防治杨树叶枯病的毒蘑菇菌株筛选及其生防机理研究
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摘要
杨树叶枯病是东北地区苗圃近年来较为流行的主要林木病害之一,由链格孢菌(Alternaria alternata)引起,对该病的防治侧重于使用化学农药。残留在土壤中的化学农药是农业面源污染物的主要来源之一,而农业面源污染是造成水体污染的重要原因。由于在防治该病的过程中,大量地使用化学农药,目前已造成了严重的环境污染与经济损失,寻求高效、安全、无污染的绿色生物农药更显得尤为迫切。毒蘑菇所含有的多种活性物质可抗菌、抗虫、抗病毒等,在生物防治领域显示出了巨大的应用开发潜力,因而以毒蘑菇为来源研究开发防治杨树叶枯病的微生物农药,具有极其重要的意义。
从8个毒蘑菇菌株中,筛选到了对叶枯病菌(A. alternata)生长抑制能力最强的菌株为绒白乳菇(Lactarius vellereus),其培养液对该病原菌菌体生长的抑制率为61.44%,对其孢子萌发的抑制率为91.45%;绒白乳菇(L. vellereus)生长的最适pH为6~7,最适生长温度为25℃;能较好地利用甘露醇、琥珀酸、甘氨酸,烟酸有明显的促生长作用。
研究了绒白乳菇(L. vellereus)抑菌活性物质的提取工艺。结果表明,绒白乳菇(L. vellereus)抑菌活性物质主要存在于发酵液中,最佳提取工艺为发酵液与正丁醇以1?3萃取3h,萃取1次,在此条件下得到的发酵液提取物对叶枯病菌(A. alternata)菌体生长的抑制率为78.95%,对其孢子萌发的抑制率为91.47%。
通过对发酵液提取物室内防治效果,以及毒性的研究,得出以下结论:该提取物能够有效控制叶枯病斑的个数及病斑的扩展速度,接菌后4h施药与施药后4h接菌的防效分别达到了88.99%和87.80%;对小白鼠进行经口灌胃染毒实验,确定了发酵液提取物的半数致死量为8570.8mg/kg,为低毒,提取物对小白鼠无触杀毒性,在杨树叶片上无残留毒性。
发酵液提取物在pH5~7、30~80℃的范围内抑菌活性稳定;浓度为0.1%的H2O2与Na2SO3不影响提取物的抑菌活性;低浓度的Cu~(2+)与Al3+对提取物的抑菌活性破坏严重,高浓度的Fe3+、Fe~(2+)与Zn~(2+)对提取物的抑菌活性有破坏作用;紫外线与贮存时间对其抑菌活性无影响。
研究了发酵液提取物对叶枯病菌(A. alternata)的生物防治机理。结果表明,该提取物能够抑制菌体4种保护酶的活性,使膜脂过氧化严重;该提取物能够引起菌体ATP酶阶段性的增强,使同一阶段的HK、PK、LDH、SDH、MDH活力与辅酶Ⅰ含量下降,干扰了糖酵解与TCA循环的顺利进行,直接以菌体电导率、呼吸强度、蛋白质含量的下降及菌体与孢子形态发生非正常的变化表现出来,最终使菌体膜系统完整性丧失。
采用生物活性跟踪技术,运用柱层析、TLC等方法对发酵液提取物中的抑菌活性成分进行了分离、纯化,得到了能完全抑制叶枯病菌(A. alternata)生长的活性成分,纯度为98.805%,综合UV、IR、EI-MS、NMR的检测结果,推断此化合物的分子式是C8H9NO,分子量为135,化学名为1-(2-吡啶基)-2-丙酮,将其命名为绒白乳菇素PP。
对绒白乳菇(L. vellereus)深层发酵生产1-(2-吡啶基)-2-丙酮的培养基配方进行了优化。结果表明,最佳发酵培养基配方为(1000mL):2g葡萄糖、50g麸皮、1.25g硫酸铵、1.25g黄豆粉、71.20mg无机盐、120mg烟酸,在此条件下测得发酵液中1-(2-吡啶基)-2-丙酮的含量为12.69mg/L。
对绒白乳菇(L. vellereus)深层发酵生产1-(2-吡啶基)-2-丙酮的培养条件进行了优化。结果表明,最佳发酵培养条件为接种量12.5%、装液量300mL/500mL、pH6、摇床转速120rpm、温度26℃、发酵天数12d,在此条件下测得发酵液中1-(2-吡啶基)-2-丙酮的含量为14.91mg/L,培养条件优化后的发酵液提取物能够完全抑制叶枯病菌(A. alternata)的生长,并使发酵周期减少了3d。
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 Cu~(2+) and Al3+ had strong destruction on the extraction; while higher concentration of Fe3+, Fe~(2+) and Zn~(2+) 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.
从8个毒蘑菇菌株中,筛选到了对叶枯病菌(A. alternata)生长抑制能力最强的菌株为绒白乳菇(Lactarius vellereus),其培养液对该病原菌菌体生长的抑制率为61.44%,对其孢子萌发的抑制率为91.45%;绒白乳菇(L. vellereus)生长的最适pH为6~7,最适生长温度为25℃;能较好地利用甘露醇、琥珀酸、甘氨酸,烟酸有明显的促生长作用。
研究了绒白乳菇(L. vellereus)抑菌活性物质的提取工艺。结果表明,绒白乳菇(L. vellereus)抑菌活性物质主要存在于发酵液中,最佳提取工艺为发酵液与正丁醇以1?3萃取3h,萃取1次,在此条件下得到的发酵液提取物对叶枯病菌(A. alternata)菌体生长的抑制率为78.95%,对其孢子萌发的抑制率为91.47%。
通过对发酵液提取物室内防治效果,以及毒性的研究,得出以下结论:该提取物能够有效控制叶枯病斑的个数及病斑的扩展速度,接菌后4h施药与施药后4h接菌的防效分别达到了88.99%和87.80%;对小白鼠进行经口灌胃染毒实验,确定了发酵液提取物的半数致死量为8570.8mg/kg,为低毒,提取物对小白鼠无触杀毒性,在杨树叶片上无残留毒性。
发酵液提取物在pH5~7、30~80℃的范围内抑菌活性稳定;浓度为0.1%的H2O2与Na2SO3不影响提取物的抑菌活性;低浓度的Cu~(2+)与Al3+对提取物的抑菌活性破坏严重,高浓度的Fe3+、Fe~(2+)与Zn~(2+)对提取物的抑菌活性有破坏作用;紫外线与贮存时间对其抑菌活性无影响。
研究了发酵液提取物对叶枯病菌(A. alternata)的生物防治机理。结果表明,该提取物能够抑制菌体4种保护酶的活性,使膜脂过氧化严重;该提取物能够引起菌体ATP酶阶段性的增强,使同一阶段的HK、PK、LDH、SDH、MDH活力与辅酶Ⅰ含量下降,干扰了糖酵解与TCA循环的顺利进行,直接以菌体电导率、呼吸强度、蛋白质含量的下降及菌体与孢子形态发生非正常的变化表现出来,最终使菌体膜系统完整性丧失。
采用生物活性跟踪技术,运用柱层析、TLC等方法对发酵液提取物中的抑菌活性成分进行了分离、纯化,得到了能完全抑制叶枯病菌(A. alternata)生长的活性成分,纯度为98.805%,综合UV、IR、EI-MS、NMR的检测结果,推断此化合物的分子式是C8H9NO,分子量为135,化学名为1-(2-吡啶基)-2-丙酮,将其命名为绒白乳菇素PP。
对绒白乳菇(L. vellereus)深层发酵生产1-(2-吡啶基)-2-丙酮的培养基配方进行了优化。结果表明,最佳发酵培养基配方为(1000mL):2g葡萄糖、50g麸皮、1.25g硫酸铵、1.25g黄豆粉、71.20mg无机盐、120mg烟酸,在此条件下测得发酵液中1-(2-吡啶基)-2-丙酮的含量为12.69mg/L。
对绒白乳菇(L. vellereus)深层发酵生产1-(2-吡啶基)-2-丙酮的培养条件进行了优化。结果表明,最佳发酵培养条件为接种量12.5%、装液量300mL/500mL、pH6、摇床转速120rpm、温度26℃、发酵天数12d,在此条件下测得发酵液中1-(2-吡啶基)-2-丙酮的含量为14.91mg/L,培养条件优化后的发酵液提取物能够完全抑制叶枯病菌(A. alternata)的生长,并使发酵周期减少了3d。
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 Cu~(2+) and Al3+ had strong destruction on the extraction; while higher concentration of Fe3+, Fe~(2+) and Zn~(2+) 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.
引文
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