ISOLATION, PURIFICATION AND INACTIVATION MECHANISM OF CASSIICOLIN PRODUCED BY CORYNESPORA CASSIICOLA

Abstract

The Corynespora leaf fall disease (CLFD) caused by Corynespora cassiicola (Ber.&Curt.) is the second important disease after South American Leaf Blight (SALB). The toxin produced by the pathogens is the important pathogenic factor. In order to obtain the cassiicolin of the pathogenic strains in domestic rubber tree, the production conditions of the toxin of HC-10 were optimized and the crude toxin was then concentrated and purified. In addition, detoxification mechanism of the antagonistic endophytic bacteria, inorganic salts and fungicides against the toxin were performed. Then the production conditions and the stability of the toxin of HC-10 were determined. The conditions of improved Fries 3 medium, 25°C, lighted 12 h, cultured 10 d, 160 rpm speed and 4 of initial pH of medium were most conducive to the formation of toxin; a certain relevance among temperature, UV and different concentrations of the toxin with the activity of toxin existed. The crude toxin was concentrated and purified by using of dialysis, freeze-dried, ammonium sulfate precipitation, as well as technologies such as ion-exchange chromatography and polyacrylamide gel electrophoresis. The molecular weight of the toxin protein was about 14.4 KDa, and 10 inorganic salts were used to inactivate the crude toxin produced by Corynespora cassiicola. The results showed that the growth of mycelial and the germination of spores were inhibited strongly by CuSO₄•5H₂O, FeCl₃ and FeSO₄•6H₂O at a concentration of 2~5 mg/mL. KMnO₄ could inactivate the toxin well at a concentration of 0.5 mg / mL with a wilting rate of 2.04%; FeCl₃ was the second with the wilting rate of 3.97%. Five fungicides were used to inactivate the toxin, and the results showed that carbendazim is the most effective fungicide with EC₅₀ =0.2243 μg/mL, followed by prochloraz amine with EC₅₀ 0.0.4797 μg/mL.

Keywords: Corynespora cassiicola, Hevea brasilensis, cassiicolin, inactivation, toxin, fungicide, rubber, carbendazim, prochloraz amine