Progress 02/06/06 to 02/05/11
Outputs
Progress Report Objectives (from AD-416) Discover, develop, and improve augmentative bioherbicides, as viable weed biocontrol products through innovative field application, formulation, and mass-production strategies. Develop methodologies to eliminate, reduce or regulate undesirable secondary metabolites from biocontrol pathogens. Discover disease-promoting or weed defense-inhibiting chemicals that synergize bioherbicide. Identify or create biocontrol pathogens with novel traits. Develop molecular markers for bioherbicide strain identification, post-release monitoring, and environmental risk assessment. Discover novel information on genetic determinants and regulation mechanisms of pathogenicity, e.g., virulence, stability, host range and phytotoxin production. Approach (from AD-416) Assess biocontrol potential of several pathogens for control of various weeds: Myrothecium verrucaria for kudzu, redvine, and trumpetcreeper, Colletotrichum truncatum for hemp sesbania, and C. gloeosprioides cassiae for sicklepod. Assess formulations, interactions with agrochemicals, and application timing under field conditions. Assess combinations of host- specific bioherbicides to broaden the weed control spectrum. Develop methodologies for mass production and formulation to improve stability and virulence of bioherbicides. Reduce or eliminate undesirable secondary metabolites (e.g. trichothecenes) from M. verrucaria through mutagenesis, fermentation modifications, growth media alterations, strain selection, use of metabolic regulators, purification, and filtration of pathogen cultures. Inhibitors and other methods to metabolically inactivate trichothecene synthesis in M. verrucaria will be examined. Monitor toxin production via HPLC, ELISA, and HPLC-MS. Assay plant tissues from laboratory, greenhouse and field tests to determine enzyme and secondary plant constituent levels related to weed defense mechanisms against pathogens. Implement biochemical analyses of biomarker defense enzymes and plant constituents to ascertain mechanism of action of the pathogen and the synergistic action of combination of herbicides and other compounds with pathogens. Develop molecular methods for strain identification and post-release monitoring during field testing. Assess the ecological competence of biocontrol agents and the influence of environmental and weed host factors in field and controlled model systems. This is the final report for the project. Substantial results were realized over the 5 years of the project. For example, fermentation methods were developed that resulted in greatly reduced toxins in the fungus Myrothecium verrucaria for controlling the invasive weed kudzu. It was discovered that toxin production in this fungus could be altered by cultural conditions, and that when grown in liquid culture, the fungus was nearly void of toxins. Carbon and nitrogen types and quantities were also found to alter toxin accumulation. It was demonstrated that tests for toxins could be done by using relatively inexpensive assay kits. The mixtures of this fungus and others could be mixed with certain glyphosate herbicides formulations, resulting in greatly improved weed control of redvine, trumpetcreeper, and Hemp sesbania. It was demonstrated that commonly available non-ionic (and one organic) surfactants also promote infection by this fungus, and that the growth hormone herbicide quinclorac exhibited unique interactions using in vitro bioassays with several weed hosts. Weed control was greater when mixtures of plant pathogenic fungi were applied in narrow row spaced soybean test plots as compared to weed control obtained in wider row spacings, and that some vegetable oils, such as corn oil, greatly improves weed control. Formulation research with Hemp sesbania and the northern jointvetch pathogen Colletotrichum gloeosporioides f. sp. aeschynomene showed that Hemp sesbania was not infected by fungal spores alone, but was highly susceptible to infection when formulated in an invert emulsion. The research over the term of this project was designed to improve the weed control efficiency of several different biological weed control plant pathogens, which may eventually result in commercial products. The major impacts, such as formulation technologies developed during this research, have resulted in adaptation of these techniques at several other research facilities in the U.S. as well as in several other countries. ARS researchers at Stoneville, MS, published some of the only results worldwide, demonstrating that when successful bioherbicidal weed control was achieved, increased crop yields resulted.
Impacts
(N/A)
Publications
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Progress 10/01/09 to 09/30/10
Outputs
Progress Report Objectives (from AD-416) Discover, develop, and improve augmentative bioherbicides, as viable weed biocontrol products through innovative field application, formulation, and mass-production strategies. Develop methodologies to eliminate, reduce or regulate undesirable secondary metabolites from biocontrol pathogens. Discover disease-promoting or weed defense-inhibiting chemicals that synergize bioherbicide. Identify or create biocontrol pathogens with novel traits. Develop molecular markers for bioherbicide strain identification, post-release monitoring, and environmental risk assessment. Discover novel information on genetic determinants and regulation mechanisms of pathogenicity, e.g., virulence, stability, host range and phytotoxin production. Approach (from AD-416) Assess biocontrol potential of several pathogens for control of various weeds: Myrothecium verrucaria for kudzu, redvine, and trumpetcreeper, Colletotrichum truncatum for hemp sesbania, and C. gloeosprioides cassiae for sicklepod. Assess formulations, interactions with agrochemicals, and application timing under field conditions. Assess combinations of host- specific bioherbicides to broaden the weed control spectrum. Develop methodologies for mass production and formulation to improve stability and virulence of bioherbicides. Reduce or eliminate undesirable secondary metabolites (e.g. trichothecenes) from M. verrucaria through mutagenesis, fermentation modifications, growth media alterations, strain selection, use of metabolic regulators, purification, and filtration of pathogen cultures. Inhibitors and other methods to metabolically inactivate trichothecene synthesis in M. verrucaria will be examined. Monitor toxin production via HPLC, ELISA, and HPLC-MS. Assay plant tissues from laboratory, greenhouse and field tests to determine enzyme and secondary plant constituent levels related to weed defense mechanisms against pathogens. Implement biochemical analyses of biomarker defense enzymes and plant constituents to ascertain mechanism of action of the pathogen and the synergistic action of combination of herbicides and other compounds with pathogens. Develop molecular methods for strain identification and post-release monitoring during field testing. Assess the ecological competence of biocontrol agents and the influence of environmental and weed host factors in field and controlled model systems. This is the final report for the project 6402-22000-056-00D terminated in August 2010. All planned field experiments were completed prior to termination. Substantial results were realized over the 5 years of the project. For example, fermentation methods were developed that resulted in greatly reduced toxins in the fungus Myrothecium verrucaria (MV) for controlling the invasive weed kudzu. We discovered that toxin production in this fungus be altered by cultural conditions, and that when grown in liquid culture, the fungus was nearly void of toxins. Carbon and nitrogen types and quantities were also found to alter toxin accumulation. We demonstrated that we could test for toxins using relatively inexpensive assay kits. We found that mixtures of this fungus and others with could be mixed with certain glyphosate herbicides formulations, resulting in greatly improved weed control of redvine, trumpetcreeper, and hemp sesbania. We also demonstrated that other commonly available non-ionic (and one organic) surfactants also promote infection by this fungus, and that the growth hormone herbicide quinclorac exhibited unique interactions using in vitro bioassays with several weed hosts. We found that weed control was greater when mixtures of plant pathogenic fungi were applied in narrow row spaced soybean test plots as compared to weed control obtained in wider row spacings, and that some vegetable oils, such as corn oil, greatly improves weed control. Formulation research with hemp sesbania and the northern jointvetch pathogen Colletotrichum gloeosporioides f. sp. aeschynomene showed that hemp sesbania was not infected by fungal spores alone, but was highly susceptible to infection when formulated in an invert emulsion. The impact of the research over the term of this project were designed to improve the weed control efficiency of several different biological weed control plant pathogens, which may eventually result in commercial products. Formulation technologies developed during this research have resulted in adaptation of these techniques at several other research facilities in the U.S. as well as in several other countries. ARS researchers at Stoneville published some of the the only results worldwide that when successful bioherbicidal weed control was achieved, increased crop yields resulted. Accomplishments 01 Bioherbicide Formulation. A reduced mycotoxin M. verrucaria mycelial formulation is efficacious against target weeds (kudzu, redvine, trumpetcreeper, hemp sesbania) when co-applied with a surfactant. Research has been focused on the surfactant Silwet L-77, but it is possible that more widely-available and less-toxic surfactants might be used instead. Five commercial products and 1 family of experimental compounds have been identified by ARS researchers at Stoneville, MS, tha improve the activity of this fungus. In greenhouse trials, some of thes products achieved the same bioherbicidal activity as Silwet L-77 even wh applied with only 20% as much of the bioherbicide. Research has been focused on the surfactant Silwet L-77, but it is possible that more wide available and less-toxic products might be used instead. Of even more importance, the demonstration that toxin production by this fungus can b greatly reduced using appropriate culturing and growth media modificatio has enabled large scale, off-site field testing, and interest by EPA and commercial entities. This research may yield insight resulting in methodology to eliminate these mycotoxins thus making M. verrucaria and similar organisms more feasible for commercialization. 02 Bioherbicide Host Range Alterations. Hemp sesbania, considered �immune� to the northern jointvetch fungal pathogen Colletotrichum gloeosporioide f. sp. aeschynomene, was found to be highly susceptible to this fungus when formulated in an invert emulsion. If successful in the field, the utility of the commercial product (Lockdown�, Agricultural Research Industries, Fayetteville, AR) would be greatly expanded, resulting in improved weed control and rice yields. 03 Kudzu Control. Field plot research on herbicidal control of kudzu has been evaluated at two locations for two years. Two classes of herbicide have constantly produced excellent control of kudzu. In the fourth year of the study we are transitioning from broadcast applications to spot treatments to achieve eradication. Observations revealed that mowing kudzu can provide greater than 50% suppression, even in the year after treatment. Subsequent work has demonstrated greater than 90% control wi a combination of mowing, bioherbicide and herbicide application. This work has fostered collaboration with the Mississippi Department of Transportation, private landowners and the Holly Springs National Forest A new experimental herbicide is under evaluation in replicated field trials. This work has fostered collaboration with the Mississippi Department of Transportation, private landowners and the Holly Springs National Forest. A new, experimental herbicide is under evaluation in replicated field trials. 04 Redvine and Trumpetcreeper Control. In field experiments, trumpetcreepe and redvine were successfully controlled by two applications (fall and spring) of M. verrucara mycelium combined with a compatible herbicide glyphosate commercial formulation (Touchdown). A single application of either Touchdown alone, the fungus alone, or an the fungus in combinatio with glyphosate did not control either weed species. The fact that the fungus is capable of controlling several different weeds in addition to kudzu improves the utility and potential marketability of this bioherbicide. 05 Natural Products Related to Effectiveness Against Disease. The microbia antibiotics helvolic and fusidic acids, produced by various fungi, were found to exhibit varying degrees of toxicity tests on weeds. Tomatine an tomatidine were found to exhibit phytotoxicity and antifungal activity using bioassays and in tests on several fungal pathogens of weeds. Thes results suggest that these compounds may play a role in plant defense against diseases or infection of plants. 06 Nutritional Factors in Bioherbicide Production and Toxin Detection. In bioherbicide production there are the competing goals of minimizing production costs and incubation time while maximizing yield. Because of the toxicological concerns with the plant killing fungus M. verrucaria, additional goal is to identify conditions that allow abundant conidia formation, but also minimize the production of toxins. ARS researchers Stoneville, MS, showed that carbohydrate and nitrogen concentrations exe control over spore production and toxin concentrations. We also demonstrated that we could test for toxins using relatively inexpensive assay kits. Additional improvements are being explored through control o pH, other nutrient selections, and lowering the limit of detection of trichothecenes. Knowledge gained through this research will improve bioherbicidal utility of M. verrucaria by lowering the limit of detectio of trichothecene mycotoxins.
Impacts
(N/A)
Publications
- Boyette, C.D., Hoagland, R.E., Weaver, M.A. 2008. Interaction of a bioherbicide and glyphosate for controlling hemp sesbania in glyphosate - resistant soybean. Weed Biology and Management. 8:19-24.
- Boyette, C.D., Bowling, A.J., Vaughn, K.C., Hoagland, R.E., Stetina, K.C. 2010. Induction of Infection in Sesbania exaltata by Colletotrichum gloeosporioides f. sp. aeschynomene Formulated in an Invert Emulsion. World Journal of Microbiology and Biotechnology. 26:951-956.
- Zablotowicz, R.M., Reddy, K.N., Weaver, M.A., Mengistu, A., Krutz, L.J., Gordon, R.E., Bellaloui, N. 2010. Cover crops tillage and glyphosate effects on chemical and biological properties of a Lower Mississippi Delta soil and soybean yield. Environmental Research. 4:227-251.
- Krutz, L.J., Shaner, D.L., Weaver, M.A., Webb, R.M., Zablotowicz, R.M., Reddy, K.N., Huang, Y., Thomson, S.J. 2010. Agronomic and Environmental Implications of Enhanced s-Triazine Degradation. Pest Management Science. 66:461-481.
- Zablotowicz, R.M., Reddy, K.N., Weaver, M.A., Mengistu, A., Krutz, L.J., Gordon, R.E., Bellaloui, N. 2009. Cover Crops, Tillage, and Glyphosate Effects on Chemical and Biological Properties of a Lower Mississippi Delta Soil and Soybean Yield. In: Tomas H. Latos, ed. Cover Crops and Crop Yields, Nova Publishers, Inc., Huntington, NY, pp. 265-289.
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Progress 10/01/08 to 09/30/09
Outputs
Progress Report Objectives (from AD-416) Discover, develop, and improve augmentative bioherbicides, as viable weed biocontrol products through innovative field application, formulation, and mass-production strategies. Develop methodologies to eliminate, reduce or regulate undesirable secondary metabolites from biocontrol pathogens. Discover disease-promoting or weed defense-inhibiting chemicals that synergize bioherbicide. Identify or create biocontrol pathogens with novel traits. Develop molecular markers for bioherbicide strain identification, post-release monitoring, and environmental risk assessment. Discover novel information on genetic determinants and regulation mechanisms of pathogenicity, e.g., virulence, stability, host range and phytotoxin production. Approach (from AD-416) Assess biocontrol potential of several pathogens for control of various weeds: Myrothecium verrucaria for kudzu, redvine, and trumpetcreeper, Colletotrichum truncatum for hemp sesbania, and C. gloeosprioides cassiae for sicklepod. Assess formulations, interactions with agrochemicals, and application timing under field conditions. Assess combinations of host- specific bioherbicides to broaden the weed control spectrum. Develop methodologies for mass production and formulation to improve stability and virulence of bioherbicides. Reduce or eliminate undesirable secondary metabolites (e.g. trichothecenes) from M. verrucaria through mutagenesis, fermentation modifications, growth media alterations, strain selection, use of metabolic regulators, purification, and filtration of pathogen cultures. Inhibitors and other methods to metabolically inactivate trichothecene synthesis in M. verrucaria will be examined. Monitor toxin production via HPLC, ELISA, and HPLC-MS. Assay plant tissues from laboratory, greenhouse and field tests to determine enzyme and secondary plant constituent levels related to weed defense mechanisms against pathogens. Implement biochemical analyses of biomarker defense enzymes and plant constituents to ascertain mechanism of action of the pathogen and the synergistic action of combination of herbicides and other compounds with pathogens. Develop molecular methods for strain identification and post-release monitoring during field testing. Assess the ecological competence of biocontrol agents and the influence of environmental and weed host factors in field and controlled model systems. Significant Activities that Support Special Target Populations Formulation research with hemp sesbania and the northern jointvetch anthracnose pathogen Colletotrichum gloeosporioides f. sp. aeschynomene (CGA), showed that hemp sesbania was not infected by CGA spores alone, but was highly susceptible to CGA when formulated in an invert emulsion. A manuscript was submitted and accepted with minor revisions. Freeze- dried MV mycelial formulations were found to be efficacious against kudzu and hemp sesbania seedlings. The dried formulation and refrigerated fresh MV formulation retained viability after several months of storage. We discovered that macrocyclic trichothocene production in MV can be altered by cultural conditions. MV grown under submerged culture was shown to be nearly void of trichothecenes. Carbon and nitrogen types and quantities were also found to alter trichothecene accumulation. MV growth and sporulation can occur under conditions that inhibit or greatly reduce accumulation of this mycotoxin. Two manuscripts were published in peer reviewed journals. Ultrastructural studies on MV, the trichothecene roridin A, and kudzu interactions continued. A manuscript summarizing this research was submitted and is being revised. Ivyleaf, moonvine, and palmleaf morningglories were found to be tolerant to MV, while pitted, multi-color, moonflower, and cypressvine morningglories exhibited more severe injury. MV combined with an invert emulsion was generally more effective than MV alone. Compatibility of MV with agrochemicals has been characterized in greater detail MV and the auxenic herbicide quinclorac were found to exhibit unique interactions using in vitro bioassays with several weed hosts.
Impacts
(N/A)
Publications
- Hoagland, R.E., Weaver, M.A., Boyette, C.D. 2008. Enzyme-linked immunosorbent assay detection of trichothecenes produced by the bioherbicide myrothecium verrucaria in cell cultures, extracts, and plant tissues. Communications in Soil Science and Plant Analysis 39:3059-3075.
- Weaver, M.A., Hoagland, R.E., Boyette, C.D., Zablotowicz, R.M. 2009. Macrocyclic Trichothecene Production and Sporulation by a Biological Control Strain of Myrothecium verrucaria is Regulated by Cultural Conditions. World Mycotoxin Journal 2(1):35-43.
- Weaver, M. A., Jin, X., Hoagland, R. E., Boyette, C. D. 2009. Improved bioherbicidal efficacy by Myrothecium verrucaria via spray adjuvants or herbicide mixtures. Biocontrol. 50: 150-156.
- Lovelace, M.L., Talbert, R.E., Hoagland, R.E., Scherder, E.F. 2009. Influence of Quinclorac Drift on the Accumulation and Movement of Herbicide in Tomato (Lycopersicon esculentum) Plants. Journal of Agriculture and Food Chemistry 57:6349-6355.
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Progress 10/01/07 to 09/30/08
Outputs
Progress Report Objectives (from AD-416) Discover, develop, and improve augmentative bioherbicides, as viable weed biocontrol products through innovative field application, formulation, and mass-production strategies. Develop methodologies to eliminate, reduce or regulate undesirable secondary metabolites from biocontrol pathogens. Discover disease-promoting or weed defense-inhibiting chemicals that synergize bioherbicide. Identify or create biocontrol pathogens with novel traits. Develop molecular markers for bioherbicide strain identification, post-release monitoring, and environmental risk assessment. Discover novel information on genetic determinants and regulation mechanisms of pathogenicity, e.g., virulence, stability, host range and phytotoxin production. Approach (from AD-416) Assess biocontrol potential of several pathogens for control of various weeds: Myrothecium verrucaria for kudzu, redvine, and trumpetcreeper, Colletotrichum truncatum for hemp sesbania, and C. gloeosprioides cassiae for sicklepod. Assess formulations, interactions with agrochemicals, and application timing under field conditions. Assess combinations of host- specific bioherbicides to broaden the weed control spectrum. Develop methodologies for mass production and formulation to improve stability and virulence of bioherbicides. Reduce or eliminate undesirable secondary metabolites (e.g. trichothecenes) from M. verrucaria through mutagenesis, fermentation modifications, growth media alterations, strain selection, use of metabolic regulators, purification, and filtration of pathogen cultures. Inhibitors and other methods to metabolically inactivate trichothecene synthesis in M. verrucaria will be examined. Monitor toxin production via HPLC, ELISA, and HPLC-MS. Assay plant tissues from laboratory, greenhouse and field tests to determine enzyme and secondary plant constituent levels related to weed defense mechanisms against pathogens. Implement biochemical analyses of biomarker defense enzymes and plant constituents to ascertain mechanism of action of the pathogen and the synergistic action of combination of herbicides and other compounds with pathogens. Develop molecular methods for strain identification and post-release monitoring during field testing. Assess the ecological competence of biocontrol agents and the influence of environmental and weed host factors in field and controlled model systems. Significant Activities that Support Special Target Populations We have proven that that the production of macrocyclic trichothocenes can be altered by changing the cultural conditions. The amount and type of carbon and nitrogen sources alters the amount of mycotoxin accumulation. Most significantly, it is now clear that growth and sporulation can occur under conditions that lead to greatly reduced mycotoxin concentrations. Ultrastructaural studies revealed that MV caused rapid protoplast detachment from cell walls, with plasmodesmata rapidly broken off and retained in cell walls of kudzu leaves, prior to the appearance of fungal growth. Roridin A (trichothecene mycotoxin, produced by unwashed MV spores, but not washed spores or mycelium) caused symptoms similar to those induced by spores plus surfactant. Data indicate penetration of phytotoxic substance(s) in fungal formulation is facilitated by the surfactant Silwet L-77, and that roridin A has phytotoxic properties similar to MV phytotoxin(s). These overall effects appear unique among known phytotoxins or mycotoxins. Freeze-dried MV mycelial formulations were found to be efficacious against kudzu and hemp sesbania seedlings. Long-term storage of this formulation and refrigerated fresh MV formulations are being evaluated. A proteolytic enzyme from MV mycelium was isolated, partially purified, and tested for a role as a virulence factor. Results of freeze-dried enzyme samples tested under greenhouse conditions, alone, and in combination with MV were not definitive on kudzu seedlings, but in hemp sesbania the enzyme combined with MV treatment caused greater fresh weight reduction and shoot growth than MV alone. Lab bioassays showed that the enzyme combined with MV, caused more maceration of etiolated excised hemp sesbania tissues than MV alone. Further experimentation is needed to verify if this enzyme is a virulence factor. We found that ivyleaf, moonvine, and palmleaf morning glories were relatively tolerant to MV, while pitted, multi-color, moonflower, and cypressvine morning glories exhibited more severe injury. Although some of these plants exhibited tolerance to MV plus Silwet L-77, MV combined with an invert emulsion increased their susceptibility. Compatibility of MV with agrochemicals has been characterized in greater detail. We demonstrated that MV is incompatible with several commercial formulations of glyphosate, but is compatible with two commercial formulations. We also demonstrated that other commonly available non- ionic (and one organic) surfactants also promote MV infection. Hemp sesbania, considered �immune� to the northern jointvetch anthracnose pathogen Colletotrichum gloeosporioides f. sp. aeschynomene (CGA), was found to be highly susceptible to CGA spores when formulated in an invert emulsion. NP 304, Component: 9, Problem Statement: b, c. Technology Transfer Number of New/Active MTAs(providing only): 1 Number of Web Sites managed: 1
Impacts
(N/A)
Publications
- Boyette, C.D. 2006. Adjuvants Enhance the Biological Control Potential of an Isolate of Colletotrichum Gloeosporioides for Biological Control of Sicklepod (Senna obtusifolia). Biocontrol Science and Technology. 16:1057- 1066.
- Boyette, C.D., Hoagland, R.E., Weaver, M.A., Reddy, K.N. 2008. Redvine (Brunnichia ovata) and Trumpetcreeper (Campsis radicans) Controlled Under Field Conditions by a Synergistic Interaction of the Bioherbicide Myrothecium verrucaria and Glyphosate. Weed Biology and Management 8:39-45.
- Boyette, C.D., Hoagland, R.E., Weaver, M.A. 2007. Effect of Row Spacing on Biological Control of Sicklepod (Senna obtusifolia) with Colletotrichum gloeosporioides. Biocontrol Science and Technology 17(9):957-967.
- Hoagland, R.E., Boyette, C.D., Abbas, H.K. 2007. Myrothecium verrucaria Isolates and Formulations as Bioherbicide Agents for Kudzu. Biocontrol Science and Technology. 17(7):721-731.
- Hoagland, R.E., Boyette, C.D., Weaver, M.A., Abbas, H.K. 2007. Bioherbicides: Research and Risks. Journal of Toxicology Toxins Reviews, Vol. 16, pp. 1-30.
- Weaver, M.A., Kenerley, C.M. 2008. Competitiveness of a Genetically Engineered Strain of Trichoderma virens. Mycopathologia 166:51-59.
- Boyette, C.D., Weaver, M.A., Hoagland, R.E., Stetina, K.C. 2008. Submerged Culture of a Mycelial Formulation of a Bioherbicidal Strain of Myrothecium verrucaria with Mitigated Mycotoxin Production. World Journal of Microbiology and Biotechnology - DOI 10.1007/s11274-008-9759-6.
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Progress 10/01/06 to 09/30/07
Outputs
Progress Report Objectives (from AD-416) Discover, develop, and improve augmentative bioherbicides, as viable weed biocontrol products through innovative field application, formulation, and mass-production strategies. Develop methodologies to eliminate, reduce or regulate undesirable secondary metabolites from biocontrol pathogens. Discover disease-promoting or weed defense-inhibiting chemicals that synergize bioherbicide. Identify or create biocontrol pathogens with novel traits. Develop molecular markers for bioherbicide strain identification, post-release monitoring, and environmental risk assessment. Discover novel information on genetic determinants and regulation mechanisms of pathogenicity, e.g., virulence, stability, host range and phytotoxin production. Approach (from AD-416) Assess biocontrol potential of several pathogens for control of various weeds: Myrothecium verrucaria for kudzu, redvine, and trumpetcreeper, Colletotrichum truncatum for hemp sesbania, and C. gloeosprioides cassiae for sicklepod. Assess formulations, interactions with agrochemicals, and application timing under field conditions. Assess combinations of host- specific bioherbicides to broaden the weed control spectrum. Develop methodologies for mass production and formulation to improve stability and virulence of bioherbicides. Reduce or eliminate undesirable secondary metabolites (e.g. trichothecenes) from M. verrucaria through mutagenesis, fermentation modifications, growth media alterations, strain selection, use of metabolic regulators, purification, and filtration of pathogen cultures. Inhibitors and other methods to metabolically inactivate trichothecene synthesis in M. verrucaria will be examined. Monitor toxin production via HPLC, ELISA, and HPLC-MS. Assay plant tissues from laboratory, greenhouse and field tests to determine enzyme and secondary plant constituent levels related to weed defense mechanisms against pathogens. Implement biochemical analyses of biomarker defense enzymes and plant constituents to ascertain mechanism of action of the pathogen and the synergistic action of combination of herbicides and other compounds with pathogens. Develop molecular methods for strain identification and post-release monitoring during field testing. Assess the ecological competence of biocontrol agents and the influence of environmental and weed host factors in field and controlled model systems. Accomplishments We have discovered an isolate of Myrothecium verrucaria (MV) that is very efficacious against kudzu and other invasive weeds, and this pathogen is the primary focus of our biological control of weeds research efforts. This strain of M. verrucaria produces a group of mycotoxins that may limit its practical usage. Several industrial entities have expressed interest in developing this pathogen commercially if mycotoxin content can be reduced to levels acceptable by Environmental Protection Agency (EPA). We have developed methodologies to reduce MV mycotoxin content to a level of EPA. acceptability for registration. The fungus Colletotrichum gloeosporioides, originally isolated from the weed species, coffee senna, can effectively control a related weed, sicklepod, when fungal spores are formulated in either unrefined corn oil or an invert emulsion. Control of hemp sesbania is also enhanced when fungal spores are applied to weeds in an unrefined corn oil emulsion. We are also currently evaluating MV formulations with reduced trichothecene content that we have developed, and have demonstrated that these formulations retain high weed control efficacy. Redvine and trumpetcreeper are increasingly problematic in no-till and low-till soybeans in the Mississippi Delta, and are highly tolerant to recommended glyphosate usage rates. We have found that sequential applications of a glyphosate product, followed by an application of MV, will effectively control these weeds. Control of kudzu and other highly invasive weeds may require a combination of management practices. Identifying the ability to co-apply MV with newer herbicides provides greater ability for use of the bioherbicide. We have also discovered a synergistic interaction occurs between certain glyphosate products and Colleototrichum truncatum for improved biological control of hemp sesbania. Laboratory studies have assessed the compatibility of MV with three herbicides currently being used for control of kudzu: aminopyralid (Milestone), metsulfuron (Escort) and flouoroxypyr (Vista). A good degree of survival of spores was found in tank mix concentrations of Milestone and Escort and was capable of growth in the presence of commercial formulation of these herbicides. Field trials are evaluating efficacy of combinations of the biocontrol fungus with these herbicides for integrated kudzu control. Technology Transfer Number of Non-Peer Reviewed Presentations and Proceedings: 6 Number of Newspaper Articles,Presentations for NonScience Audiences: 7
Impacts
(N/A)
Publications
- Boyette, C.D., Jackson, M.A., Bryson, C.T., Hoagland, R.E., Connick Jr, W. J., Daigle, D.J. 2006. Sesbania exaltata biocontrol with colletotrichum truncatum microsclerotia formulated in 'pesta' granules. Biocontrol - Online DOI 10.1007/s/10526-006-9031-7.
- Boyette, C.D., Hoagland, R.E., Weaver, M.A. 2007. Biocontrol efficacy of colletotrichum truncatum for hemp sesbania (sesbania exaltata) is enhanced with unrefined corn oil and surfactants. Weed Biology and Management 7:70- 76.
- Hoagland, R.E., Weaver, M.A., Boyette, C.D. 2007. Myrothecium verrucaria fungus: A Bioherbicide and Strategies to Reduce Its Non-Target Risks. Allelopathy Journal 19(1): 179-192.
- Williams, R.D., Hoagland, R.E. 2007. Phytotoxicity of mimosine and albizziine on seed germination and seedling growth of crops and weeds. Allelopathy Journal. 19(2):423-430
- Boyette, C.D., Reddy, K.N., Hoagland, R.E. 2006. Biocontrol of kudzu (Pueraria lobata), redvine (Campsis radicans), and trumpetcreeper (Brunnichia ovata) is synergized by glyphosate. Biocontrol Science and Technology 16:1067-1077.
- Boyette, C.D., Hoagland, R.E., Abbas, H.K. Evaluation of the bioherbicide Myrothecium verrucaria for weed control in tomato (Lycopersicon esculentum) . Biocontrol Science and Technology 17:171-178. 2007.
- Lovelace, M.L., Talbert, R.E., Scherder, E.F., Hoagland, R.E. 2007. Effects of multiple applications of simulated quinclorac drift rates on tomato. Weed Science 55:167-169.
- Weaver, M.A., Lyn, M.E. 2007. Compatability of a Biological Control Agent with Herbicides for Control of Invasive Plant Species. Natural Areas Journal 26:264-268.
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Progress 10/01/05 to 09/30/06
Outputs
Progress Report 1. What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? Why does it matter? Management of invasive weeds is a necessary but expensive challenge. Weed control with synthetic chemicals currently exceeds $4 billion annually, excluding immense indirect costs to the producer, consumer and the environment, and has also resulted in development of resistant weed biotypes. Over the past decade, many chemical herbicides have been lost due to lack of re-registration, competition from other products, and the development of genetically modified crops with resistance to broad- spectrum herbicides such as glyphosate. The majority of herbicide usage is for agronomic areas or turf, but there are few herbicides registered for (or being developed for) smaller markets or niche weed problem areas such as control of kudzu, and invasive weeds in non-cropland areas. Furthermore,
chemical weed control is not an option in organic cropping systems and in sensitive natural habitats. Herbicides have not been successful in managing many invasive plant species, such as kudzu, an important host of Asian soybean rust. Classical pathogen mediated biocontrol of weeds has also had only limited success; due in part to a dependence on optimal weed pathogen environment interactions and the small market potential associated with highly specific (one weed species) biocontrol agents released with this approach. An augmentative deployment strategy for a bioherbicide can achieve effective control through multiple applications. Broad-spectrum bioherbicides improve a product's marketability, and by embracing an augmentative deployment strategy the agent can achieve effective control through repeated applications. The fungus Myrothecium verrucaria (MV) is a bioherbicide with high potential. It is one of the most effective biocontrol agents against kudzu, and controls many
other invasive plant species, but it also produces mycotoxins. A major goal of this project is to make safe formulations of MV with enhanced efficacy and reduced toxicity to enable product development. The assessment of the risks associated with deployment of biocontrol agent requires knowledge of its epidemiology, post-release survival, spread, and interaction with the natural ecological microbial community. Sensitive detection of a pathogen at sites distant from deployment or long after application requires molecular tools, which have only recently been developed. Information generated from this research will expand current knowledge of weed:pathogen interactions, while addressing needs of federal and regional research priorities, regulatory agencies (e.g. EPA, APHIS), the agrochemical industry, consultants, environmental scientists, farmers, land managers, and the general public. Relevance to ARS National Program Action Plan: National Program 304 Crop Protection and
Quarantine: This program falls within Component B (Weed Science), Sub-component IX (Biological Control of Weeds). This project will focus on: 2.A (Agent discovery, selection and risk assessment); 2.B (Efficacy and mass production of augmentative agents); 2. C (Field evaluation); and 2.D (Combining biocontrol agents). This plan may also contribute to Component I: Preplant Soil Fumigation Alternatives of NP 308 "Methyl Bromide Alternatives." 2. List by year the currently approved milestones (indicators of research progress) Year 1. (FY 2006) 1. Initiate/develop/optimize trichothecene-deficient MV formulations. 2. Conduct MV/glyphosate (and other herbicide) compatibility assessments. 3. Develop MV trichothecene inhibition assays. 4. Initiate/develop MV mutants with reduced trichothecene levels. Year 2. (FY 2007) 1. Complete MV trichothecene-deficient fermentation studies. 2. Develop micro-encapsulation methodologies for MV. (Milestone modification request was approved by NPL
to direct encapsulation research on MV; also approved request to move milestone from FY 2006 to FY 2007). 3. Establish research to characterize MV genotypic/phenotypic diversity. 4. Identify biomarkers for following fate of MV in the environment. (Milestone modification request was approved by NPL to direct biomarker research on MV; also approved request to move milestone from FY 2006 to FY 2007). 5. Trichothecene-deficient mutants identified. 6. Initiate and complete cloning of MV Tri-genes. Year 3. (FY 2008) 1. Improved MV formulation assessment completed. 2. MV on-site field assessment on kudzu completed. 3. Trichothecene biosynthesis inhibitors identified. 4. MV mycotoxin and pathogenicity diversity characterized. 5. Milestone modification requested and approved by NPL to direct research towards MV encapsulation; also approved to move from 2008 to 2009 in order to obtain two sets of field data. Year 4. (2009) 1. Field assessments of MV formulations and encapsulation
completed. 2. MV fermentation/inhibitor systems developed. 3. MV:weed defense interactions characterized. 4. Complete post-release MV monitoring and risk assessment. 5. Tri-gene expression documented. 4a List the single most significant research accomplishment during FY 2006. The new project was only approved in February 2006. Laboratory, greenhouse, and field experiments have been initiated in each of the projected milestones. 5. Describe the major accomplishments to date and their predicted or actual impact. We have discovered and patented several plant pathogens and biologically- based compounds which are highly effective in controlling several agronomic, horticultural, and exotic invasive weed species. For example, we have shown that the fungus Myrothecium verrucaria can effectively control kudzu and other invasive weeds in field trials. We have developed and patented formulations, such as invert, oil emulsions, and granular, which greatly enhance the biocontrol efficacy of
bioherbicides. For example, we have demonstrated that the fungus Colletotrichum truncatum is highly effective in controlling the weed hemp sesbania in soybean, cotton, and rice fields when formulated with corn oil or invert emulsions, or with pre-emergence applications of fungus-infested wheat- gluten granules (Pesta) or rice grain formulations. Various surfactants, such as Silwet L-77 enhanced the virulence of C. truncatum for hemp sesbania control, and C. gloeosporioides for sicklepod control. We have discovered important biochemical defense mechanisms that are triggered in several weeds when the plants are challenged by pathogens such as Myrothecium verrucaria, Alternaria cassiae, A. crassa and C. truncatum, and herbicides such as glyphosate. Understanding such mechanisms is paramount to finding compounds that can inhibit weed defenses and promote pathogenesis and thereby increase pathogen efficacy. We have discovered that low levels of glyphosate can synergize diseases of
several different pathogen:weed host systems. The impact of some of these discoveries could result in improved bioherbicide efficacy and ultimately to commercial products, and to a reduction in the overall usage of chemical herbicides. 6. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end- user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? Assisted in organization of USDA-ARS sponsored Kudzu Biocontrol Workshop, Stoneville, MS. Attended by NPL and several divergent researchers and stakeholders, e.g., forestry, DOT, and homeowners. Two members of the research unit made oral presentations concerning biocontrol of kudzu and risk assessment. Compiled proceedings and submitted final report to NPL May, 2006. Met with representatives from Novozymes Biologicals, Inc. to discuss bioherbicide commercialization;
oral overview presentation of SWSRU bioherbicide research. Met with representative of Prophyta Ltd. to discuss commercial development of Myrothecium verrucarria. Presented posters at the Weed Science Society of America, New York, New York. Presented posters at the Mississippi Weed Science Society, Stoneville, MS. Mentored student presented poster at the Mississippi Academy of Science, Jackson, MS (Won second place). Attended and presented invited poster at 2006 Agronomic Crops Field Day, Delta Research and Extension Station, Stoneville, MS. 7. List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: List your peer reviewed publications below). Boyette, C.D., Hoagland, R.E., and Weaver, M.A. 2006. Interaction of the fungus Colletotrichum truncatum and glyphosate for controlling hemp sesbania in glyphosate-resistant soybean. Phytopathology. 96:S15. Weaver M.A., Hoagland, R.E., and Boyette, C.D.
2006. Compatibility of the bioherbicide Myrothecium verrucaria with selected pesticides. Phytopathology. 96:S121.
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