Performing Department
(N/A)
Non Technical Summary
Crazy Root disease (Agrobacterium rhizogenes; CR) has recently been recognized as an emerging disease, particularly in greenhouse hydroponic tomato production. There is currently no commercial product for control of CR disease. Consequently, growers must sterilize or throw away all infected materials leading to added expenses and operational downtime. Use of biocontrol in greenhouses continues to increase; however, a significant problem in the expanding biologicals market is inconsistent performance of biological products. 3Bar Biologics is bringing to market a novel beneficial microbe delivery system, to improve suboptimal, inconsistent performance of biological products. The delivery system is easily activated on-site by the grower, by simply pushing a button to combine the stabilized bacteria inoculum with the liquid growth medium. 3Bar's disposable bioreactor technology opens up the potential for growers to apply more effective microbial strains with CR control capability. Discovery research at The Ohio State University has identified several Pseudomonas strains capable of CR control. The Phase I project aims to prove feasibility to prevent CR disease in hydroponic tomato production using the identified Pseudomonas strains delivered with 3Bar's system. The proposed work addresses three technical objectives: i) maximize cellular yields in the system with identified Pseudomonas strains, ii) characterize compatibility of new formulations for feasible use in hydroponics systems, and iii) evaluate efficacy in greenhouse hydroponic tomato production. The ultimate goal is to develop a broad-spectrum biocontrol product for use against multiple plant diseases, particularly Crazy Root and fungal root rots, and at the same time provide "probiotic" benefits to plants to increase vegetable yield and quality in hydroponic systems. The proposed project is directly aligned with USDA NIFA Challenge Area 1) Global Food Security, and Priority 8.2.3) to research bio-based approaches for plant protection against abiotic and biotic stresses.
Animal Health Component
50%
Research Effort Categories
Basic
0%
Applied
50%
Developmental
50%
Goals / Objectives
The ultimate goal of the project is to develop a broad-spectrum biocontrol product for use against multiple recurrent diseases in hydroponic vegetable production systems. Common diseases of hydroponic systems include Crazy Root disease (Agrobacterium rhizogenes), fungal root rots (caused by Pythium, Fusarium, and Phytophthora spp.), and diseases of the leaves (Powdery Mildew; Oidium lycopersicum) and fruits (Gray Mold; Botrytis cinerea). Most methods for disease control involve routine use of chemical pesticides and sterilization agents; however, the hydroponic vegetable production industry is beginning to shift its focus toward the use of biocontrol agents that limit the spread of diseases, are safer for workers and consumers, and are easily produced and applied. Currently, there are few effective biocontrol-based products available to control diseases on hydroponically produced vegetables. To meet this overall goal, application of Pseudomonas spp. will be used for control of plant diseases in hydroponic systems. Pseudomonads (Pseudomonas spp.) have been widely studied for their biocontrol activity against numerous plant pathogens (including, most notably Pythium spp. and Phythopthora spp.), are excellent root colonizers, and can sometimes exhibit plant growth promoting activity. Despite the significant body of research on the benefits of Pseudomonas spp. over the past 30 years, very little successful commercialization has occurred due to the lack of effective formulation and delivery technologies for these and other gram-negative, non-spore forming microbes. To address this issue with commercialization of pseudomonads and other non-spore forming beneficial bacteria, 3Bar Biologics developed and patented (US 10,774,298) a novel beneficial microbe delivery system for improving microbial product viability in the supply chain.Using target Pseudomonas spp. strains and 3Bar's beneficial microbe delivery system, the specific goals of the Phase I project include:1) Maximize cellular yields of the solid and liquid phases of the delivery system. Technical objectives to meet this goal include: i) development of freeze-dried formulations of each target strain stable at room temperature for at least 6 months, ii) improved delivery system design including gas permeability, liquid volumes, and amount and type of growth media for increasing bacterial cellular yields, and iii) evaluation of growing bacteria consortium in the delivery system. Efforts will result in a second-generation delivery system for hydroponic use with improved growth performance for target strains or combined consortia.2) Characterize the stability and compatibility of new formulations for feasible use in hydroponic systems. Working directly with industry partners to understand hydroponic system operation and common chemical products used (e.g., nutrient solutions), the technical objective is to develop a chemical compatibility chart for nutrient solutions and other products that are compatible with the bacteria strains and fits grower practice.3) Evaluate efficacy in tomato hydroponics trials on Crazy Root disease. The technical objectives to meet this goal involvetesting in OSU's pilot-scale hydroponics system i) individual strains under direct disease pressure, ii) combinations of strains (consortia) for synergistic effects to determine efficacy, and iii) individual strains for yield improvement (no pathogen pressure). All efficacy trialswill be conducted using the candidate formulations and delivery system. Resulting efficacy data will support the feasibility to select Pseudomonas strains for further development of a biopesticide to control Crazy Root disease, with the intent in Phase II to expand development for control of fungal root rot diseases.
Project Methods
Methods for each goal/objective and project task. Objective 1: Maximize cellular yields of the solid and liquid phases of the delivery system. Task 1: Develop dry formulation of Pseudomonas spp. strains. Building upon previous efforts, lead candidate formulations will be developed for the target strains of Pseudomonas (e.g., 1B1, 93G8, and 48G9). Current commercial practice involves freeze drying gram-negative bacteria. Key design variables appropriate for formulation include consideration of the culture medium (e.g., nutrient composition, conditioning additives), growth stage when the culture is harvested, and additive type and amount (e.g., lyoprotectant) in formulation. Completion of this task will result in dry formulations for each of the target strains. Task 2: Develop improved delivery system design. The following experiments will be used to improve upon the current MVP design with the goal of increasing cellular yields by at least 10-fold in the liquid phase and extend the window of use beyond 28 days.Experiment 1: Delivery system design and size. The next generation prototype utilizes single-use polyethylene bags ("bladders") and is being redesigned to improve performance for production scale-up and automated manufacturing. Gamma-irradiated polyethylene bags commonly used in food packaging are low-cost, available through multiple suppliers, and in a range of film thicknesses, gas permeability properties, and sizes (1 L to 1000 L). The experiment aims to evaluate different film thicknesses, gas permeability properties, liquid volumes and headspaces to see if the cellular yields can be increased and maintained for 90 days or longer after activation.Experiment 2: Amount and type of growth media. This experiment will evaluate the amount and type of growth media to increase cellular yields in the delivery system. To start, tryptic soy broth (TSB; a common nutrient-rich growth media) will be autoclaved and aseptically filled into sterile bladders for testing. Bladders of different sizes (10 L, 20 L) and liquid fill volumes (50%, 75%, 90%) will be evaluated with different nutrient strengths (10%, 50%, and 100% TSB) to determine the impact of head space and nutrient strength on cellular yields. Additionally, other nutrient growth medias will be identified and efforts to evaluate initiated to determine the feasibility of increasing cellular yields while minimizing material costs associated with media. Experiment 3: Evaluate a consortium of beneficial microorganisms in the delivery system. The purpose of this task is to evaluate the utility of the delivery system to deliver not only a single strain of Pseudomonas but also a consortium of known beneficial microorganisms. To do so, we will evaluate combinations of the three target Pseudomonas strains (1B1, 93G8, 48G9) in the same bioreactor system. The dry formulated microbes will be added together in different combinations, inserted into the cap, and attached to the main delivery system according to current manufacturing practice.All tests for Experiments 1, 2, and 3 will include three different strains of Pseudomonas (1B1, 93G8, and 48G9). Delivery systems will be activated and sampled at 1, 2, 7, 14, 21, 28, 60, and 90 days post activation (DPA). Each experiment will be run twice. Viable cell counts from each sample will be determined by dilution plating and enumeration using standard microbiology techniques. Overall, growth rates and cellular yields (both individually and through co-culturing) of activated delivery systems will be determined. The completion of this task will result in a second-generation delivery system for hydroponic use with improved growth performance for target strains, either select single strains or combined consortia.Objective 2: Characterize the compatibility of new formulations for use in hydroponic systemsTask 3: Evaluate compatibility of strains with nutrient solutions (fertilizers) and other products applied within recirculation system. 3Bar will work directly with Village Farms to understand hydroponic system operation and common chemical products used by the industry requiring compatibility with the beneficial microbes. Some nutrient solutions contain micronutrients (such as copper, a known biocide) that may be detrimental to the bacteria. Samples of bacterial cultures will be loaded into 96-well plates and exposed to different organic and inorganic nutrient solutions commonly used in the industry. To do so, 100µL of test solution will be serially diluted in 2-fold increments from 1X to 1/8X of the recommended concentration. To these, an equal volume of bacterial culture will be added. Plates will be incubated at room temperature for 24 hr, after which viable cell counts from each sample will be determined by dilution plating and enumeration using standard microbiology techniques. Completion of this task will allow for the development of a chemical compatibility chart for nutrient solutions and other products with a range of active ingredients. Objective 3: Evaluate efficacy in hydroponic trials.Task 4: Conduct trials of new formulations in a hydroponic system. We will further test the strains using 3Bar Biologics' second-generation delivery system. Our initial test will be in three parts: 1) testing individual strains under direct disease pressure in pilot scale hydroponic units in the Taylor laboratory using bacteria grown in 3Bar Biologic's delivery system, and 2) testing a combination of strains (consortia) to determine their efficacy in controlling CR, and 3) testing individual strains for yield improvement (no pathogen pressure). Experiment 1: Pilot scale testing of strains. The sterilizable test system allows direct application of microbes and pathogens under actual hydroponic conditions. The infection process was optimized with A. rhizogenes and can achieve nearly 95% disease efficiency. Each tray will be pretreated with the test Pseudomonas strains (individually or in combination) grown in 3Bar Biologic's delivery system. Two-week-old plants grown in oasis cubes or soilless media will be placed in the individual rockwool cubes. Plants will then be treated with an adequate dose of A. rhizogenes to ensure disease at 6, 12, 24, 48 and 72 hours after treatment. Plants will be examined for the presence of CR and the efficacy of treatment determined. Several controls will be utilized, including mock inoculated, non-treated, and a non-disease causing variant (i.e. cured) strain of A. rhizogenes. All trials will be done with four replicates (4-tables) and repeated three times. Experiment 2: Combination testing of strains for synergistic effects. To further examine the potential of Pseudomonas to inhibit CR disease, combinations of Pseudomonas will be tested to determine if there is any improvement in pathogen control above single strain application. Double and triple combinations will be tested to determine if there is any change in biocontrol activity.Experiment 3: Yield testing. Yield testing will be conducted using Pseudomonas grown in 3Bar's delivery system with application for growth promotion potential in the absence of any disease pressure. Yield testing will be conducted using Bato BucketTM system used for growing vine tomatoes for fruit production. Tomato plants (var. Rebelski -F1) will be germinated in rock wool cubes and transferred to the individual Bato Buckets and inoculated with Pseudomonas. We will initially apply Pseudomonas once every month to the root system of the plant. Plants will be measured for shoot height and leaf surface area. Plants will be trellised and allowed to produce fruit for three months. Ripe fruit will be harvested throughout the experiment and weighed. At the end of three months, all fruit will be harvested and weighed. We expect to be able to complete 2 additional trials over the duration of the Phase I funding.