2009 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.
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.
Nutritional Factors in Bioherbicide Production. In bioherbicide production there are the competing goals of minimizing production costs and incubation time while maximizing yield. Because of the toxicological concerns with M. verrucaria, an additional goal is to identify conditions that allow abundant conidia formation, but also minimize the production of mycotoxins. It was recently proven that carbohydrate and nitrogen forms and concentration exert control over sporulation and mycotoxin concentrations. Knowledge gained through this research will improve bioherbicidal utility of M. verrucaria by lowering the limit of detection of trichothecene mycotoxins.
Kudzu Control. Kudzu is a problem weed in many areas of the southeastern U.S. and is difficult to control. Field plot research on herbicidal control of kudzu has been evaluated at two locations for two years. Two pyrimidine, and one sulfonourea herbicide have constantly produced excellent control of kudzu. In the third year of the study we are transitioning from broadcast applications to spot treatments to achieve eradication. Recent observations have 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.
Redvine and Trumpetcreeper Control. Redvine and Trumpetcreeper are fairly tolerant to recommended usage rates of glyphosate and are becomind very serious weed problems GMO soybeans, especially in non-tilled fields. In field experiments, trumpetcreeper and redvine were successfully controlled by two applications (fall and spring) of Myrothecium verrucaria mycelium (with greatly reduced mycotoxin amounts) combined with a compatible glyphosate commercial formulation. A single application of either Myrothecium verrucaria alone or in combination with glyphosate did not control either weed species. The fact that M. verrucaria is capable of controlling several different weeds in addition to kudzu improves the utility and potential marketability of this bioherbicide.
Natural Products Related to Virulence. Natural disease defense mechanisms present in plants, including weeds, can adversely affect the biological control efficacy of bioherbicides.The microbial antibiotics helvolic and fusidic acids, produced by various fungi, were found to exhibit varying degrees of phytotoxicity using in vitro weed bioassays. Tomatine and tomatidine were found to exhibit phytotoxicity and antifungal activity using bioassays and in vitro tests on several fungal pathogens of weeds. These results suggest that these compounds may play a role in plant defense against pathogens or act a virulence factors in infection of plants.
Bioherbicide Formulation. Myrothecium verrucaria is only effective as a pathogen when co-applied with a surfactant (e.g. Silwet L-77). Five commercial products and one family of experimental compounds have been identified that improve the activity of M. verrucaria. 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 itis possible that more widely-available and less-toxic products might be used instead.
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. 39:3057-3073. DOI: 10.1080/00103620802432923.
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.