Recipient Organization
MONTANA STATE UNIVERSITY
(N/A)
BOZEMAN,MT 59717
Performing Department
Plant Sciences and Plant Pathology
Non Technical Summary
Organic growers in Montana use complex systems with crop rotation, green manures, and tillage to control weeds. In 2017, the Organic Advisory and Education Council (OAEC) approached us and sought collaboration in a project to develop bioherbicides for their two most recalcitrant weeds, field bindweed and Canada thistle. These deep-rooted perennial weeds (both listed in the world's top ten worst weeds) evade current management practices. Through OAEC and their organizing farmers in Montana, samples of diseased bindweed and thistle from organic fields have been sent to our laboratory for analysis in the hope of finding herbicidal plant pathogens.We developed a safe and novel selection system to enhance bioherbicidal fungi that was successfully demonstrated for biocontrol of a parasitic weed of maize, millet, sorghum, and recently reported wheat. This soil-borne weed, witchweed (Striga hermonthica) is a production limiting scourge among smallholder farmers in Kenya (www.toothpickproject.org). We propose to use the same bioherbicide technology (enhanced virulence of locally sourced host specific strains of Fusarium oxysporum. Although these endemic fungi cause wilt disease of these weeds, it is necessary to improve their virulence by selecting (without recombinant genetics) strains that excrete two natural non-toxic amino acids lysine and methionine, or lysine and threonine in the case of field bindweed. The amino acids produced by the fungi inhibit the weed as the fungus infects the stem and root tissue of the weed. These enhanced fungi when coated on crop seeds can inoculate the soil with the bioherbicidal fungi. Conventional farmers as well as organic farmers will benefit from development of these bioherbicides.
Animal Health Component
60%
Research Effort Categories
Basic
20%
Applied
60%
Developmental
20%
Goals / Objectives
Objective 1a: Obtain strains of fungi that attack C. Thistle or bindweed.A primary aim of this project is to establish that Montana soils host strains of Fusarium oxysporum that are pathogens against our two most troublesome invasive weeds, Canada thistle and field bindweed. Currently we have 40 fungal isolates with preliminary identification as Fusarium oxysporum. We have been able to isolate Fusarium oxysporum from seed heads of these plants and from sick plants provided by growers. We have proceeded to test such isolates of the fungi for their pathogenicity. The isolation of the fungi from the field will continue in order to obtain virulent endemic isolates.Milestone: Isolate from 40 samples per year some 80 fungal isolates and greenhouse test for virulence.Objective 1b. Virulence EnhancementThis objective includes the enhancement of Montana isolates by selecting for amino acid excretion using analog selection procedures as described above and (83,93). Bioassays for these amino acids with knockout auxotrophic strains of E.coli will be used to quantify the level of excretion. Selected excreting strains will be reselected several times as done by Lynn Anderson and Eylul Kaya (80).Milestone: Year: Enhance virulence of isolates, at least 10 from each host.Objective 2a. Testing for field efficacy.A sub-objective is to develop a field protocol testing protocol that can span over several years and several locations on both organic and conventionally managed landscapes. Randomized block design will involve at least four replications, with the performance data analyzed using standard statistical packages (analysis of variance programs). MSU offers statisticians to design field experiments and subsequent analytical packages. We will also establish a greenhouse testing protocol that can be used to differentially determine the most virulent isolates.Milestone: Field test isolates for effectiveness when delivered as fresh inoculum. This will require at least three plots per year on organic and conventional property.Objective 2b. Testing for distribution.A second sub-adjective is to create an alternative method of distributing the bioherbicidal fungi. Our experience with a carrier seed method of fusarial distribution is based on the observation by many pathologists that Fusarium oxysporum may be host specific attacking and damaging one host, but it is capable of colonizing root systems of many plant species when their seed coated with the fungus. The seed can be of a cover crop or of an actual crop such as wheat or a legume. Experiments will be done to determine what percentage of seed need to be coated to establish the fungi in the soil. This “soil vaccination” approach may be effective in providing long term inoculum against these weeds. The fungi are already isolated with the Nash-Snyder medium (77) or Komada’s medium (65), but their additional marker is amino acid analog resistance. Their populations can be followed in the soil quantitatively.Milestone: Test crop (wheat and legume) seed coating for field efficacy of bioherbicide field inoculation.Objective 2c. Design bioherbicide protocol.A third sub-objective is to design bioherbicide protocols that can be used by both organic farmers and conventional farmers in transition to organic production. These will be delivered at field days and via e-mail to cooperating organic and transitional growers for feedback.Milestone: Deliver effective protocol for use of bioherbicides.Objective 2d. Production Delivery System:A seed coating system will be tested for delivery of the bioherbicides. To quote Rocha et al. in 2019 (90), “Tested in more than 50 plant species with seeds of different dimensions, forms, textures, and germination types (e.g., cereals, vegetables, fruits, pulses, and other legumes), seed coating has been studied using various species of plant growth-promoting bacteria, rhizobia, Trichoderma, and to a lesser extent mycorrhizal fungi. Most of the studies regarding microbes applied via seed coating are aimed at promoting crop growth, yield, and crop protection against pathogens. The right combination of biological control agents applied via seed coating can be a powerful tool against a wide number of diseases and pathogens. Notwithstanding the promising results, there are still challenges mainly related with the scaling up from the laboratory to the ?eld and proper formulation, including developing ef?cient microbial combinations and coating materials that can result in extended shelf-life of both seeds and coated plant beneficial microbes. These limitations need to be addressed and overcome in order to allow a wider use of seed coating as a cost-effective delivery method in sustainable agricultural systems.” In our own work with bioherbicidal fungi, we found that carboxymethyl cellulose was an effective coating with Fusarium oxysporum spores (99).Milestone: Design and prove efficacy of organic and conventional crop seed coating system and offer for commercialization.Objective 3. Determine registration requirements.Production and registration requirements are to be designed and coordinate with registration agencies and commercial applicants, with organic certification requirements and reasonable cost requirements. Given our experience developing the toothpick technology in Kenya, by designing and equipping a fungal production facility for primary inoculum, and designing a second fresh inoculum system for use in villages, we are prepared to design protocols that will lead to registration and commercialization of bioherbicides for Canada thistle and bindweed. Here, the requirements for organic bioherbicide production are even more demanding and we will work directly with Dr. Georgana Webster (MDA – Organic Agricultural Specialist) and Dr. Michael Braverman (USDA IR4 specialist) to design trials to reduce the possibility of inadequate data submission.Milestone: Determine production capabilities with commercial interests, to prepare for registration.
Project Methods
Methods: Field Survey and Soil Survival of Weed Pathogens: 1. Year 1, 2 and 3: In 2018-9, an e-mail solicitation for diseased Canada Thistle (CT) and Field Bindweed (FB) samples were successful in getting more than ten samples mailed into the laboratory from Northern Montana. In addition, we traveled to several farms to pick up samples of wilted plants. Most samples were of wilted CT collected and sent into the lab in late summer. This year we have resolicited, again by email, for more samples Our objective will be to have samples from 40 new locations per year. The search for effective bioherbicides will continue through several seasons until effective bioherbicides are found. Samples will be processed with selective media (Nash-Snyder's (77) and then Komada's (66)) for the wilt pathogens Fusarium oxysporum. At this stage, a quick screening for pathogenicity against CT or FB will eliminate many fungi. Only those that are pathogenic will be further processed. These fungi will be identified with use of cultural characteristics and microscopy (69, 79). Cultures will be purified by using the nif minus (chlorate survival) mutation method of John Leslie (69), or by the amino acid analog selection method (both methods are designed to select for single nuclei from this multinucleate fungus). The analog selection is an innovative way to enhance virulence and concomitantly select for single nuclei (80).2. Greenhouse testing of potential plant pathogens for CT and FB in done by inoculating spore suspensions into stem wounded plants in three replicate trials. Initiation of wilt symptoms occurs under slight water stress, and plants are compared control sterile inoculations and to a reference strain inoculated concomitantly.3. Strain preservation will involve two methods: Toothpick preservation, on dried toothpicks after five days growth on agar, and by -80C preservation on agar slanted vials. A third method is with preservation granules.4. Field testing of isolates will be done in organic certified field plots as has been done in 2019 in Hinsdale, Montana in patches of CT. and FB with three or more replications of each sample. Field studies, so far, have been done with cooked organic rice (organic long grain white rice as a fungal substrate) put in certified organic fields, or at an MSU campus pasture infested with Canada thistle. Further trials will be in plots near Bozeman where patches of CT are located, (not necessarily on organically farmed land). Field observations will involve following progress for two years. Plants will be rated on a 1-5 scale for disease severity. Koch's postulates (verification analyses) will be done with selective media.Methods: Selection for enhanced virulence: (Years 1, 2 and 3). We determined that certain amino acids inhibit C.T. and F.B. (See illustration in section above) and selection of Fusarium oxysporum strains that excrete these amino acids is possible by exposing the fungi to toxic amino acid analogs. Examples are the lysine analog (amino ethyl-cysteine), and methionine analog (seleno-methionine). At levels of 10mg/l to 1000 g/l, these are toxic when the fungi are grown in minimal salts media (Czapek's plus vitamins). Individual mutants can sometimes be observed on agar media to produce enough of these amino acids to feed proximal non mutant fungi. Repeated use of the analog procedure, stepping up the concentration of analogs to the point of saturation can result in greater excretion levels, as determined with analysis done with knock-out strains of E.coli, or with strains of Lactobacillus developed for such purposes (83, 93). The amino acids inhibitory to F.B. are lysine and threonine. The objective is to develop fungi that excrete these amino acids as they infect the weed, localizing the production in the field rather than in a large chemical plant.Methods: Seed Coating: Year 2 and 3. The toothpick method, developed where smallholder farmers in Kenya use a fungal laden toothpick to inoculate their own cooked rice (80), was successful in small acreages and designed especially for farmers who hand-plant their crops. However, fungi applied as a coated crop seed treatment is a more appropriate technology for large acreages. We are testing seed coatings (there are traditional and organic coatings available) to see which seeds and which coatings will be most effective at delivering fungal inoculum into the soil. Our original work on this technology twenty years ago was with carboxy methyl cellulose as a seed coat, patented for use with fungi including Fusarium oxysporum (96, 99, 100). Seed coating is a developing technology. Refer to a summary covered in an extensive review of seed coating by Rocha, et.al. (90 in 2019). We need to determine what percent of crop seeds and what crops will be optimal (it could be as low as 1%). The objective is to distribute the inoculum (soil vaccination), into the soil for long term establishment Methods: Determining presence or absence of toxins: Year 3 and 4. Some Fusarium strains (not strains of Fusarium oxysporum) are known to produce toxins, and any release of such pathogens will only be possible after the strains have been tested by an independent laboratory that specializes in toxin analysis. Our strains used in Kenya were found to be toxin-free (strains in rice inoculum were found to be free of the six common fusarial toxins (deoxynivalenol (DON), 3-acetyldeoxynivalenol (3-ADON), 15-acetyldeoxynivalenol (15-ADON), nivalenol (NIV), zearalenone (ZEA), and fumonisin) by two laboratories, the Mycotoxicology Laboratory at Virginia Tech and University of Nairobi, Kenya (80). Dr. Schmale at the Virginia Tech has offered to analyze our fusaria on a contract basis. Dr. Like Folkers (114, 116) in the Netherlands has determined that genes coding for production of these toxins are not found in the genomes of Fusarium oxysporum. We have the current APHIS PPQ permits necessary to safely transfer cultures to these laboratories.Methods: Production Engineering: Years 3-5. Fungal fermentation systems have been developed for biocontrol agents (52,57,58). In terms of producing primary inoculum for seed coatings we are working closely with the Toothpick project in Kenya where they are coating seeds with Foxy T14 for Striga biocontrol. It is possible to obtain chlamydospore concentrations as high as 100 million per gram, and these can be diluted to much lower levels to deliver 10, 100, and 1000 spores per seed treated. The dynamics of using coated seed in Striga infested fields are expected to be quite different than those where green manure crops are used to establish the fungi in dryland agriculture. Many patents that involve these methodologies (9, 17, 61,82, and 108) will have to be licensed, but most are no longer in effect. Production of chlamydospores of Fusarium oxysporum are likely to require some innovation, and they are likely to be determined as part of the necessary technology transfer aspect of commercialization.Methods: Registration of and Patenting of Bioherbicides: Year 4 and 5. Establish a clear understanding and collaboration with registration officials concerning what the field trial and toxicological data requirements are for registration to expedite the registration process. In 2019, our project has been selected as the test case for a Southern Africa Regional Group of nations, to possibly standardize bioherbicide registration on a regional basis. Our training of scientists from 11 additional countries was initiated in part, in recognition of the Nagoya Protocol (germplasm from countries cannot be taken without fair compensation). Interstate transfer of biological agents is also regulated. We are in contact with the officials at the Montana Department of Agriculture (G. Webster), and with the IR4 Agency of the USDA (M. Braverman) to establish the design of experiments leading to the efficacy data for these bioherbicides.