Performing Department
(N/A)
Non Technical Summary
This project will develop and qualify a system that is capable of treating soybean farming operations where susceptibility to SCN is the primary yield limiting pest. Soybean cyst nematode (Heterodera glycines; SCN) is the major pathogen of soybean causing over $1 billion in crop losses annually. SCN is now considered endemic to North America and is spreading as more acres of land are planted in soybean. Widely prevalent strategies for SCN management include crop rotation and the use of resistant soybean cultivars. These approaches have met with only limited success in reducing the populations of parasitic nematodes in the field. The recent loss of SCN resistance in Ohio-grown soybean highlights the importance of generating new methods for SCN control such as biocontrol. Examples of biocontrol with nematicidal activity are found in the scientific literature including numerous strains of bacteria. However, commercial applications by the large seed companies are limited to spore-forming Bacillus strains. While these spore-formers have some limited ability to survive conventional on-seed application processes and supply chain conditions, they can be limited in their effectiveness against target pests. Environmental stress and abuse associated with manufacturing and distribution typically results in microorganism death and/or dormancy, which decrease viability and physiological activity of the microorganism at the time of application. Thus, current microbial products have decreased effectiveness and reliability.3Bar Biologics is creating a disruptive new system to deliver beneficial microorganisms - a low cost, disposable bioreactor that protects the microorganisms until the farmer is ready to activate the product prior to application. On-site growth of the beneficial microorganisms short-cuts the conventional supply chain, resulting in fresher, more viable microorganisms delivered to the field. This bioreactor opens up the potential for farmers to apply more effective microorganism strains with SCN control capability.The work plan addresses three technical objectives to create a functional prototype for commercialization of strains with demonstrated activity on SCN, to i) maximize cellular yields in the device with a number of proven beneficial strains, ii) characterize stability and compatibility of new formulations, and iii) evaluate efficacy against SCN in the field.This project is directly aligned with USDA NIFA Challenge Area 1) Global Food Security, and Priority 8.2.4) to research biobased approaches for plant protection against abiotic and biotic stresses.
Animal Health Component
0%
Research Effort Categories
Basic
0%
Applied
40%
Developmental
60%
Goals / Objectives
The overall goal of the proposed project is to establish a new biopesticide product for soybean cyst nematode (SCN). The new biopesticide product builds on our minimum viable prototype bioreactor system for delivery of Pseudomonas spp. as a beneficial microbial inoculant. By providing production agriculture with more and better microbial products, our dependence on more costly chemical pesticides will be reduced, increasing the efficiency of production and lowering input costs to growers.Specific to the Phase I portion of this effort, two overarching goals include: 1) create a bioreactor system with adequate performance and stability that is capable of being commercialized at scale, and 2) qualify additional Pseudomonas spp. strains with SCN suppressive capability in the system. Each goal and supporting project objectives are discussed below.Goal 1: Create a bioreactor system capable of being commercialized at scaleThe technical objectives include:Objective 1: maximize cellular yields of the solid and liquid phases of the bioreactorObjective 2: characterize the stability and compatibility of new formulationsThe tasks for the first objective include testing key design variables impacting the number and stability of bacterial cells produced by the bioreactor, in order to maximize cellular yield and viability of inocula produced. This includes testing various beneficial Pseudomonas spp. strains from The Ohio State University's (OSU) collection, both as single strains and as consortia in the system.The task for the second objective involves testing the stability of the bioreactor contents to changes in supply chain conditions (primarily temperature) to evaluate storage stability lifetime. Also, the second objective involves evaluating the ability of various beneficial strains to tolerate mixing with various classes of fertilizers and pesticides that customers might wish to tank mix and/or co-apply with them.Goal 2: Qualify performance of bacteria strains with SCN suppressive capabilities in system Objective 3: evaluate relative efficacy on SCN in greenhouse and field situationsThe tasks for the third objective involve screening a subset of formulations for SCN suppression, enhanced plant growth, and yield under greenhouse and field conditions. This will allow an assessment of which strain(s) is most competitive in field situations at controlling SCN populations.Through these tasks, we will determine the formulation parameters, which will form the basis for biopesticide registration with US EPA. Thus, the proposed Phase I work will lead directly to Phase II trials and product evaluations to support biopesticide registration, which will involve extensive field-based testing beyond Ohio of the new formulations arising from Phase I. The Phase II work will accurately quantify field performance in a large-scale, regional testing program as well as further replicated trials in university field plots.
Project Methods
Phase I efforts involve individual tasks that map to each of the three project objectives.Objective 1: Maximize cellular yields of the solid and liquid phases of the bioreactor. Task 1: Optimize key design variables of bioreactor based on cell counts. The purpose of this task is to improve upon the current bioreactor design with the goal of 1) extending shelf stability in solid phase beyond 6 months and 2) increasing cellular yields by at least 10-fold in both solid and liquid phases. This task will inform lead design(s) of a more robust, second-generation bioreactor for testing targeted SCN-inhibitory Pseudomonas strains (88A6, 48C10, 38D4, 89F1).Modified formulations: Modified formulations of the current soybean substrate will be evaluated: i) addition of nutrient source (tryptic soy broth (TSB), to increase cellular yields), ii) addition of glycerol (stabilizing agent), iii) addition of both nutrient source and glycerol. Tests will involve the currently used Pseudomonas strain (Wood3). Bacterial culture will be prepared and inoculated onto the solid substrate according to current manufacturing practice. Bioreactors will be maintained at room temperature (25oC), with activation at T=0 (initial), 2, 4, 8, 12, and 24 weeks. Bioreactors will be sampled and cell counts measured at 48 hrs and 14 days post activation. Active cell counts from each sample will be determined using the drop plate method.Advanced encapsulation formulation: Encapsulation in alginate beads has been shown to enhance survival of gram-negative bacteria for a year or longer at room temperature. Certain additives (e.g., starch, glycerol, skim milk) may further enhance stability. Alginate beads will be generated and loaded with bacterial culture and the following additive combinations: i) starch, ii) skim milk, iii) glycerol, iv) starch and skim milk, and v) all three additives. Stability of the cells with time will be measured by dissolving the alginate beads and measuring the cell count.Task 2: Evaluate survival and cellular yields of additional strains and consortia in the bioreactor. The purpose of this task is to extend the utility of the second-generation bioreactor design to deliver targeted SCN-inhibitory Pseudomonas strains (88A6, 48C10, 38D4, 89F1) with the goal of achieving equivalent or improved product stability and cellular yields of the lead design(s) as compared to the current Wood 3 strain. Tests will involve SCN-inhibitory strains both individually and in combination in the lead design(s), following the same procedure as Task 1. For treatments involving multiple strains (consortia), the identity of the cultured bacteria will be confirmed by re-isolation, colony morphology matching, and, as needed, 16S sequence-based identification. Completion of this task will support selection of individual strain(s) and/or consortia that appear relatively robust in the bioreactor system to move forward with in field testing.Objective 2: Characterize the stability and compatibility of new formulations.Task 3: Evaluate stability of inoculant strains in the packaged bioreactors. The purpose of this task is to evaluate lead design(s) at both ambient and accelerated temperature degradation conditions with the goal of establishing a data set to inform product recommendations regarding storage conditions and expected shelf life. Tests will involve storage of bioreactors at either 5oC, 25oC, or 40oC, with activation of the bioreactor at T=0 (initial), 4, 8, 12, and 24 weeks. Activated bioreactors will be maintained at room temperature, with samples taken and cell counts measured at 48 hrs and 14 days post activation. The experiment will be repeated twice. Note: this task will be continued as part of a Phase 2 project aiming to assess the maximum viability period of the product up to two years post manufacturing.Task 4: Evaluate tank-mix compatibility in vitro. The purpose of this task is to systematically evaluate the current Wood 3 strain and targeted SCN-inhibitory strains for compatibility with various chemistries, with the goal of establishing a chemical compatibility chart to inform product recommendations regarding tank mixes. Common starter fertilizer mixes planned for testing include: 6-24-6, 5-15-20, 10-34-0. Common pesticides planned for testing include: Captan, Fludioxinil, Metalaxyl, Tebuconazole, Thiram, Clothianidin, Imidacloprid, Glyphosate, and 2,4-D. Test compounds will be serially diluted in 96 well-plates in 2-fold increments from 4X to 1/8X of the recommended tank-mix concentration for each substance. To these, an equal volume of bacteria inoculum will be added at the labeled rate for tank mixing (i.e. 3 x 106 cells per ml). Plates will be incubated at room temperature for 48 hrs, after which 100 microliters of media will be added to the wells of the plate. Turbidity will be assessed after 48 hrs to determine the level of tolerance of cells to the various chemicals. The experiment will be repeated twice.Objective 3: Evaluate relative efficacy on SCN in greenhouse and field situations. Task 5: Conduct trials of new formulations in the greenhouse and field. The purpose of this task is to evaluate targeted SCN-inhibitory strains for efficacy against SCN under controlled conditions, with the goal to demonstrate product efficacy comparable to laboratory-based results reducing SCN numbers by nearly 40%. Tests will be conducted in two parts: 1) individual strains under direct SCN pressure in the greenhouse, and 2) individual and a combination of SCN-inhibitory strains under microplot field conditions. In each case, treatments will include SCN-inhibitory strains in fresh bacteria culture or activated from the bioreactor, and applied as either a soil drench or seed treatment. In addition, the current Wood3 strain is included as a negative control (it is not SCN-inhibitory, but does promote plant growth). A water drench and "blank" bioreactor will be included as treatment controls. A block design with multiple replicates (20) will be used.Greenhouse: Greenhouse trials will evaluate SCN efficacy and plant growth promotion of soybean. Soybean seeds (cv. Lee 74) will be germinated in a sand-turface potting mix. At 2 week growth stage, pots will be inoculated with bacteria and 3-4 days later inoculated with SCN eggs. After 45 days, mature female cysts will be counted to assess efficacy of treatments against SCN. Separate soybean plants (grown in 4 inch pots and BACCTO potting mix) will be used to assess plant growth promotion at 45 days by measuring plant height, stem width, fresh root weight, dry root weight, root length, and shoot length.Microplots: A microplot test system located at the Ohio Agricultural Research and Development Center's (OARDC) Wooster, Ohio campus will be utilized. Each microplot consists of a large PVC tube (10 inch diameter by 12 inch deep) buried 11 inch in the soil. Each tube will be placed with eight soybean seeds (cv. Lee 73), inoculated with bacteria treatments (either as soil drench or seed treatment), and 3-4 days later inoculated with 80,000-100,000 SCN eggs. Nematodes will be harvested and counted 30 days after inoculation and at the end of the season. For multiple-strain (consortia) treatments, soil-DNA extraction and strain-specific primers will assess presence/absence of strains in the individual and consortia treatments. Stand counts and end of season yield will also be measured.Completion of the greenhouse and microplot trials will provide an initial assessment of biopesticide potential of the SCN-inhibitory strains. Results will help inform selection of strains to move forward in a Phase 2 project for further product development and field trials.