Phytotoxicity and mammalian cytotoxicity of macrocyclic trichothecene mycotoxins from Myrothecium verrucaria.

Macrocyclic trichothecene toxins produced by Myrothecium verrucaria (a phytopathogen of interest in biological weed control) and the non-trichothecene toxin atranone B from Stachybotiys atra were tested for phytotoxicity in duckweed (Lemna pausicostata L.) plantlet cultures and kudzu (Pueraria lobata L.) leaf disc assays, and for mammalian cytotoxicity in four cultured cell lines. Roridin E and H, epi-isororidin E, and verrucarin A and J were phytotoxic (half-maximal effect in the concentration range 0.1-9.7 microM on duckweed and 1.5->80 microM on kudzu) and cytotoxic to mammalian cell lines (half-maximal inhibition of proliferation in the concentration range 1-35 nM). Trichoverrins A and B and atranone B were moderately phytotoxic (half-maximal effect in the concentration range 1 9-69 microM on duckweed and 13->80 microM on kudzu) and weakly cytotoxic with mammalian cell lines (half-maximal inhibition of proliferation in the concentration range 0.3->2 microM).

Macrocyclic trichothecenes are undetectable in kudzu (Pueraria montana) plants treated with a high-producing isolate of Myrothecium verrucaria.

Myrothecium verrucaria was found to be an effective pathogen against kudzu grown in the greenhouse and the field. M. verrucaria produced large amounts of macrocyclic trichothecenes when cultured on solid rice medium, including epiroridin E (16.8 mg/g crude extract), epiisororidin E (1 mg/g), roridin E (8.7 mg/g), roridin H (31.3 mg/g), trichoverrin A (0.6 mg/g), trichoverrin B (0.1 mg/g), verrucarin A (37.4 mg/g), and verrucarin J (2.2 mg/g). Most of these toxins were also isolated from M. verrucaria spores and mycelia grown on potato dextrose agar medium, including epiroridin E (32.3 mg/g), epiisororidin E (28.6 mg/g), roridin E (0 mg/g), roridin H (60 mg/g), trichoverrin A (1.3 mg/g), trichoverrin B (1.8 mg/g), verrucarin A (13.8 mg/g), and verrucarin J (131 mg/g). When M. verrucaria was cultured on liquid media, the numbers but not the amounts of toxins decreased. Only epiroridin E (28.3 mg/g), epiisororidin E (29.6 mg/g), verrucarin B (195 mg/g) and verrucarin J (52.6 mg/g) were measured when the fungus was cultured on cornsteep medium. On soyflour-cornmeal broth M. verrucaria produced several toxins, including epiroridin E (58.1 mg/g), epiisororidin E (5.8 mg/g), verrucarin B (29.9 mg/g) and verrucarin J (32 mg/g). In contrast, no macrocyclic trichothecenes were detected by HPLC analysis of plant tissues of kudzu, sicklepod, and soybean treated with aqueous suspensions of M. verrucaria spores formulated with a surfactant. Chloroform-methanol extracts of kudzu leaves and stems treated with M. verrucaria spores were less cytotoxic to four cultured mammalian cell lines than the corresponding extracts from control plants. Purified macrocyclic trichothecenes (verrucarin A and T-2 toxin) were very cytotoxic to the same cell lines (< or = 2 ng/ml). These results show that neither intact macrocyclic trichothecenes nor toxic metabolites could be detected in plant tissues after treatment with M. verrucaria spores. These results argue for both safety and efficacy for the use of M. verrucaria in biological control of kudzu and other noxious weeds, and support proceeding to animal feeding trials for further evaluation of safety.