Tropical soda apple is an invasive weed that imposes a severe economic constraint on the livestock industry, threatens pristine natural areas, and imminently menaces croplands in the Southeast. The technology described herein will spur the development of an innovative, effective, environmentally beneficial, nonchemical control for this noxious weed. This research will promote the development of the world's first virus-based commercial bioherbicide that will help control this weed and provide for the economic viability of livestock production. Reduction in the use of chemical herbicides and the alleviation of the weed's encroachment into natural areas and croplands are other benefits. OBJECTIVES: The overall objective of this project is to establish the feasibility of developing Tobacco mild green mosaic tobamovirus (TMGMV) as an EPA-registered bioherbicide to control Solanum viarum (tropical soda apple; TSA), an economically and ecologically damaging invasive weed. TMGMV is a naturally occurring (i.e., not genetically altered), economically insignificant plant virus, which is found in Florida, several other states in the USA, and in many other countries. We have demonstrated that TMGMV is capable of killing TSA plants. We have also shown that this virus can be used as a bioherbicide to control TSA infestations in the field. The virus can be easily produced on a laboratory-scale in susceptible tobacco plants, extracted from tobacco leaves, and applied in the field with readily available tools to control TSA In small areas (1 to 10 m2). These studies indicate that it should be possible to produce the virus on an industrial scale, but the scientific and technical feasibility of such production needs to be established. It is also unknown whether the virus could be made into a stable, standard formulation for use to meet the market needs, i.e., to treat several thousands of acres on demand. To answer these questions, we will examine the feasibility of producing TMGMV in quantities of 250 to 500 grams of active ingredient per production batch, establish efficacy, handling, and application criteria, thereby assessing the feasibility of transitioning the prototype into to a first-generation product. In summary, we will determine the technical and practical realities of implementing the bioherbicide under commercial, user-driven TSA-management programs. The specific objectives are to: 1) develop and validate methods to produce TMGMV in a 3-acre field-grown tobacco crop and maximize the virus yield through optimization of plant inoculation, plant production, leaf age-harvest relationships, and leaf harvest and processing; 2) set standards for a commercial prototype bioherbicide by determining the optimum active-ingredient (a.i.) concentration, formulation type, handling, storability, application, and disinfestations; 3) develop and validate field-application parameters for the prototype bioherbicide including the amount of the bioherbicide that will treat an acre, best methods of application for spot treatment and large-area treatment, application volume in GPA, and the practicability of using TMGMV in state-wide TSA-management programs in Florida; 4) determine the possibility of movement of TMGMV from experimental and production plants (tobacco) to two susceptible species planted as sentinels near the experimental/production plants; and 5) determine the possible persistence of TMGMV at the experimental/production site. A final report will be prepared which will set the stage to develop a Phase II Proposal. APPROACH: A 3-acre field will be set up at the University of Florida Plant Science Research and Education Unit (PSREU, a subcontractor), to grow irrigated tobacco crops. Tobacco plants will be grown from seed in a greenhouse and inoculated when the seedlings are at 4- to 6-leaf stage by spraying TMGMV inoculum evenly over the foliage with an air-brush. Plants will be transplanted, grown, and managed according to the recommended production and crop protection practices. The optimum leaf age for harvest, number of harvests per plant per growth-cycle, and the effect of flowering on virus yield will be determined. Using the best conditions for maximum virus yield, the leaf-biomass yield per harvest, total leaf yield per acre, virus yield per acre of tobacco, total materials, time, and labor required for harvest; and the cost of production will be estimated. The harvested leaves will be transported to the subcontractor laboratories in the Plant Pathology Department and stored in a cool, dry place until processed. The virus will be extracted from the leaf tissue and processed into a wettable powder. This virus preparation will be packaged in a plastic satchel that will be storable at ambient conditions inside air-conditioned buildings. As a precaution, it may be stored in a refrigerator for prolonged storage. The material will be applied with high-pressure, hand-held sprayers (for spot application) or tractor-mounted spray booms (for large areas) or wiper applicators. Unused material will be destroyed by inactivating the virus by adding laundry detergent or bleach (e.g., Clorox) in sufficient amount of water. The product will be standardized with respect to units of active ingredients per gallon or per acre. Application volume and practical tools needed to obtain best levels of control will be established. The bioherbicide's efficacy will be determined from the level of TSA kill under field conditions, with 95-100% as the target. The possibility of TMGMV spreading to nontarget plants at the production sites will be monitored by planting two TMGMV-susceptible species, Capsicum annuum and Petunia x hybrida, as sentinel species. The experimental and production plots will be surrounded by narrow bands of these sentinel species and the possible movement of TMGMV from inoculated tobacco plants to the sentinels will be monitored for symptom development over the entire duration of the study. If symptoms develop, the plant tissues will be assayed for TMGMV by ELISA and back inoculation to TSA and tobacco plants. If no symptoms develop during of this study, a sampling of the sentinel plants will be subject to ELISA test at the conclusion of the study to confirm the lack of TMGMV's spread. The possibility of persistence and survival of TMGMV at the production site will be determined at the conclusion of the study by assaying soil samples from several locations within the site. ELISA and plant inoculations (on TSA and tobacco) will be used. If the virus is detected at significantly high levels (e.g., high proportions of positive samples), the soil will be tilled once or several times at 1- or 2-month intervals until no traces of the virus are found
