Bioassay evaluation, GC-MS Analysis and Acute Toxicity Assessment of Methanolic Extracts of Trichoderma species in an Animal Model

Fungi of the genus Trichoderma have been marketed for the control of crop pests, weeds, and diseases. However, some Trichoderma species may produce toxic secondary metabolites; the existence of these compounds in bioformulated products, along with their relative risk, should receive due attention to ensure human safety. In this study, we investigated the in vitro antagonistic potential of T. asperellum AU131 and T. longibrachiatum AU158 as a biocontrol agent against F. xylarioides and the associated antagonistic mechanism with bioactive substances. Swiss albino mice were used to evaluate the in vivo toxicity and pathogenicity of T. asperellum AU131 and T. longibrachiatum AU158 methanolic crude extracts and spore suspensions, respectively, in a preliminary safety assessment for use as biofungicides. Metabolite profiling of the crude extracts were performed by Gas Chromatography-Mass Spectrometry (GC-MS). The agar diffusion assay of the crude extracts from both T. asperellum AU131 and T. longibrachiatum AU158 were the most effective at a concentration of 200g/mL, causing 62.5%, and 74.3% inhibition, respectively. A GC-MS analysis of crude extracts from both bioagents identified 23 volatile secondary metabolites classified as alcohols, acids, sesquiterpenes, ketones and aromatic compounds. The oral administration of crude extracts (doses of 600, 1200, 2000 and 5000 mg/kg body weight) and spore suspensions (doses of 1 x 106, 1 x 107, 1 x 108 and 1 x 109 spores/mL) to female Swiss albino mice over 14 days did not show any significant signs of toxicity, mortality or changes to body weight. The highest dose, 5000 mg/kg, can be used to determine the no observed effect level of crude extracts and spore suspensions of T. asperellum AU131 and T. longibrachiatum AU158 when calculating safety margins. It can be concluded that these two Trichoderma species can serve as a biocontrol agents against F. xylarioides and the mechanism for this function was due to the secondary metabolites with effective bioactive substance. Moreover, the tested spore suspensions and crude extracts were not pathogenic or toxic, respectively, when administered to Swiss albino mice at various doses.