橡胶树白粉病生防菌研究
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摘要
从橡胶树根际土壤分离出菌株276株,并从中筛选出对橡胶树白粉病具有较好拮抗作用的菌株2株,标记为S43、S55。对两株拮抗菌进行形态观察、培养性状、生理生化性状分析及16S rDNA序列分析,确定了2株拮抗菌的分类地位,均为枯草芽孢杆菌。
菌株S43、S55的不同浓度发酵液对白粉病菌孢子抑制试验和盆栽试验的结果表明：随着发酵液浓度的增加,孢子萌发抑制率和防效均提高。当两者发酵液浓度为20%时即可很好的抑制白粉病孢子萌发,萌发抑制率均达80%,盆栽试验的防效也达到80%。
将拮抗菌株的发酵液和菌悬液通过三种处理方法(接种白粉菌前24h喷施、接种白粉菌时喷施和接种白粉菌后48h喷施)喷施于橡胶树叶片。通过接种S43、S55培养液,橡胶树叶片内的PPO、POD活性比对照大一倍,由此说明菌株S43、S55可诱导植物体产生防御酶。同时,分别测得拮抗菌对白粉病的防效。三种处理方法菌株S43发酵液防效分别为84.62%、76.53%、43.32%,而菌悬液防效较差,其防效分别为57.32%、56.56%、21.02%；三种处理方法菌株S55发酵液防效分别为82.35%、80.57%和57.94%,而菌悬液防效也较差,防效分别为57.76%、57.74%、32.29%。结果表明：(1)接种前24h喷施发酵液与接种时同时喷施发酵液和菌悬液的防效显著高于接种后48h喷施发酵液和菌悬液；(2)喷施发酵液比菌悬液效果好；(3)菌株S43和S55的发酵液对白粉病都具有很好的防效,但S43发酵液比S55发酵液的防效好。
通过采用伤叶和不伤叶接种方式研究菌株S43和S55在橡胶叶片上的定殖规律。研究表明,编号菌株S43*和S55*在叶片上接种一天后定殖量最大,但随着时间的推移其定殖量逐渐减少,并在15d后基本趋于稳定。在接种初期,伤叶处理的菌株定殖量比不伤叶处理的菌株定殖量大一倍,但两种方式处理后的菌株定殖量最终无显著差异。
对拮抗菌株S43发酵条件进行初步优化,优化培养基为：蔗糖1.5%、蛋白胨0.66%,酵母浸膏0.33%；发酵条件为：pH为7.0、摇床转速150r/min、温度30℃,种子培养液接种量2%、培养基与空气的比为2：5、最佳接种种龄为14-30h；最佳发酵时间36-72h。
276bacterial strains were separated from rhizosphere soil of rubber tree plantation. And two of them (S43and S55) have obvious antagonism against rubber tree Powdery Mildew. Based on the analysis of morphologic observation, cultural characteristics, physiological and biochemical properties, and the sequential analysis of16S rDNA, S43strain and S55were identified as Bacillus subtilis.
By means of Oidium heveae Steinm's spore test and potted seedlings experiment, the results showed that the inhibition rate to the spore germination and control effect were improved with the increasing of the concentration of fermented filtrates.20%of fermentation filtrates of strain S43and S55had effective inhibition activity against powdery mildew spores germination, with the inhibition rate of80%. Meanwhile, the control effect of the pot experiment also reached80%. This showed that the fermentation filtrates of strain S43and S55had good prevention effect to powdery mildew.
Spraying fermentation filtrates or bacterial suspension24h beforen48h after or while with powdery mildew inoculation. PPO, POD activity in the Rubber tree leaves inoculated with strain S43and S55had nearly doubled compared with the control, indicating that the strain S43and S55can induce the production of defence enzyme in plant. The control effect of S43fermented filtrates with three kinds of inoculation treatments were84.62%,76.53%and43.32%respectively as well as the inhibition rates were57.32%,56.56%and21.02%respectively with bacteria suspension of which the control effect was poorer. With the same methods of S55, the inhibition rates were82.35%、80.57%and57.94%respectively as well as the inhibition rates were57.76%、57.74%and32.29%respectively with bacteria suspension of which the control effect was poorer. The results were as follows:1. spraying24h before or while inoculation, the inhibition rate had obviously higher than that of48h after inoculation;2. fermentation filtrates had better control effect than that of bacterial suspension;3. compared with S55, fermentation filtrates of S43had better control effect to powdery mildew.
Colonization of marked strain S43and S55was researched in Rubber tree by inoculation with injuring leaves and no injuring leaves. The results showed that the marked strain S43*and S55*could set the largest colonization in the rubber leaves on the first day after inoculation. While as time went on, the population of setting reduced gradually and the population of the marked strain was almost stable after15days. The population of marked strain by treatment with injuring leaves was twice than that of with no injuring leaves at the early inoculation, but the amount of colonization of the marked strain showed no significant difference at last.
The preliminary optimizing conditions for the antagonistic strain S43were as follows:the optimal medium:sucrose1.5%, peptone0.67%and yeast exact0.33%; the fermentation conditions:temperature30℃, pH7.0, rotation speed150r/min; inoculation amount2%, medium and air ratio2:5, the best inoculation age of strain14-3Oh and the optimum fermentation time36--72h.
菌株S43、S55的不同浓度发酵液对白粉病菌孢子抑制试验和盆栽试验的结果表明：随着发酵液浓度的增加,孢子萌发抑制率和防效均提高。当两者发酵液浓度为20%时即可很好的抑制白粉病孢子萌发,萌发抑制率均达80%,盆栽试验的防效也达到80%。
将拮抗菌株的发酵液和菌悬液通过三种处理方法(接种白粉菌前24h喷施、接种白粉菌时喷施和接种白粉菌后48h喷施)喷施于橡胶树叶片。通过接种S43、S55培养液,橡胶树叶片内的PPO、POD活性比对照大一倍,由此说明菌株S43、S55可诱导植物体产生防御酶。同时,分别测得拮抗菌对白粉病的防效。三种处理方法菌株S43发酵液防效分别为84.62%、76.53%、43.32%,而菌悬液防效较差,其防效分别为57.32%、56.56%、21.02%；三种处理方法菌株S55发酵液防效分别为82.35%、80.57%和57.94%,而菌悬液防效也较差,防效分别为57.76%、57.74%、32.29%。结果表明：(1)接种前24h喷施发酵液与接种时同时喷施发酵液和菌悬液的防效显著高于接种后48h喷施发酵液和菌悬液；(2)喷施发酵液比菌悬液效果好；(3)菌株S43和S55的发酵液对白粉病都具有很好的防效,但S43发酵液比S55发酵液的防效好。
通过采用伤叶和不伤叶接种方式研究菌株S43和S55在橡胶叶片上的定殖规律。研究表明,编号菌株S43*和S55*在叶片上接种一天后定殖量最大,但随着时间的推移其定殖量逐渐减少,并在15d后基本趋于稳定。在接种初期,伤叶处理的菌株定殖量比不伤叶处理的菌株定殖量大一倍,但两种方式处理后的菌株定殖量最终无显著差异。
对拮抗菌株S43发酵条件进行初步优化,优化培养基为：蔗糖1.5%、蛋白胨0.66%,酵母浸膏0.33%；发酵条件为：pH为7.0、摇床转速150r/min、温度30℃,种子培养液接种量2%、培养基与空气的比为2：5、最佳接种种龄为14-30h；最佳发酵时间36-72h。
276bacterial strains were separated from rhizosphere soil of rubber tree plantation. And two of them (S43and S55) have obvious antagonism against rubber tree Powdery Mildew. Based on the analysis of morphologic observation, cultural characteristics, physiological and biochemical properties, and the sequential analysis of16S rDNA, S43strain and S55were identified as Bacillus subtilis.
By means of Oidium heveae Steinm's spore test and potted seedlings experiment, the results showed that the inhibition rate to the spore germination and control effect were improved with the increasing of the concentration of fermented filtrates.20%of fermentation filtrates of strain S43and S55had effective inhibition activity against powdery mildew spores germination, with the inhibition rate of80%. Meanwhile, the control effect of the pot experiment also reached80%. This showed that the fermentation filtrates of strain S43and S55had good prevention effect to powdery mildew.
Spraying fermentation filtrates or bacterial suspension24h beforen48h after or while with powdery mildew inoculation. PPO, POD activity in the Rubber tree leaves inoculated with strain S43and S55had nearly doubled compared with the control, indicating that the strain S43and S55can induce the production of defence enzyme in plant. The control effect of S43fermented filtrates with three kinds of inoculation treatments were84.62%,76.53%and43.32%respectively as well as the inhibition rates were57.32%,56.56%and21.02%respectively with bacteria suspension of which the control effect was poorer. With the same methods of S55, the inhibition rates were82.35%、80.57%and57.94%respectively as well as the inhibition rates were57.76%、57.74%and32.29%respectively with bacteria suspension of which the control effect was poorer. The results were as follows:1. spraying24h before or while inoculation, the inhibition rate had obviously higher than that of48h after inoculation;2. fermentation filtrates had better control effect than that of bacterial suspension;3. compared with S55, fermentation filtrates of S43had better control effect to powdery mildew.
Colonization of marked strain S43and S55was researched in Rubber tree by inoculation with injuring leaves and no injuring leaves. The results showed that the marked strain S43*and S55*could set the largest colonization in the rubber leaves on the first day after inoculation. While as time went on, the population of setting reduced gradually and the population of the marked strain was almost stable after15days. The population of marked strain by treatment with injuring leaves was twice than that of with no injuring leaves at the early inoculation, but the amount of colonization of the marked strain showed no significant difference at last.
The preliminary optimizing conditions for the antagonistic strain S43were as follows:the optimal medium:sucrose1.5%, peptone0.67%and yeast exact0.33%; the fermentation conditions:temperature30℃, pH7.0, rotation speed150r/min; inoculation amount2%, medium and air ratio2:5, the best inoculation age of strain14-3Oh and the optimum fermentation time36--72h.
引文
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