腐烂茎线虫不同群体同工酶表型与生物学特性研究
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摘要
甘薯茎线虫病是我国甘薯生产上重要病害之一,该病病原腐烂茎线虫(Ditylenchus destructor)为我国检疫性病害。腐烂茎线虫最早在马铃薯上发现,该线虫除了危害甘薯外,还危害花生、茄子、薄荷、甜菜和草莓等作物。本实验以来自国内的10个腐烂茎线虫群体为研究材料,做了以下几方面的研究：
对国内不同地理来源的10个腐烂茎线虫群体进行同工酶表型研究。结果表明：1)在对DeJL和DeLJ 2个群体的同工酶梯度分析中发现,碾样200、400、600条线虫的10μl提取液中,只有碾样400条线虫提取液对DeJL和DeLJ表型研究均有较好的染色效果；2)10个群体苹果酸脱氢酶(MDH)酶谱分为2个酶型,酶型Ⅰ的相对迁移率(Rf值)约为0.21、0.26；酶型Ⅱ的Rf值约为0.21、0.26、0.31,而酯酶染色不成形；3)结合MDH表型和核糖体ITS区核酸序列分析,将腐烂茎线虫划分为3个不同遗传类型。
对国内不同地理来源的10个腐烂茎线虫群体在两个中抗甘薯茎线虫病品种(苏薯9号、宁薯9-9)上测定腐烂茎线虫群体致病力、繁殖力、雌雄比(性比)、甘薯薯块鲜重损失量。结果表明：1)在P=0.05水平上,各群体间的致病力、繁殖力、性比差异显著,其中致病力最高的群体来自河北廊坊DeHB,对苏薯9号的致病力为75%(即苏薯的病情指数为75%)；2)在苏薯9号和宁薯9-9上,各线虫群体致病力与线虫繁殖力呈正相关；3)偏相关分析显示,在苏薯9号上不同线虫群体繁殖力与性比呈负相关,相关系数低,只为-0.213,而在宁薯9-9上不同群体繁殖力与性比呈显著正相关,相关系数高,为0.901,说明寄主品种的改变对线虫群体性别分化影响显著；4)在抗甘薯茎线虫病和保鲜力方面,宁薯9-9分别比苏薯9号高33.48%和19.86%。
将腐烂茎线虫2个基因型进行杂交,在48个正反交组合中,33个杂交组合未产生后代,15个组合产生的F1代死亡,2个组合"DeLYxDeYL"和"DeSDxDeHB"产生活力很弱的F1代幼虫,继代培养未能产生可延续的后代；所有的杂交组合中所产生的F1代均为幼虫且其数量在P0.05水平上均显著低于亲本对照组；对"DeLYxDeYL"和"DeSDxDeHB"2个组合的F1代进行ITS-PCR扩增产物和RsaⅠ、HaeⅢ、HinfⅠ3种内切酶的酶切(RFLP)分析显示,F1代ITS区PCR产物与其母本条带长度较一致,约为800bp,2个杂交代与其亲本的酶切图谱在3个酶切位点上差异显著；对同一基因型内的线虫种群间杂交发现,杂交均能成功且产生可育的后代,对其F1代同样进行ITS区扩增产物和RFLP分析,发现同一基因型内杂交后代ITS区扩增产物和RFLP图谱与其亲本一致。说明我国甘薯腐烂茎线虫种群间分化现象严重,变异复杂,即基因型A4个种群分别与基因型B 6个种群间杂交不亲和,存在生殖隔离现象,我国甘薯腐烂茎线虫很可能是一个包含两种不同线虫的复合种。
对5个不同地理来源的腐烂茎线虫群体分别接种到甘薯(待其发病腐烂时做以下实验处理)、甘油和细沙3种介质中,在-20℃和-70℃温度下经过不同的处理时间测定其存活率来研究腐烂茎线虫耐寒力,并探索腐烂茎线虫在低温冷冻下的保存方法。腐烂茎线虫在-20℃和-70℃低温下,随着处理时间的延长,不同群体线虫的存活率都有所下降,其中DeWY和DeLJ群体在不同处理时间下的存活率均高于其它3个群体,且在P0.01水平下均达到极显著差异,初步证实这2个群体有很强的耐寒力。试验发现在细沙和不同甘油浓度条件下,所有线虫群体在低温处理下均未查到活虫。腐烂茎线虫在低温下的生存状态依赖于原寄主材料,甘薯薯块是腐烂茎线虫在低温下抵御逆境最好的屏障,腐烂茎线虫侵染的薯块放在低温下保存,在遗传学研究等方面保存原始线虫种源有应用价值。
Ditylenchus destructor is one of the important diseases in sweet potato production of our country. Ditylenchus destructor is the pathogeny, and it is not allowed to enter into our country by quarantine. It was founded in potato at first, but the main host is sweet potato in our country. In addition of peanuts, eggplant, mint, sugar beet and strawberries and other crops.10 Chinese populations of Ditylenchus destructor were studed as research materials to do research on such several aspects:
Two isozymes from 10 populations of sweet potato stem nematode, Ditylenchus destructor, were analyzed; the results show that among the 10 populations, there were three main bands of malate dehydrogenase (MDH), which were corresponding to two different phenotypes. RF-was about 0.21,0.26 and the other was 0.21,0.26 and 0.31.Three genetic types of the 10 populations were divided by isozyme phenotypes.
The pathogenicity, fecundity and sex ratio of the 10 populations were significant at the P0.05 level; the highest pathogenicity population was from DeHB.There were positive relationships between fecundity and pathogenicity on the Sushu No.9 and Ningshu 9-9. Partial correlation analysis showed that there was negative relationship between fecundity and sex ratio of the 10 populations on the Sushu No.9 and significant positive relationship on the Ningshu 9-9, their coefficients were-0.213,0.901,respectively, indicating that the sex differentiation of D. destructor is significantly affected by the different varieties of sweet potato.In addition, the Ningshu 9-9 was 33.48% higher than the Sushu No.9 in the resistant to D. destructor, and 19.86% higher in preservation.
Species hybridization with 10 populations (Ditylenchus destructor) from the diferent geographical areas,68.75% of the hybrids were not successful in 48 positive and negative, 31.25% of the hybrids had been dead or dispread after a generation or more, all of the F1 Hybrids were significant lower than control group in the average number of the generation at P0.05 level; The.hybrids of all combination didn't continue to live after several generations, that was to say there was incompatibility between genetic A and B in Ditylenchus destructor species;Combating to ITS-PCR production, there showed a main band in the two of F1 generations,which length of ITS-PCR production was about 800bp; There were a difference between F1 hybrids and the parents on the ITS-PCR-RFLP study; by using of biological hybrid experiments and ITS-PCR-RFLP method,we confirmed there was differentiation serious to potato nematode populations in China, complex variation, which was a certain degree of reproductive isolation, there are likely two different nematode species.
The cold tolerance of 5 populations of Ditylenchus destructor was studied in this research. The results demonstrated that the vitality of all the populations decreased after being treated with-20℃and -70℃in infected sweet, but they could still maintain certain propagating ability. DeLJ and DeWY and treatment groups at different time of survival are higher than the other three groups, and there were extremely significant differences at P0.01 level between two groups confirmed that demonstrated a very strong cold tolerance. However, in the sand and glycerol, all populations of Ditylenchus destructor could not survive at low temperature. It is confirmed that the survival of Ditylenchus destructor depends on the sweet potato in the low temperature, which can be seen sweet potato (host) are decomposed the best barrier against low temperatures to Ditylenchus destructor; This research has important significance in provenance original study of genetics eara through preserved effected sweet potato in low temperature.
对国内不同地理来源的10个腐烂茎线虫群体进行同工酶表型研究。结果表明：1)在对DeJL和DeLJ 2个群体的同工酶梯度分析中发现,碾样200、400、600条线虫的10μl提取液中,只有碾样400条线虫提取液对DeJL和DeLJ表型研究均有较好的染色效果；2)10个群体苹果酸脱氢酶(MDH)酶谱分为2个酶型,酶型Ⅰ的相对迁移率(Rf值)约为0.21、0.26；酶型Ⅱ的Rf值约为0.21、0.26、0.31,而酯酶染色不成形；3)结合MDH表型和核糖体ITS区核酸序列分析,将腐烂茎线虫划分为3个不同遗传类型。
对国内不同地理来源的10个腐烂茎线虫群体在两个中抗甘薯茎线虫病品种(苏薯9号、宁薯9-9)上测定腐烂茎线虫群体致病力、繁殖力、雌雄比(性比)、甘薯薯块鲜重损失量。结果表明：1)在P=0.05水平上,各群体间的致病力、繁殖力、性比差异显著,其中致病力最高的群体来自河北廊坊DeHB,对苏薯9号的致病力为75%(即苏薯的病情指数为75%)；2)在苏薯9号和宁薯9-9上,各线虫群体致病力与线虫繁殖力呈正相关；3)偏相关分析显示,在苏薯9号上不同线虫群体繁殖力与性比呈负相关,相关系数低,只为-0.213,而在宁薯9-9上不同群体繁殖力与性比呈显著正相关,相关系数高,为0.901,说明寄主品种的改变对线虫群体性别分化影响显著；4)在抗甘薯茎线虫病和保鲜力方面,宁薯9-9分别比苏薯9号高33.48%和19.86%。
将腐烂茎线虫2个基因型进行杂交,在48个正反交组合中,33个杂交组合未产生后代,15个组合产生的F1代死亡,2个组合"DeLYxDeYL"和"DeSDxDeHB"产生活力很弱的F1代幼虫,继代培养未能产生可延续的后代；所有的杂交组合中所产生的F1代均为幼虫且其数量在P0.05水平上均显著低于亲本对照组；对"DeLYxDeYL"和"DeSDxDeHB"2个组合的F1代进行ITS-PCR扩增产物和RsaⅠ、HaeⅢ、HinfⅠ3种内切酶的酶切(RFLP)分析显示,F1代ITS区PCR产物与其母本条带长度较一致,约为800bp,2个杂交代与其亲本的酶切图谱在3个酶切位点上差异显著；对同一基因型内的线虫种群间杂交发现,杂交均能成功且产生可育的后代,对其F1代同样进行ITS区扩增产物和RFLP分析,发现同一基因型内杂交后代ITS区扩增产物和RFLP图谱与其亲本一致。说明我国甘薯腐烂茎线虫种群间分化现象严重,变异复杂,即基因型A4个种群分别与基因型B 6个种群间杂交不亲和,存在生殖隔离现象,我国甘薯腐烂茎线虫很可能是一个包含两种不同线虫的复合种。
对5个不同地理来源的腐烂茎线虫群体分别接种到甘薯(待其发病腐烂时做以下实验处理)、甘油和细沙3种介质中,在-20℃和-70℃温度下经过不同的处理时间测定其存活率来研究腐烂茎线虫耐寒力,并探索腐烂茎线虫在低温冷冻下的保存方法。腐烂茎线虫在-20℃和-70℃低温下,随着处理时间的延长,不同群体线虫的存活率都有所下降,其中DeWY和DeLJ群体在不同处理时间下的存活率均高于其它3个群体,且在P0.01水平下均达到极显著差异,初步证实这2个群体有很强的耐寒力。试验发现在细沙和不同甘油浓度条件下,所有线虫群体在低温处理下均未查到活虫。腐烂茎线虫在低温下的生存状态依赖于原寄主材料,甘薯薯块是腐烂茎线虫在低温下抵御逆境最好的屏障,腐烂茎线虫侵染的薯块放在低温下保存,在遗传学研究等方面保存原始线虫种源有应用价值。
Ditylenchus destructor is one of the important diseases in sweet potato production of our country. Ditylenchus destructor is the pathogeny, and it is not allowed to enter into our country by quarantine. It was founded in potato at first, but the main host is sweet potato in our country. In addition of peanuts, eggplant, mint, sugar beet and strawberries and other crops.10 Chinese populations of Ditylenchus destructor were studed as research materials to do research on such several aspects:
Two isozymes from 10 populations of sweet potato stem nematode, Ditylenchus destructor, were analyzed; the results show that among the 10 populations, there were three main bands of malate dehydrogenase (MDH), which were corresponding to two different phenotypes. RF-was about 0.21,0.26 and the other was 0.21,0.26 and 0.31.Three genetic types of the 10 populations were divided by isozyme phenotypes.
The pathogenicity, fecundity and sex ratio of the 10 populations were significant at the P0.05 level; the highest pathogenicity population was from DeHB.There were positive relationships between fecundity and pathogenicity on the Sushu No.9 and Ningshu 9-9. Partial correlation analysis showed that there was negative relationship between fecundity and sex ratio of the 10 populations on the Sushu No.9 and significant positive relationship on the Ningshu 9-9, their coefficients were-0.213,0.901,respectively, indicating that the sex differentiation of D. destructor is significantly affected by the different varieties of sweet potato.In addition, the Ningshu 9-9 was 33.48% higher than the Sushu No.9 in the resistant to D. destructor, and 19.86% higher in preservation.
Species hybridization with 10 populations (Ditylenchus destructor) from the diferent geographical areas,68.75% of the hybrids were not successful in 48 positive and negative, 31.25% of the hybrids had been dead or dispread after a generation or more, all of the F1 Hybrids were significant lower than control group in the average number of the generation at P0.05 level; The.hybrids of all combination didn't continue to live after several generations, that was to say there was incompatibility between genetic A and B in Ditylenchus destructor species;Combating to ITS-PCR production, there showed a main band in the two of F1 generations,which length of ITS-PCR production was about 800bp; There were a difference between F1 hybrids and the parents on the ITS-PCR-RFLP study; by using of biological hybrid experiments and ITS-PCR-RFLP method,we confirmed there was differentiation serious to potato nematode populations in China, complex variation, which was a certain degree of reproductive isolation, there are likely two different nematode species.
The cold tolerance of 5 populations of Ditylenchus destructor was studied in this research. The results demonstrated that the vitality of all the populations decreased after being treated with-20℃and -70℃in infected sweet, but they could still maintain certain propagating ability. DeLJ and DeWY and treatment groups at different time of survival are higher than the other three groups, and there were extremely significant differences at P0.01 level between two groups confirmed that demonstrated a very strong cold tolerance. However, in the sand and glycerol, all populations of Ditylenchus destructor could not survive at low temperature. It is confirmed that the survival of Ditylenchus destructor depends on the sweet potato in the low temperature, which can be seen sweet potato (host) are decomposed the best barrier against low temperatures to Ditylenchus destructor; This research has important significance in provenance original study of genetics eara through preserved effected sweet potato in low temperature.
引文
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