| Rice blast is one of rice in our country the safe production of the most serious diseases, how to select the natural plant source pesticide instead of chemical pesticides to control rice blast is a hot spot in the field of pesticide research and development. The previous studies in our lab showed that the antibacterial activity of citral to rice blast fungus was good, And by electron microscopy showed that citral can destroy the cell wall of Magnaporthe grisea, but the specific role of target sites is not yet clear. Therefore, this study adopts enzyme activity assay were used to determine the activity of citral on rice blast fungus cell wall key enzyme and clear of citral on rice blast fungus cell wall effect larger key enzymes; Secondly, using PCR technique to explore the differences of citral to rice blast bacteria cell wall target gene expression, and explore the citral of rice blast fungus cell wall targets. In order to provide reference for the rational design of a new type of high efficiency and low toxicity new fungicide with novel target site. The main research results are as follows:1. The main components of Litsea cubeba oil citral has good biological activity of Magnaporthe grisea. The growth rate and spore germination method were used to determine the effects of citric on the growth and spore germination of the rice blast fungus and the length of the tube, The results show that, The inhibition rate of mycelium growth of rice blast fungus was positively correlated with the concentration of citral; The rice blast fungus spore germination rate decreased with increasing concentration of citral, germ tube length is similar. With EC50 were 40.28 and 37.12μg/mL in the growth of the mycelium and spore germination of the rice blast fungus.2. The citral can significantly improve the chitinase of Magnaporthe grisea in the cell wall of activity. Effects of citral on Magnaporthe grisea chitinase activity was determined by enzyme method, The Results show that chitinase activity decreased atfirst and rises again, and finally reduce, 24 h after tends to be stable. When the citral concentration of 100 μg/mL, in effect to rice blast fungus mycelium after 0.5 and 1 h,chitinase activity were control group of 0.80 and 1.14 times. When the treatment time was increased to 12 h and 24 h, the enzyme activity was 2.14 and 1.38 times as compared with that of the control. When the treatment time was increased to 12 h and24 h, the enzyme activity was 2.14 and 1.38 times as compared with that of the control. The concentration of 20 μg/mL reagent in role in grisea 1 h and 12 h, the activity of chitinase in mycelia as control group 1.54 and 1.76 times. When agent concentration was 20 μg/mL in effect on citral at 3 h, the activity of chitinase in mycelia reached the maximum 2.38 times of the control group.3. Citral can not improve significantly the activity of β-(1,3)-GA in Magnaporthe grisea. Effects of citral on β-(1,3)-GA in Magnaporthe grisea activity was determined by β-(1,3)-GA Reagent kit. The Results show that, Citral although can improve the rice blast fungus β-(1,3)-GA activity, but from the perspective of the determination of period of time, the rice blast fungus β-(1,3)-GA activity has been and there is no remarkable difference in the control group. The concentrations of citral is 100 μg/mL in the role in hyphal 3 h and 9 h, β-(1,3)-GA activity respectively control group 1.36 and 1.29 times. The enzyme activity was 1.40 and 1.34 times as compared with that of the control group in the treatment of hyphae 12 h and 24 h. When the concentrations of citral is 20 μg/mL in effect on hyphae at 6 h and 12 h, β-(1,3)-GA activity for control group 1.27 and 1.63 times, when the treatment time was increased to 24 h, the activity of the enzyme was 1.29 times of that of the control.4. The same concentration of citral in the role of the hyphae of Magnaporthe grisea in different time, the chitinase gene expression increased the magnitude of the overall performance is a trend of increased first and then decreased. Determination results show that, with concentrations of citral agent after induced by Magnaporthe grisea 6, 12, 24 h, chitinase gene expression were significantly increased, With the increase of time, the amplitude of the increase of gene expression was first increased and then decreased. Citral concentration of 100 μg/mL, in the rice blast fungus mycelium after 6 h and 12 h treatment, the chitinase gene expression amount group25.31 and 46.05 times. And in the treatment of 24 h, the increase amplitude of 31.03 times of the control group; When the concentrations of citral is 200 μg/mL, in Magnaporthe grisea hypha 6 h and 12 h after induction, chitinase gene expression respectively in the control group of 52.51 and 38.23 times. And in the treatment of24 h, the increase amplitude of 34.66 times of the control group.5. The different concentrations of citral in the same time induced by Magnaporthe grisea, chitinase gene expression showed first increased and then decreased with the change in the overall volume. At the same time, with the increase of the concentration of citric aldehyde(0, 25, 50, 100, 200 μg/mL), Chitinase gene expression rate gradually increased, to a certain degree, show a downward trend. In concentrations of citral is 25 and 100 μg/mL role in Magnaporthe grisea after 12 h, chitinase gene expression were divided into the control group 19.31 and 46.05 times. When the concentration of the reagent increased to 200 μg/mL, the expression amount of h was38.23 times of that of the control group; When concentrations of citral respectively 50 and 100 μg/mL after 6 h mycelium treatment, chitinase gene expression compared to the control group of 20.72 and 25.31 times. When the concentration increased to 200μg/mL in effect on the mycelium after 6 h, the expression level was 52.51 times higher than that of control group. The results showed that in the determination of concentration and time range, the high concentration of citral in a relatively short time induced by Magnaporthe grisea, chitinase gene expression was increased greatly. |
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