Extraction And Toxigenic Characteristic Of Citrus Brown Spot Toxin And Its Effect On Defensive Enzymes Of Citrus

Citrus brown spot is a fungus disease caused by Alternaria alternata. The site infected by the pathogen produce brown necrotic spots, so it is called brown spot disease. The pathogen mainly damage leaves, branches and fruit of citrus, may lead to leaves and fruit drop and even cause the plant die.These have a strong impact on the quality and yield of citrus. Now agricultural and chemical methods are the main measures to control it.In 2010, the disease was first offocially reported in China. In 2011, the disease was occurred seriously in Wanzhou, Chongqing and the pathogen was identified as A. alternata. There have been a lot of researches on biological characteristic and genetic of the pathogen, but rare research on the toxin produced by the pathogen. A large number of studies have shown that toxin produced by pathogen is the main pathogenic factor, and toxins can be used as a selection agent for screening of resistant varieties. So it is necessary to figure out the extraction methods, cultural conditions and pathogenesis of the toxin. In this paper, extraction, bioassay of toxin produced by the pathogen, toxigenic characteristic and its effect on defensive enzymes of diffent varieties of citrus were researched systematicly. The primary results were as follows:(1)In order to get crude toxin, five kinds of extractants, including acetone, methyl alcohol, ethyl acetate, trichloromethane and carbon tetrachloride were used to extract culture filtrate. After extracting, the bioassay of water phase and oil phase was analyzed respectively. The result shows that the toxins were soluble in polar organic solvents easily, among them methyl alcohol’s extraction showed the best solubility. It was identified that the toxin was a small molecule material and with a big polarity,5.1-5.4.(2)By using needle puncting, the pathogenicity and toxin-producing capability of 28 isolates had compared, and toxin-producing of SP-36 and CJBHJF-2 were the hightest. SP-36 and CJBHJF-2 strains were selected for toxigenic characteristic. The results showed that the two strains could not produce toxin in Cazpek’s medium, CJBHJF-2 strain could not produce toxin in PSK medium, and the most suitble medium wes improved Richard’s medium. The two strains could produce toxin in the range of 10℃～35℃ and the suitable temperature for their toxin-producing was 28℃. these two strains could not produce toxin at pH 2, but they could produce toxin in the range of pH 3～7. The most suitable pH value for SP-36 and CJBHJF-2 toxin-producing was pH 4 and pH 5, respectively. Toxin-producing of the two strains increased and then decreased in the period of 5-30 d. Their toxin-producing were the highest at 10 d. Under the condition of continuous illumination or oscillation conditions, their toxin-producing were relatively higher. The maximum toxin production was obtained at 28℃, pH 4～5, under oscillation, continuous illumination condition, and the optimum culture time was 10 days, the suitable medium was Improved Richard’s.(3)In the period of 3～72 h, the activities of CAT, POD, SOD, PPO, PAL varied with citrus varieties after treated with toxin. The changes of activities of those defensive enzymes appeared earlier and faster in the resistant varieties than those in susceptible ones. Activity of CAT increased slowly and then decreased slowly at firstly, and incresed and then decresed rapidly later on, with the first peak of CAT activity was at 24 h. But susceptible and modarate resistance varieties had no big changes along the treatment. The peak of POD activity in resistant varieties appeared at 6 h. And the peak of susceptible and modarate resistance varieties appeared at 24 h and 12 h, respectively. The trends of SOD activity were the same as CAT, with the first peak of SOD activity in resistent varieties at 3 h, and the peak of susceptible and modarate resistance varieties at 24 h. PPO activity of resistance and modarate resistance varieties was higher than that of susceptible varieties. PPO activity was fast increased in the period of 6-12 h. The results showed that PPO had effect on defensive reaction in the early time. PAL activity of different varieties of citrus increased and then decreased. The peak of PAL activity of resistance variety was at 6 h, the peak of susceptible and medarate resistance varieties appeared at 12 h. The results showed that defense enzymes of resistant varieties were active at different times in the course of disease or in the same period or even at the same time, to protect against toxin.