Studies On Biocontrol Mechanisms Of The Mycoparasite Agent Trichothecium Roseum

Trichothecium roseum was isolated from loose smut fungi (Ustilago tritici(Pers.)Rostr.) during wheat loose smut pathogen was isolated from infected wheat head. T. roseum could grow well on PDA medium from which nutrition had been used up by the inoculated Ustilago tritici. This primarily indicated that T. roseum was a mycoparasitical fungus. Light microscope and electronic scanning microscope were carried out towards mycoparasitical interections between T. roseum and several selected fungal pathogens, the results indicated that can either coil around hyphae of host fungi, or penetrate hyphal cells of pathogens by forming hooks, haustoria and appressoria - like structures which invariably lead to hyphal cell cytoplasm concentration and disruption. In addition, the culture filtrate of zymolytic medium via T. roseum can strongly inhibit the growth of Verticillium dahliae.From the culture filtrate of the Trichothecium roseum, An extra-cellular chitinase was purified to SDS-PAGE homogenous by ammonium sulfate fraction, DEAE-Sepharose FF chromatography and CM-Sepharose FF chromatography The molecular weight of the chitinase was about 39 kDa. The optimum temperature was 40℃. The optimum activity was at pH 4.0-7.0. Besides, antibody was produced using the purified chitinase.The antifungal mechanism of mycoparasitic fungi involves fungal cell wall degrading enzymes such as chitinases. Trichothe~ium roseum is an important mycoparasite fungus with significant antifungal ability, but studies on chitinase genes of T. roseum were poor. Here, we report a novel chitinase cDNA isolated from T. roseum by PCR amplification based on conserved chitinase sequences. Southern blot analysis suggested that a single copy of the gene exists in the genome of T. roseum. The deduced open reading frame of 1,143 nucleotides encodes a protein of 380 amino acids with a calculated molecular weight of 41.6 kDa. The fusion chitinase expressed in Escherichia coli has been purified by single-step chromatography. It has a pI of pH 5.4, and expresses a thermal stability, but is insensitive to pH in a broad pH range. According to expectation, E. coli efficiently yielded a high amount of active chitinase. Remarkably, the fusion chitinase offered high antifungal activity.Trichothecenes are a group of biologically active mycotoxins produced by several fungi and have a cytotoxinc effect on eucaryotic by resisting protein synthesis. Here, the authors reported that a mycotoxin was extracted and purified from a mycoparasite fungi, Trichothecium roseum isolated from natural cotton field, and showed obviously production amount difference when T. roseum was cultured respectively in the two kinds of relatively identical culturing conditions with only adding chitin or not. The results were that 0.11 mg and 0.04mg TCN mycotoxins were purified by filtering through filter paper, Kieselgel 60 Silica gel chromatography, reverse-HPLC and silica gel thin layer chromatography. It was obviously that chitin lead to the production's difference. Antifungal assay by TLC-BIOASSAY showed that the toxin could resist pathogens growth and development.Based on these results, we can conclude that both chitinase and TCN toxin confer the antifungal activity.