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United States Department of Agriculture

Agricultural Research Service

2008 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
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.

1. Development of trichothecene-free MV formulation

We have previously discovered an isolate of Myrothecium verrucaria 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, (e.g. trichothecenes) 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 E.P.A. We have developed a method utilizing liquid fermentation to produce in order to reduce MV trichothecene content to a level of E.P.A. acceptability for registration, and have demonstrated this formulation to retain high efficacy for controlling kudzu and hemp sesbania. These studies underscore the importance of cultural conditions and formulation on bioherbicide safety and efficacy. Results have been published in World Journal of Microbiology and Technology.

NP 304, Component: 9, Problem Statement: b, c.

2. Occurrence of Natural Variation in MV

The efficacy of several formulations of MV isolates and sectors were comparatively evaluated versus the MV (wild-type). We showed that considerable variation in MV virulence can occur in MV in sectors occurring on agar cultures. Results were published in Biocontrol Science and Technology. These studies indicate that MV is capable of producing mutations under natural conditions. A second publication (Technology Transfer) assessed research, safety, and environmental considerations of bioherbicides, including our strain of MV being developed for kudzu control, was published in Toxin Reviews.

NP 304,Component: 9, Problem Statement: b.

3. Improving bioherbicide efficacy through synergistic interactions with glyphosate Redvine and trumpetcreeper are becoming increasingly problematic in no-till and low-till soybeans in the Mississippi Delta, and are highly tolerant to recommended glyphosate usage rates.

We have previously shown that a synergistic interaction occurs between certain glyphosate products and Colleototrichum truncatum for hemp sesbania control. We have found that MV combined with a commercial glyphosate product will effectively control identifying the ability to co-apply MV with newer herbicides provides greater ability for use of the bioherbicide. These studies indicate that bioherbicide efficacy can be enhanced through synergistic interactions with certain chemical herbicide (glyphosate) interactions, and that control of highly invasive weeds may require a combination of management practices. Results have been published in Weed Biology and Management.

NP 304, Component: 9, Problem Statement: b, c.

4. Enhancing bioherbicide performance by formulation and agronomic practices

The fungus Colletotrichum gloeosporioides, originally isolated from coffee senna, was shown to weakly virulent against sicklepod, a much more important weed species. However, we found that this fungus will effectively control sicklepod when fungal spores are formulated in either unrefined corn oil or an invert emulsion. Soybean row spacing was also shown to influence bioherbicidal efficacy of this bioherbicide. Effective control of sicklepod required two formulated biohebicide applications in wide (40 inch) rows, but a single application controlled weeds just as effectively in narrow (20 inch) rows. These studies indicate that bioherbicide formulation and crop row spacing can affect mycoherbicidal efficacy of this and possibly other bioherbicide performance. Results have been published separately in Biocontrol Science and Technology.

NP 304, Component: 9, Problem Statement: b, c.

6.Technology Transfer

Number of the New MTAs (providing only)1
Number of Web Sites Managed1
Number of Non-Peer Reviewed Presentations and Proceedings5

Review 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. 26:313-342.

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., and 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. 24(11):2721-2726.

Last Modified: 8/31/2015
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