentrations; 1% (standard recommended concentration) and 3%. Sodium hypochlorite (chlorine bleach) was used at 10% (1:10 dilution of 5% stock, which is the standard for bleach sterilization in laboratories and greenhouses) and 15%. The active concentrations of sodium hypochlorite in our experiments were therefore 0.5% sodium hypochlorite (considered 10% bleach solution since its concentration is only 10% of standard bleach) and 0.75% sodium hypochlorite (considered 15% bleach solution since it is 15% the concentration of standard bleach). The purpose of the times and treatments used was to compare the effectiveness of virucide treatments, as well as the length of time and type of treatment necessary for each virucide to effectively destroy potyviruses and prevent their transmission during pruning operations. Treatment times were chosen based on knowledge of current tomato greenhouse practices, and an effort to keep treatment times realistically short, yet sufficiently long to destroy virus particles. One month after inoculation, plants were scored for visible virus symptoms (data not shown), and samples were collected from the third compound leaf (up from the base of the plant) and tested by enzyme linked immunosorbent assay (ELISA) using the Agdia PVY detection kit (Agdia Inc., Elkhart, IN) using standard procedures as recommended by the manufacturer. The entire experiment was conducted twice, with the number of infected plants for each treatment confirmed by ELISA presented in Figure 1. Significant effects were determined through ANOVA using th