烟草黑胫病诱抗物质的筛选及机理研究
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摘要
本文研究和评价了水杨酸、二氢茉莉酸丙酯、脱落酸、乙烯利、稻瘟灵、苯丙噻二唑、噻菌灵7种药剂对烟草黑胫病和烟草花叶病的效果，从中选择出了对烟草黑胫病效果较好的药剂－二氢茉莉酸丙酯（PDJ），并测定了PDJ对黑胫病菌的直接作用；在上述试验的基础上，进行了系统抗性试验和不同诱导时间后的接种试验；研究了不同药剂联合作用对烟草黑胫病的控制效果，明确了不同药剂之间的联合作用性质；对筛选出的PDJ诱导烟草抗黑胫病的机理进行了初步探讨。
对烟草浸种和苗期喷雾处理结果表明，供试药剂在适宜的诱导时间及浓度下均能诱导烟草对黑胫病的抗性，降低发病率和病情指数，但效果之间存在较大差异；其中PDJ效果最为突出，用100mg/L苗期喷雾处理，诱导后3d、7d接种防病效果都在60%以上，浸种处理效果达70.83%，但对烟草生长有一定的抑制作用。供试7种药剂对花叶病的控制效果一般，其中PDJ和PBZ相对较高，效果仍然在50%以下。
测定了PDJ对烟草黑胫病菌的直接作用，包括对菌丝生长、孢子囊产生、游动孢子释放及休止孢萌发的作用，结果表明PDJ浓度在200mg/L以下对病原菌的以上生理过程均没有明显的抑制作用，表明PDJ对烟草黑胫病的控制效果是由于诱导了烟草自身的抗病性，而非直接作用于病原菌。
通过局部叶片施药，在未施药部位接种，观察黑胫病的发生发展情况的方法测定了PDJ诱导烟草的系统抗性。结果表明，下部第3片叶施药，第4、第5和第6片接种叶病斑比对照都明显减小，由此说明PDJ诱导烟草对黑胫病的抗性具有系统性。
对PDJ处理后不同时间接种黑胫病的发病率、病情指数和防治效果进行了测定，结果表明，药后3d接种对黑胫病的诱抗效果最好，随着用药时间的延长，诱抗效果逐渐降低，但药后15d仍具有51.04%的诱抗效果，至21d时诱抗效果只有12.50%，表明PDJ诱导烟草对黑胫病菌的抗性大约可以持续15d左右。
测定了PDJ与其它几种药剂联合作用对烟草黑胫病的效果，结果表明PDJ与其它6种药剂混用除了与ABA混用有一定的增效作用外，其它均表现为相加作用。
对几种抗病相关酶活性测定结果表明，施用PDJ可以提高地上部和根部PAL、POD的活性，但根部PAL和POD活性高峰的出现要迟于地上部；接种后烟草PAL和POD活性有较大幅度的提高，但是施用PDJ的烟苗两种酶活性有较长时间的高表达，不施药接种的烟苗酶活性只有短暂的高表达，后迅速降低。PDJ处理对烟草体内CAT活性影响不大。
对不同处理烟草叶片病原相关蛋白电泳图谱的分析表明，PDJ处理可以诱导烟草体内某些蛋白的表达，其中Rf值为0.11、0.28和0.57的蛋白表达较为强烈；接种也可以导致烟草体内PR蛋白的表达，但是施药和不施药处理之间差异较大，两种处理都有5条谱带表达，但表达量不同，PDJ处理后接种的烟草5条谱带表达都比不施药接种的烟草强烈。
分析了PDJ处理对烟草体内osmotin和CDPK2基因表达的影响，结果表明PDJ处理可以诱导烟草osmotin基因的表达，对CDPK2的表达没有影响。接种也可以诱导osmotin基因的表达，但施药和不施药的烟苗表达情况不一致，施药后接种的烟苗表达时间更长。
Seven chemicals were used to control tobacco black shank and tobacco mosaic, and their effects were evaluated; the best one was selected and its effect to Phytophthora parasitica Var nicotianae was studied.In order to testify the result, a systemic experiment was conducted. In addition to these, the interaction between PDJ and other chemicals was studied and the primary studies on the mechanism of PDJ inducing the resistance of tobacco were also undertaken.
The results of seed soaking and seedling spraying showed that all of these seven chemicals could induce the resistance of tobacco to black shank, but their effects were different. The effect of PDJ was the best among them, which could reach above 60% when tobacco seedling was sprayed with 100 mg/L PDJ. And the effect was higher when the tobacco seeds were soaked with 100mg/L PDJ,which could reach 70.83%.As for tobacco mosaic, these seven chemicals didn't have notable effects. Although PDJ had higher effect than the others, its effect was not prominent.
We studied the effect of PDJ on Phytophthora parasitica Var nicotianae, including on the development of mycelium, the producing of sporangium, the releasing of swarm spore and the germination of dormant spore. It showed that PDJ had little influences on the four life processes. Therefore we draw the conclusion that PDJ could induce tobacco's resistance to black shank.
In order to testify the conclusion, we conducted a systemic
experiment, that is to say, we treated the lower leaf with PDJ and inoculated the upper leaves. It showed that 100 mg/L PDJ and 200 mg/L PDJ could significantly reduce the diameter of lesions, and the suppressions of the lesion diameter on the fourth leaf were 65.38% and 57.69% respectively. As for the fifth leaf and the sixth leaf, the suppressions of 100mg/L and 200 mg/L PDJ were both about 60%.Basing on this results, we concluded that PDJ could induce the resistance of tobacco, and this resistance was systemic.
The results of different inoculation time showed that PDJ had the best effect to black shank when we inoculated tobacco three days after PDJ-treatment. And the effect reduced gradually and it could last about 15 days.
The joint effects of PDJ with other six chemicals were almost additive; expect that PDJ incorporating with ABA showed a little synergistic effect.
PDJ treatment could increase the activities of peroxidase (POD) and phenylalanine ammonialyase (PAL), and it had little influence on catalase (CAT).But the POD and PAL activities pinnacle of root were later than that of leaves. Inoculation also could induce the activities of POD、PAL and CAT, but this trends were different between PDJ treatment and CK(water treatment).The activities of POD、PAL and CAT in PDJ-treatment-and-inoculation tobacco were higher and could last more days.
The result of SDS-PAGE analysis showed that PDJ could induce some pathogenesis-related proteins，among them Rf＝0.11、0.28 and 0.57 proteins had a higher expression quantities. Inoculation also could induce PRP, but the concentration of PRP bands are different.All these five protein bands had a higher expression quantities in PDJ-treating tobaccos than in water－treating tobaccos.
The study on the influence of PDJ to osmotin and CDPK2 genes showed that PDJ could induce the expression of osmotin gene, and it had no influence on CDPK2.Inoculation also could induce osmotin expression, but in PDJ-treating tobacco osmotin genes could express more days.
对烟草浸种和苗期喷雾处理结果表明，供试药剂在适宜的诱导时间及浓度下均能诱导烟草对黑胫病的抗性，降低发病率和病情指数，但效果之间存在较大差异；其中PDJ效果最为突出，用100mg/L苗期喷雾处理，诱导后3d、7d接种防病效果都在60%以上，浸种处理效果达70.83%，但对烟草生长有一定的抑制作用。供试7种药剂对花叶病的控制效果一般，其中PDJ和PBZ相对较高，效果仍然在50%以下。
测定了PDJ对烟草黑胫病菌的直接作用，包括对菌丝生长、孢子囊产生、游动孢子释放及休止孢萌发的作用，结果表明PDJ浓度在200mg/L以下对病原菌的以上生理过程均没有明显的抑制作用，表明PDJ对烟草黑胫病的控制效果是由于诱导了烟草自身的抗病性，而非直接作用于病原菌。
通过局部叶片施药，在未施药部位接种，观察黑胫病的发生发展情况的方法测定了PDJ诱导烟草的系统抗性。结果表明，下部第3片叶施药，第4、第5和第6片接种叶病斑比对照都明显减小，由此说明PDJ诱导烟草对黑胫病的抗性具有系统性。
对PDJ处理后不同时间接种黑胫病的发病率、病情指数和防治效果进行了测定，结果表明，药后3d接种对黑胫病的诱抗效果最好，随着用药时间的延长，诱抗效果逐渐降低，但药后15d仍具有51.04%的诱抗效果，至21d时诱抗效果只有12.50%，表明PDJ诱导烟草对黑胫病菌的抗性大约可以持续15d左右。
测定了PDJ与其它几种药剂联合作用对烟草黑胫病的效果，结果表明PDJ与其它6种药剂混用除了与ABA混用有一定的增效作用外，其它均表现为相加作用。
对几种抗病相关酶活性测定结果表明，施用PDJ可以提高地上部和根部PAL、POD的活性，但根部PAL和POD活性高峰的出现要迟于地上部；接种后烟草PAL和POD活性有较大幅度的提高，但是施用PDJ的烟苗两种酶活性有较长时间的高表达，不施药接种的烟苗酶活性只有短暂的高表达，后迅速降低。PDJ处理对烟草体内CAT活性影响不大。
对不同处理烟草叶片病原相关蛋白电泳图谱的分析表明，PDJ处理可以诱导烟草体内某些蛋白的表达，其中Rf值为0.11、0.28和0.57的蛋白表达较为强烈；接种也可以导致烟草体内PR蛋白的表达，但是施药和不施药处理之间差异较大，两种处理都有5条谱带表达，但表达量不同，PDJ处理后接种的烟草5条谱带表达都比不施药接种的烟草强烈。
分析了PDJ处理对烟草体内osmotin和CDPK2基因表达的影响，结果表明PDJ处理可以诱导烟草osmotin基因的表达，对CDPK2的表达没有影响。接种也可以诱导osmotin基因的表达，但施药和不施药的烟苗表达情况不一致，施药后接种的烟苗表达时间更长。
Seven chemicals were used to control tobacco black shank and tobacco mosaic, and their effects were evaluated; the best one was selected and its effect to Phytophthora parasitica Var nicotianae was studied.In order to testify the result, a systemic experiment was conducted. In addition to these, the interaction between PDJ and other chemicals was studied and the primary studies on the mechanism of PDJ inducing the resistance of tobacco were also undertaken.
The results of seed soaking and seedling spraying showed that all of these seven chemicals could induce the resistance of tobacco to black shank, but their effects were different. The effect of PDJ was the best among them, which could reach above 60% when tobacco seedling was sprayed with 100 mg/L PDJ. And the effect was higher when the tobacco seeds were soaked with 100mg/L PDJ,which could reach 70.83%.As for tobacco mosaic, these seven chemicals didn't have notable effects. Although PDJ had higher effect than the others, its effect was not prominent.
We studied the effect of PDJ on Phytophthora parasitica Var nicotianae, including on the development of mycelium, the producing of sporangium, the releasing of swarm spore and the germination of dormant spore. It showed that PDJ had little influences on the four life processes. Therefore we draw the conclusion that PDJ could induce tobacco's resistance to black shank.
In order to testify the conclusion, we conducted a systemic
experiment, that is to say, we treated the lower leaf with PDJ and inoculated the upper leaves. It showed that 100 mg/L PDJ and 200 mg/L PDJ could significantly reduce the diameter of lesions, and the suppressions of the lesion diameter on the fourth leaf were 65.38% and 57.69% respectively. As for the fifth leaf and the sixth leaf, the suppressions of 100mg/L and 200 mg/L PDJ were both about 60%.Basing on this results, we concluded that PDJ could induce the resistance of tobacco, and this resistance was systemic.
The results of different inoculation time showed that PDJ had the best effect to black shank when we inoculated tobacco three days after PDJ-treatment. And the effect reduced gradually and it could last about 15 days.
The joint effects of PDJ with other six chemicals were almost additive; expect that PDJ incorporating with ABA showed a little synergistic effect.
PDJ treatment could increase the activities of peroxidase (POD) and phenylalanine ammonialyase (PAL), and it had little influence on catalase (CAT).But the POD and PAL activities pinnacle of root were later than that of leaves. Inoculation also could induce the activities of POD、PAL and CAT, but this trends were different between PDJ treatment and CK(water treatment).The activities of POD、PAL and CAT in PDJ-treatment-and-inoculation tobacco were higher and could last more days.
The result of SDS-PAGE analysis showed that PDJ could induce some pathogenesis-related proteins，among them Rf＝0.11、0.28 and 0.57 proteins had a higher expression quantities. Inoculation also could induce PRP, but the concentration of PRP bands are different.All these five protein bands had a higher expression quantities in PDJ-treating tobaccos than in water－treating tobaccos.
The study on the influence of PDJ to osmotin and CDPK2 genes showed that PDJ could induce the expression of osmotin gene, and it had no influence on CDPK2.Inoculation also could induce osmotin expression, but in PDJ-treating tobacco osmotin genes could express more days.
引文
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