Study on the Screening of Biocontrol Toxic Mushroom Strains Inhibiting Poplar Leaf Blight and Its Acting Mechanism
|School||Harbin Institute of Technology|
|Keywords||Lactarius vellereus Alternaria alternata bioactive component purification and separation biocontrol|
Poplar leaf blight caused by Alternaria alternata is one of the important diseases in northeast China which is more epidemic in nursery at present. The prevention and treatment to the blight focused on the use of chemical pesticides. The reagent chemicals residual in the soil is one of the major sources that caused agricultural non-point pollution, while agricultural non-point pollution is the important reason for water pollution. Since a lot of reagent chemicals were used in the course of prevention and cure, it has already caused the serious environmental pollution and economic loss. It looks urgently necessary to explore biopesticide which is highly effective, safe and without pollution of environment. Toxic mushroom strains have showed great application and development value in the field of biocontrol, that some kinds of bioactive components from toxic mushroom strains had the ability of anti-microbe, anti-insect, anti-virus etc. So it is most important to take toxic mushroom strains as materials to carry the study on exploring biopesticide in the control of poplar leaf blight.Lactarius vellereus with the best inhibiting effect either on the growth or sprouting of A. alternata was isolated from 8 toxic mushroom strains. The suppressing rate of 61.44% to the growth of A. alternata mycelia and the inhibiting rate of 91.45% to the germination of A. alternata spore were presented by the strains’culture liquor. The results indicated that the optimal pH value was 6 to 7, the optimal temperature was 25℃. L. vellereus strain could take good use of mannitol, succinic acid and glycine etc. Effect of nicotinic acid on the enhancement of L. vellereus growth was obvious.The extracting procedure of the anti-A. alternata bioactive components from L. vellereus was studied. The results showed that the anti-A. alternata bioactive components mainly existed in fermenting liquor; the optimum extracting conditions were showed as follows: the ratio of fermenting liquor to n-Butanol 1:3, the extracting time 3h, the extracting times once. The suppressing rate of 78.95% to the growth of A. alternata mycelia and the inhibiting rate of 91.47% to the germination of A. alternata spore were presented by the fermenting liquor extraction, which was received under the upper optimum extracting conditions.Study on the indoor control effect and toxicity of the fermenting liquor extraction was carried out. The results showed that the number and epidemic speed of disease spots was controlled effectively by the extraction; the biocontrol efficacy of application 4h later after inoculation and inoculation 4h later after application was determined, the former was 88.99% and the latter was 87.80%; the LD50 of the fermenting liquor extraction in the oral acute toxic examination was 8570.8mg/kg, which was less toxic, the extraction had no contact toxicity to mice and no residue on poplar leaves.The anti-A. alternata activities of the extraction were stable in the pH value of 5 to 7 and the temperature range of 30℃to 80℃; the inhibition ability of extraction was not affected with H2O2 and Na2SO3 with the concentration of 0.1%. Lower concentration of Cu2+ and Al3+ had strong destruction on the extraction; while higher concentration of Fe3+, Fe2+ and Zn2+ had destructive effect, the inhibition ability of the extraction was not affected with ultraviolet radiation and storage-time.The biocontrol mechanism of fermenting liquor extraction to A. alternata was studied. The results showed that the extraction could inhibit the activities of 4 protective enzyme, and could make membrane lipid being peroxidized seriously; the extraction could induce ATP enzyme to be increased periodically, the period was that the activities of HK, PK, LDH, SDH, MDH and the contents of coenzyme I were led to decrease by the extraction; the glycolysis and TCA cycle was disturbed, this was showed as follows: the conductivity ratio, respiration intensity and protein content trended towards decreasing ultimately, the mycelium and morphology was changed irregularly; Ultimately, the integrality of membrane system was destroyed thoroughly.By means of the techniques of tracking bioactivity, silica gel column chromatogram and TLC, a bioactive substance which could thoroughly inhibit the growth of A. alternata was separated out of L. vellereus fermenting liquor; its purity was 98.805%; its structure was analysed by UV, IR, EI-MS, 1H-NMR and 13C-NMR, its molecular formula is C8H9NO, its molecular weight is 135, its chemical name is 1-（2-pyridinyl）-2-propanone, it is named Lactarin PP.The culture medium of submerged fermentative production of 1-（2- pyridinyl）-2-propanone by L. vellereus were studied. The results showed that optimal medium（1000mL） was composed of glucose 2g, bran 50g, （NH4）2SO4 1.25g, soybean powder 1.25g, inorganic salt 71.20mg and niacin 120mg; under this conditions, the content of 1-（2-pyridinyl）-2-propanone in fermenting liquor was 12.69mg/L.The fermenting conditions of submerged fermentative production of 1-（2-pyridinyl）-2-propanone by L. vellereus were studied. The optimal culture conditions involved: the shaking-flask experiments were done in 500mL, flasks containing 300mL medium with 12.5% inoculation quantity, the pH value was 6, the orbital shaking was at 120rpm, the temperature was 26℃, the period was 12d; under this conditions, the content of 1-（2-pyridinyl）-2-propanone in fermenting liquor was 14.91mg/L. After the culture conditions were optimized, the extraction from fermenting liquor could thoroughly inhibit the growth of A. alternata, and 3 days of the period were shortened.