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United States Department of Agriculture

Agricultural Research Service

2010 Annual Report

1a.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.

1b.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.

3.Progress Report
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.

1. 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, that improve the activity of this fungus. In greenhouse trials, some of these products achieved the same bioherbicidal activity as Silwet L-77 even when 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 widely-available and less-toxic products might be used instead. Of even more importance, the demonstration that toxin production by this fungus can be greatly reduced using appropriate culturing and growth media modification 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.

2. Bioherbicide Host Range Alterations. Hemp sesbania, considered ‘immune’ to the northern jointvetch fungal pathogen Colletotrichum gloeosporioides 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.

3. Kudzu Control. Field plot research on herbicidal control of kudzu has been evaluated at two locations for two years. Two classes of herbicides 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 with 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.

4. Redvine and Trumpetcreeper Control. In field experiments, trumpetcreeper 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 combination 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.

5. Natural Products Related to Effectiveness Against Disease. The microbial antibiotics helvolic and fusidic acids, produced by various fungi, were found to exhibit varying degrees of toxicity tests on weeds. Tomatine and tomatidine were found to exhibit phytotoxicity and antifungal activity using bioassays and in tests on several fungal pathogens of weeds. These results suggest that these compounds may play a role in plant defense against diseases or infection of plants.

6. 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, an additional goal is to identify conditions that allow abundant conidia formation, but also minimize the production of toxins. ARS researchers at Stoneville, MS, showed that carbohydrate and nitrogen concentrations exert 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 of 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 detection of trichothecene mycotoxins.

Review 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. 8:18-24. doi:10.1111/j.1445-6664.2007.00269.x.

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.

Last Modified: 11/26/2015
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