Microbial or biochemical agents that control plant diseases by inducing systemic resistance to pathogen infection could play a significant role in crop disease management. Bacillus mycoides, isolate J, BmJ, represents the first such agent that acts by foliar (rather than root) application and that does not cause leaf tissue damage in any plants. Disease control with this strain was previously demonstrated in sugar beets. Research in a Phase I SBIR project demonstrated control of multiple diseases in cucumber, significantly expanding the commercial potential. In the phase II SBIR project, MMP will advance BmJ commercialization by demonstrating cost effective disease control in cucumber and melon crops, developing commercial formulations, expanding potential markets with tests in additional crop, disease systems and conducting further research on the mechanisms by which BmJ induces disease resistance in plants. OBJECTIVES: Montana Microbial Products is commercializing an isolate of Bacillus mycoides, BmJ, that controls crop diseases by inducing systemic resistance to pathogen infection in plants. Phase I SBIR research demonstrated that BmJ induced resistance controls fungal, bacterial and viral diseases in multiple plant/pathogen systems. The potential of this discovery for disease control in agricultural crops is both scientifically significant and commercially important. The proposed phase II project will enhance our fundamental understanding of BmJ induced residence, and commercialize the cucurbit pathogen control demonstrated in phase I. To advance these goals, the phase II work plan will have three specific objectives: Bring BmJ to commercial introduction in management of one or more pathogens in cucurbit crops; Demonstrate BmJ induced resistance and disease control in additional plant/pathogen systems; Elucidate the signal between BmJ and the leaf that triggers resistance induction. APPROACH: Phase I results demonstrated control of three important diseases in cucurbits: Anthracnose caused by the fungus Colletotrichum obiculare; target spot caused by the fungus Corynespora cassicola; and angular leaf spot, caused by the bacterium, P. syringae pv, lachrymans. Subsequent work demonstrated control of cucumber mosaic virus. In phase II, MMP will conduct a progression of field trials to define commercial application strategies and demonstrate commercial value of BmJ for disease control in cucurbits. MMP will work with the Jacobsen group in the Plant Pathology Department at Montana State University to evaluate bacterial and fungal disease control in additional crops to increase our understanding of the biology and expand market potential. Finally, MMP and the Jacobsen group will conduct a series of experiments, which we believe will identify the signal molecule or event that mediates the initial interaction between BmJ and the leaf. This will enhance our scientific understanding of induced resistance by foliar applied agents, guide potential improvements in BmJ production or formulation and provide the basis for an alternative product or delivery system for induced resistance disease control. Most importantly, elucidating the signal that triggers induced resistance would solidify the intellectual property of surrounding this discovery. To meet the specific phase II objectives described above, MMP will conduct a work plan with four principal tasks: BmJ fermentation and formulation scale up. Develop flowable solid and liquid end use formulations for commercial use. Cucurbit field trial program. Conduct replicated small plot and commercial field trials with extension and private cooperators in different growing regions to develop commercial application strategies and demonstrate efficacy to growers. Screening BmJ in additional crop/pathogen systems. Conduct greenhouse tests for control of tomato and potato diseases in cooperation with the Plant Pathology Department at Montana State University. Where disease control is demonstrated, conduct replicated plot field trials. Conduct experiments to identify the resistance induction signal. This work will fractionate BmJ cultures and test resistance induction using innovative screening assays for hydrogen peroxide production and pathogenesis related proteins to identify the molecular signal that mediates the initial interaction between BmJ and the leaf which triggers the resistance induction pathways. PROGRESS: 2005/09 TO 2006/08 In year one, MMP made progress in each of the three principal tasks. Fermentation, formulation scale up demonstrated commercial feasibility of the processes developed in phase 1. Fermentations at 800 liters provided spore yields equal to yields obtained in laboratory bench fermentors. MMP can deliver application rates tested in field trials at prices competitive with low end chemical fungicides. MMP produced Kg quantities of wettable powder formulations, which provided easy handling and uniform application with both research and commercial spray equipment. MMP also developed a liquid formulation; stability studies showed no short term loss in spore viability, studies continue. Research trials in cucurbits showed significant reductions in leaf damage from Anthracnose, Colletotrichum lagenarium, in cucumber and melons and angular leaf spot, Pseudomonas lachrymans, in cucumbers. In the Anthracnose trial, BmJ was inferior to the combination of Azoxystobin, Chlorthanonil, however BmJ applied with a one-half rate these chemicals gave equivalent control. Trials for downey and powdery mildew gave control comparable to commercial Bacillus products but inferior to chemical fungicides. Greenhouse screening indicated potential control of bacterial spot, Xanthomonas campestris, in tomatoes and peppers, and preliminary trials had shown potential in potatoes and pecans. In a bacterial spot trial in tomatoes, the combination of BmJ and Dithane significantly reduced both disease incidence and severity compared with untreated controls with results statistically equivalent to the standard commercial treatment of Dithane, Bravo, copper. A pepper trial conducted in Montana also showed disease control superior to copper based products. Preliminary results in pecans showed that BmJ alone reduced foliage damage from pecan scab as well as the standard chemical combinations. Preliminary results in potatoes conducted in Montana showed reduction in early blight in the midrange of chemicals tested in the trial. Final yield results are not yet available. First year cucurbit results show potential for BmJ as a stand alone or rotation product for controlling Anthracnose and angular leaf spot in both conventional and organic crops. Powdery and downey mildew results to date suggest potential markets limited to organic or specific rotation niches in conventional cucurbits. Results for bacterial spot show excellent commercial potential in tomatoes and peppers where there is increasing resistance of bacterial spot to copper based bactericides. Trials have shown commercial potential for control of pecan scab and for early bight in potatoes. MMP will conduct additional trials in tomatoes and peppers in the fall of 2006 in Florida and Georgia. In parallel work (not funded by the USDA phase II project) MMP obtained an Experimental Use Permit for grower trials in sugar beets. MMP plans to expand the EUP for tomatoes and peppers and possibly potatoes and pecans to enable grower trials in parallel with continued research trials during year 2. IMPACT: 2005/09 TO 2006/08 BmJ will provide growers with a safe effective disease control product with a unique mode of action. A pulmonary toxicity pathogenicity study contracted by MMP (independent of the USDA Phase II project) showed no adverse effects. Year 1 research trials have shown commercial potential in two important cucurbit diseases, in bacterial spot in tomatoes and peppers, in pecan scab and in early blight in potatoes. BmJ has potential as a stand alone product in some applications and as a combination or rotation product with chemical fungicides in control programs. Due to the unique mode of action, BmJ also has a potential role in resistance management for chemical fungicides. BmJ is a naturally occurring isolate produced and formulated by MMP to allow use on organic crops. MMP plans to apply for organic use approval providing the rapidly growing organic production with a new disease control product. BmJ is a filamentous bacterium difficult to grow as a commercially suitable product. MMP developed and has now demonstrated a commercial scale fermentation process and end product formulation that performs well in commercial spray equipment. Based on cost estimates from commercial scale fermentations and formulation, MMP can sell BmJ profitably at prices that compete with older low cost chemical fungicides and at a substantial discount to newer chemistries. This will allow incorporation of BmJ into a variety of different disease control strategies in multiple crops.
