Title: Enzyme-Linked Immunosorbent Assay Detection of Trichothecenes Produced by the Bioherbicide Myrothecium verrucaria in Cell Cultures, Extracts, and Plant Tissues Authors
Submitted to: Communications in Soil Science and Plant Analysis
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: April 9, 2008
Publication Date: November 17, 2008
Citation: 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. Interpretive Summary: An isolate of the fungus Myrothecium verrucaria (MV) is being developed as a biological herbicide to control kudzu and other invasive weeds. Because this strain produces specific compounds (trichothecenes) that are toxic to mammals, we are evaluating numerous cultural modifications and/or formulations of this fungus, to obtain a product void of these toxins. Results using an ELISA (enzyme linked immunosorbent assay) method indicate it is a rapid, sensitive, and specific assay for detecting these compounds in MV cultures and in plant tissues treated with trichothecenes. Results also indicated that these toxins were absorbed by kudzu and hemp sesbania seedling tissues when applied to shoots or leaves, and that there was some movement of these toxins within the plant.
Technical Abstract: Commercially available enzyme linked immunosorbent assay (ELISA) plates for trichothecene detection, possessing cross-reactivity with several trichothecene mycotoxins (e.g., verrucarin A, and J, roridin A, L-2, E, and H), were tested for their ability to detect trichothecenes produced by a strain of Myrothecium verrucaria (MV) in cell cultures, and in plant tissues (hemp sesbania and kudzu) treated with purified roridin A, or ethyl acetate fractions of MV cultures. Evaluations of ELISA assays showed linear responses for standards of verrucarin A and roridin A over a concentration range of 0.2 to 20 ppb. Ethyl acetate or aqueous extractions were used to obtain samples from MV cultures and plant tissues for testing. Trichothecenes were detected in conidia and mycelia of MV, and in agar upon which wild-type MV was grown, indicating secretion into the growth media. Two sectors, isolated from the original wild-type, also tested positive for trichothecenes. Purified roridin A and concentrated extracts containing trichothecenes from MV spore cultures exhibited phytotoxicity (growth inhibition or necrosis) when applied to excised shoots of hemp sesbania seedlings and intact kudzu leaf tissues. Evidence of some translocation of trichothecenes from the application point in kudzu was found, but translocation to the upper shoot portion of hemp sesbania was not detected at the lowest limit of detection in this assay (0.14 ppb). This assay is also being employed to identify induced mutants and/or other naturally occurring sectors deficient in tricothecene mycotoxin production. Results indicated that ELISA is a sensitive and rapid assay method to quantify trichothecenes produced by this bioherbicidal fungus and in certain plant tissues treated with trichothecenes.