The Chemical Characteristics And Pathogenic Mechanism Of Cryphonectria Parasitica Toxin

On the base of production conditions of the toxin from Cryphonectria parasitica, the pathogenic activity and its host range of the toxin was determined by bioassay methods. The resistance of different chestnut varieties was identified by using the toxin. The toxin was purified by silica gel column chromatography and HPLC, and was identified by the analysis of mass spectrography (MS), nuclear magnetic resonance (NMR) and infrared spectrum (IR). The pathogenesis of C. parasitica toxin was studied by physiological and biochemical determination, ultrastructural observation at plant and cell level. And Cp-â…¡toxin was taken as inducible factor to induce resistant callus. The results were as follows.1. C. parasitica could produce phytotoxin in fluid media. The conditions of toxin production of C. parasitica were optimized. Results showed the liquid medium had the strongest toxin-producing capacity among 7 media. The optimal cultural conditions for producing toxin were cultured period at temperature 26℃, 18 d, and pH 6. Peptone and dextrin were the best nitrogenous source and carbonic sources respectively. The twigs of Chinese chestnut were selected to test the toxicity of crude toxin, which showed the cultured crude toxin had definite toxicity. C. parasitica toxin was a non-host-specific toxin. It had toxic activity to many plants from different families (33 species of 22 families). It could cause leaf blight and withered.2. The strain of C. parasitica was cultivated by liquid fermentation. The crude material with wilt activity to chestnut browse was obtained by extracting antagonistic components with petroleum ether from the fermentation broth. Three compounds were obtained by silica gel column chromatography by using V(chloroform ):V(petroleum ether ):V(alcohol)=6:2:2 as eluent. The results showed the first compound (Cp-â… ) had higher wilt activity than others. The chemical formula of the pathogenic fraction was identified by MS, NMR and IR. Cp-â… was C₁₆H₂₂O₄ (M=278), Cp-â…¡was C₉H₁₉ON (M=157), Cp-â…¢was C₇H₁₄ON (M=128). The structure of Cp-â… ,Cp-â…¡,Cp-â…¢were as follows: 3. The method of excised leaf test was used to study the effect of Cp-â…¡toxin from C. parasitica on Castanea mollissima Blume. including four varieties with different resistance, specially on its activities of the enzymes superoxide dismutase (SOD), peroxidase (POD), ascorbate peroxidase (APX), catalase (CAT), polyphenol oxidase (PPO), phenylalanine pmmonia lyase (PAL). The results suggested: the activities of the protective enzymes, including SOD, POD, CAT, APX, PPO and PAL, increased notably and the change extent of the enzymes activities of the resistant variety was the largest after treated with the Cp-â…¡toxin concentration from 25μg/mL to 50μg/mL. With increasing concentration of the Cp-â…¡toxin, the activate oxygen metabolism system was destroyed, the balance of the protective enzyme system was broken. The enzymes activities were observed to decrease in all varieties. Finally, the antioxidant enzyme system was damaged, and the excessive accumulation of the active oxygen free radicals was enhanced when the treatment concentration of Cp-â…¡toxin was 200 ug/mL. The APX, PPO and PAL activities decreased with its accumulation, and eventually caused plant damage. The regularity change of physiological and biochemical of host caused by Cp-â…¡toxin could be used a marker to assess the resistant.4. To study the ultra-structure of leaf and stem tissue of resistant and susceptible Chinese chestnut, the toxin of C. parasitica with wilt activity to Chinese chestnut browse was obtained by isolation and purification from its fermentation broth. The Results indicated that the damage on leaf tissue included the transformation of cell wall, the plasmolysis, the swollen and disruption of chloroplast, mitochondria and nuclei the transparency of in ternalmatrix, the disordering of chloroplast lamellae, and the expanding and the disappearing of mitochondria ridges. The lamellae and membrane of chloroplasts are more sensitive to the toxin and change earlier. The damages on membrane systems of the cells are lighter and occur later in the R-type than the S-type one. The stem was treated with Cp-â…¡toxin, the results showed that plasmolysis and chloroplast deformation only took place, the ultra-structure of Hongguang stem tissue was badly damaged, including the deformed and broken cell wall, the broken plasmalemma, the misshaped chloroplast, decomposed chloroplast lamellae and broken envolopen of chloroplast. Beiyu 2.5. The callus of two chestnut cultivars, Beiyu 2 and Hongguang, which were susceptive and resistant to C. parasitica respectively, were compared at ultra-structural level after callus were treated with Cp-â…¡toxin. The callus were used to determine the activities of chitinase andβ-1,3-glucanase. The results indicated that plasma membrane was more sensitive to Cp-â…¡toxin than other organs. The changes of Hongguang exhibited earlier than Beiyu 2. The activities of chitinase andβ-1,3-glucanase in the callus cells treated with the Cp-â…¡toxin were increased to the higher level at earlier time point in resistant cultivars than these in the susceptible cultivars. Chitinase andβ-1,3-glucanase activities of the resistant varieties and the susceptible ones were higher than the control, suggested that treated with Cp-â…¡toxin could induce resistance to C. parasitica through activating chitinase andβ-1,3-glucanase. The resistant callus was obtained by taking Cp-â…¡toxin (50μg/mL) as inducible factor, and susceptible variety was induced easier by analysis of resistance.