C. Douglas Boyette

Biological Control of Kudzu (Pueraria lobata) with an Isolate of Myrothecium verrucaria

An isolate of the fungus Myrothecium verrucaria ( MV ) was evaluated for biocontrol potential against kudzu ( Pueraria lobata ). In greenhouse tests, MV was highly virulent against kudzu in the absence of dew when conidia were formulated in 0.2% Silwet L-77 surfactant (SW). Inoculum concentrations S 2 2 10 7 conidia ml -1 were required to satisfactorily control plants in the third leaf stage and larger. In controlled environment experiments, kudzu mortality was greater at higher temperatures (25-40°C) than at lower temperatures (10-20°C), although pathogenesis and mortality occurred at all temperatures tested. In field tests, transplanted kudzu seedlings in the 2-3 leaf growth stage treated with MV at 2 2 10 7 conidia ml -1 in 0.2% SW, exhibited leaf and stem necrosis within 24 h following inoculation, with mortality occurring within 96 h. After 7 days, 100% of inoculated kudzu plants were killed in plots treated with the fungus/surfactant mixtures. Similar results were observed in a naturally occurring kudzu population, where 100% control occurred within 14 days after inoculation with 2 2 10 7 conidia ml -1 in 0.2% SW. In summary, MV effectively controlled kudzu in the absence of dew over a wide range of physical and environmental conditions and under field conditions. These results indicate that, when properly formulated, MV has potential as a valuable bioherbicide for controlling kudzu.

Glyphosate and bioherbicide interaction for controlling kudzu (Pueraria lobata), redvine (Brunnichia ovata), and trumpetcreeper (Campsis radicans)

Abstract In controlled environment experiments, the bioherbicidal fungus Myrothecium verrucaria (Alb. & Schwein.) Ditmar:Fr. was tested alone, in combination with, prior to, and following treatment with glyphosate [N-(phosphonomethyl)glycine] for control of kudzu [Pueraria lobata (Willd.) Ohwi], redvine [Brunnichia ovata (Walt.) Shinners], and trumpetcreeper [Campsis radicans (L.) Seem. ex Bureau] at temperatures of 20, 30, and 40°C. At all temperatures, kudzu was most adversely affected by the fungus, followed by trumpetcreeper and redvine, as indicated by greater mortality and dry weight reductions. Trumpetcreeper and redvine mortalities and dry weight reductions significantly increased when the fungus was applied 2 days after the glyphosate treatment. Application of the fungus combined with or prior to glyphosate treatment resulted in reduced weed control. Although pathogenesis and mortality also occurred at 20°C, disease development was favored by higher temperatures (30 and 40°C). Infected weeds of each species exhibited similar disease symptomatology within 12 h following treatment at incubation temperatures of 30 and 40°C. Disease symptomatology was characterized by necrotic flecking on leaves that coalesced into large lesions. Symptoms progressed, initially infecting cotyledons and leaves, and later (within 48 h) producing stem lesions. The fungus sporulated profusely on infected tissue and was easily reisolated. These results suggest that timing of glyphosate application in relation to combined treatment with the bioherbicide M. verrucaria can improve the control of kudzu, redvine, and trumpetcreeper.

BIOHERBICIDES: RESEARCH AND RISKS

Many microbes have bioherbicidal activity, and several phytopathogenic fungi and bacteria have been patented as weed-control agents. The phytotoxic components of most agents have not been elucidated, but some phytotoxins and other secondary compounds produced by such microbes may be toxic to mammalian systems. Furthermore, few rigorous assessments have addressed uptake, translocation, metabolism, and persistence of these phytotoxins (some of which have not been identified), or the environmental effects of repeated augmentative applications of these microorganisms on long-term impact, environmental fate, or interactions with other microbial communities. Generally, there is a lack of definitive research on the overall toxicological risk of bioherbicidal microorganisms to the degree achieved or required for synthetic herbicides. This article presents a brief overview of bioherbicides (microorganisms and/or their phytotoxins), with emphasis on the toxicity of certain bioherbicides, and general considerations of risks associated with bioherbicidal use. A subsequent article presents some of our research results and future research directions on efforts to develop the bioherbicidal fungus Myrothecium verrucaria as a safe and efficacious bioherbicide have been published elsewhere.

Evaluation of the bioherbicide Myrothecium verrucaria for weed control in tomato (Lycopersicon esculentum)

Abstract An isolate of the fungus Myrothecium verrucaria was evaluated for its biocontrol potential against common purslane, horse purslane, spotted spurge, and prostrate spurge, all serious weed pests in commercial tomato fields in the southeastern US. In greenhouse and field tests, M. verrucaria was highly virulent against these weeds when applied as conidial sprays formulated in 0.2% Silwet L-77 surfactant, even in the absence of dew. In field test plots naturally infested with these weeds, seedlings in the two-to-three leaf growth stage treated with M. verrucaria at 2×107 conidia mL−1 in 0.2% Silwet, exhibited leaf and stem necrosis within 24 h following inoculation, with mortality occurring within 96 h. After 7 days, M. verrucaria had killed 90–95% of both purslane species and 85–95% of both spurge species. Tomatoes that were transplanted into plots treated with M. verrucaria remained healthy and vigorous throughout the growing season. Since M. verrucaria effectively controlled several common weeds under field conditions, this fungus appears to have potential as an effective bioherbicide for pre-plant weed control in production systems with transplanted tomato.

Myrothecium verrucaria isolates and formulations as bioherbicide agents for kudzu

Abstract The fungus Myrothecium verrucaria (MV) has previously been shown to have potential as a bioherbicide for kudzu (Pueraria lobata) control. It has also been shown that MV wild-type (MV-wt) often forms sectors, when grown on various nutrient media. Experiments compared MV-wt and MV sector efficacy when grown on agar or on rice grains. In greenhouse evaluations of sectors, applied as foliar sprays in water or in other formulations (corn oil, surfactant, and corn oil plus surfactant) for efficacy against kudzu seedlings, some sectors possessed bioherbicidal activity equal that of MV-wt, but others exhibited lower activity. Without a dew period, aqueous formulations of MV-wt, a yellow sector, and a white sector provided zero control, but all three isolates were active without a dew period when formulated in corn oil, Silwet L-77 surfactant, and in surfactant plus corn oil. Generally, the yellow sector was less effective than the other two isolates in any formulations, and the MV-wt and white sector provided approximately 100% mortality of the test plants. Dew (10 h) increased weed control to 100, 33, and 65%, respectively, for MV-wt, the yellow sector and the white sector. All isolates provided nearly 100% control in the oil and surfactant formulations with a dew period compared to treatments receiving no dew. Soil incorporation studies were also performed to compare MV-wt efficacy of preparations grown on agar versus growth on rice grains. Higher efficacies (1.75–3.3-fold increase) were obtained from rice grain preparations compared to preparations grown on agar, when preparations were incorporated at several rates into soil prior to planting. Cell-free extracts of the MV-rice cultures were also phytotoxic to kudzu seedlings up to the eight- to 10-leaf growth stage. Thus, formulation, growth media, and the application method are important determinants in the efficacy of MV and MV sectors on kudzu seedlings.

Hemp sesbania (Sesbania exaltata) control in rice (Oryza sativa) with the bioherbicidal fungus Colletotrichum gloeosporioides f. sp. aeschynomene formulated in an invert emulsion

Abstract In greenhouse and field experiments, an invert emulsion (MSG 8.25) was tested with dried, formulated spores of the bioherbicidal fungus Colletotrichum gloeosporioides f. sp. aeschynomene, a highly virulent pathogen of the leguminous weed Aeschynomene virginica (northern jointvetch), but considered ‘immune’ against another more serious leguminous weed, Sesbania exaltata (hemp sesbania). A 1:1 (v/v) fungus/invert emulsion mixture resulted in 100% infection and mortality of inoculated hemp sesbania seedlings over a 21-day period under greenhouse conditions. In replicated field tests of the fungus/invert formulation conducted in Stuttgart, AR, and Stoneville, MS, hemp sesbania was controlled 85 and 90%, respectively. These results suggest that this invert emulsion expands the host range of C. gloeosporioides f. sp. aeschynomene, with a concomitant improvement of the bioherbicidal potential of this pathogen.

Adjuvants enhance the biological control potential of an isolate of Colletotrichum gloeosporioides for biological control of sicklepod (Senna obtusifolia)

Abstract In greenhouse experiments, unrefined corn oil, Silwet L-77, and an invert emulsion were tested as adjuvants with the mycoherbicidal fungus Colletotrichum gloeosporioides, a weakly virulent pathogen of sicklepod (Senna obtusifolia). A 1:1 (v/v) fungus/corn oil tank mixture containing 0.2% (v/v) Silwet L-77 surfactant reduced the dew period requirements for maximum weed infection and mortality from 16 to 8 h, and delayed the need for free moisture for greater than 48 h. This formulation also resulted in the ability of the pathogen to infect and kill weeds in larger (>5 leaf) growth stages. The invert emulsion resulted in similar effects upon these parameters. These results suggest that invert emulsions, unrefined corn oil and Silwet L-77 surfactant greatly improve the bioherbicidal potential of this pathogen for control of sicklepod, a serious weed pest in the southeastern US.

Effect of row spacing on biological control of sicklepod (Senna obtusifolia) with Colletotrichum gloeosporioides

Abstract In field experiments conducted over 3 years, the mycoherbicidal fungus Colletotrichum gloeosporioides, formulated either in 20% (v/v) unrefined corn oil and 0.2% Silwet L-77 surfactant or with an invert emulsion, provided season-long control of Senna obtusifolia in narrow (51 cm) rows of soybean. However, in wide (102 cm) rows, one application of either formulation failed to provide season-long control of S. obtusifolia, and two applications were required to achieve season-long weed control. In narrow (51 cm) rows, one application of the fungus either in unrefined corn oil or an invert emulsion controlled S. obtusifolia an average of >90%, and a second application was not required for season long weed control. Soybean yields in wide-row plots treated with two applications of either the fungus/corn oil or fungus/invert emulsion, or with a single application of the fungal treatments in narrow-row soybean plots, were not significantly different from weed-free control plots, or from plots treated with the herbicide chlorimuron. These results suggest that row spacing can affect mycoherbicidal efficacy of this fungus for controlling S. obtusifolia.

Immunohistochemical investigation of the necrotrophic phase of the fungus Colletotrichum gloeosporioides in the biocontrol of hemp sesbania (Sesbania exaltata; Papilionaceae)

UNLABELLED PREMISE OF THE STUDY Fungal plant pathogens exert much of their effect on plant cells through alterations in the host cell walls. However, obtaining biochemical proof for this change is difficult because of the relatively small number of cells that are affected by the pathogen relative to the bulk of host tissue. In this study, we examined the differences in host wall composition between infected and uninfected areas of seedlings of the weed hemp sesbania (Sesbania exaltata) that were treated with the biocontrol agent Colletotrichum gloeosporioides. • METHODS To determine the changes in cell wall composition, we used semi-thin sections and a battery of antibody probes that recognize components of the cell wall and immunogold-silver cytochemistry to visualize the probes. • KEY RESULTS A loss of specific plant cell wall polysaccharides in the region surrounding the primary fungal infection and the creation of a defensive layer by the plant to limit the fungal invasion were the two most obvious changes noted in this study. At the invasion site, there was significant loss of rhamnogalacturon-1 (RGI) and esterified and de-esterified homogalacturonan (HG)-reactive epitopes from the cell walls. In contrast, boundary tissue between the vascular tissue and the fungal lesion reacted more strongly with antibodies that recognize arabinogalactan proteins (AGPs) and xyloglucans than in unaffected areas. • CONCLUSIONS These data strongly indicate a role of pectinases in the invasion of the biocontrol agent and the importance of extensins, AGPs, and xyloglucans as defense by the host.

Bioherbicidal activity from washed spores of Myrothecium verrucaria

The fungal plant pathogen, Myrothecium verrucaria, is highly virulent to several important weed species and has potential utility as a bioherbicide. However the production of macrocyclic trichothecene mycotoxins by this fungus presents significant safety concerns. It was discovered that trichothecenes are removed from M. verrucaria spores by repeated washes with water. These washed spores retained bioherbicidal efficacy against kudzu when tested in field trials and on sicklepod when tested under greenhouse conditions. Changes in the growth medium combined with washing spores with water resulted in greater than 95% reduction in roridin A and verrucarin A. Washing spores reduced trichothecene concentrations in spore preparations with no significant effect on plant biomass reduction, thus demonstrating the possibility of M. verrucaria formulations with improved safety to researchers, producers and applicators.