Enzyme-Linked Immunosorbent Assay Detection of Trichothecenes Produced by the Bioherbicide Myrothecium verrucaria in Cell Cultures, Extracts, and Plant Tissues

A rapid technique for trichothecene detection was needed in screening tests of the potential bioherbicide Myrothecium verrucaria (MV), in order to select strains, mutants, or formulations that were void of or that possessed low amounts of these undesirable mycotoxin compounds. Commercially available enzymelinked 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, 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 MV sectors (morphological variants of 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 Received 4 June 2007, Accepted 9 April 2008 Address correspondence to Robert E. Hoagland, USDA-ARS, Southern Weed Science Research Unit, P.O. Box 350, Stoneville, MS 38776, USA. E-mail: [email protected] Communications in Soil Science and Plant Analysis, 39: 3057–3073, 2008 ISSN 0010-3624 print/1532-2416 online DOI: 10.1080/00103620802432923 3057 D o w n l o a d e d B y : [ U S D A N a t l A g r i c u l t u l L i b ] A t : 1 8 : 0 6 2 9 J a n u a r y 2 0 0 9 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 trichothecene 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.