外来入侵烟粉虱的种群动态、遗传分化及对噻虫嗪的抗性分子机理研究
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摘要
据报道,我国现有入侵生物400余种,仅松材线虫(Bursaphelenchus xylophilus)、烟粉虱(Bemisia tabaci)、斑潜蝇(Liriomyza sativae)、美国白蛾(Hyphantria cunea)、稻水象(Lissorhoptrusoryzophilus Kuschel)等11种外来有害生物,在我国造成的年经济损失就达574.3亿元人民币。其中,B型烟粉虱是世界性重要害虫,也是近年来入侵我国的外来有害生物,自90年代中后期入侵我国以来,在许多地区暴发危害,造成了严重经济损失,现已成为我国蔬菜、花卉和棉花等经济作物上的主要害虫。本论文主要对B型烟粉虱的种群动态、遗传分化及其对噻虫螓抗药性的分子机理等进行了研究。
2002-2004年对山西省烟粉虱的寄主植物范围和危害情况调查结果:山西省现有烟粉虱寄主植物27科103种(变种),其中葫芦科、十字花科、茄科和豆科等危害较为严重,且以晋南地区受害最为严重,寄主种类较多。
通过对12种主要寄主植物上烟粉虱田间种群动态调查研究,得知不同寄主植物上烟粉虱种群数量达到高峰的时间有所差别,波动的幅度也不一致。烟粉虱在12种寄主植物上的种群数量大小比较依次为:油葵>西葫芦>棉花>大豆>南瓜>茄子>丝瓜>番茄>辣椒>黄瓜>菜豆>玉米,说明烟粉虱对其寄主植物具有一定的自然选择性。另外,采用扩散系数(C=S~2/(?)),对烟粉虱在棉花、油葵和大豆三种作物田的空间分布型进行了初步分析,结果表明均为聚集分布型。
经反复实验,探索了一套简便、可行、纯度高、带型完整的多头和单头烟粉虱DNA基因组的提取方法。该方法提取的DNA,适用于如AFLP等对模板纯度和质量要求较高的分子标记技术研究,同时也适用于其它小型昆虫DNA的提取。
通过DNA模板浓度、Mg~(2+)浓度、dNTP用量以及其它反应参数等的摸索、调整,建立了一套完善的烟粉虱AFLP分子标记技术体系。
采用AFLP分子标记体系,对同一地区不同寄主上烟粉虱种群的遗传分化研究结果表明:不同寄主烟粉虱种群之间已经存在一定的遗传分化,但分化的程度比较小;同一寄主烟粉虱种群内的遗传分化则很低。
采用AFLP分子标记体系,对来自美国、以色列、巴基斯坦、澳大利亚、肯尼亚、西班牙和中国等7个国家24个地区的27个烟粉虱种群进行了不同地理种群间遗传分化及系统发育地位研究。结果表明:烟粉虱不同地理种群间存在着丰富的遗传多样性。聚类分析结果显示,中国的烟粉虱种群可能存在多个来源,同时表明中国目前至少存在B型、Q型和非B/Q等4种生物型。采用AFLP分子标记体系,对来自我国浙江(ZJ1-China)、山东(SD2-China)、北京(BJ3一China)、上海(SH1-China)、山西(SX1-China)和河南(HN-China)等地区的烟粉虱种群内遗传分化研究结果表明,同一地区烟粉虱种群内的遗传分化和杂合度均较低,说明B型烟粉虱在中国的发展历史较短,还没有积累更多的遗传变异。另外,从测试结果看,几个种群的平均杂合度大小依次为:北京(BJ3-China)>山东(SD2-China)>浙江(ZJ1-China)>上海(SH1-China)>山西(SX1-China)>河南(HN-China)。该结果将在揭示烟粉虱的入侵路径方面起着一定作用。
用噻虫嗪对烟粉虱进行室内继代抗性汰选,并采用改进的琼脂保湿浸叶法进行生物测定,获得了抗性倍数为39.96倍的抗性品系和相对敏感品系。
采用AFLP分子标记技术,对噻虫嗪烟粉虱抗性品系的遗传分化进行了研究,在聚类树中首
Bemisia tabaci (Gennadius) is an important invasive species that has caused severe damage in the world. B. tabaci was recorded for the first time on cotton in China in 1949, but significant damages caused by this insect had not been noticed until 1990s. In the past several years, the importance of this whitefly in China has increased spectacularly, which has infested in more than 20 provinces in China on cotton, some ornamental plants and most of the vegetable crops, in most of these place, which has resulted in a significant decrease in yields. However, little have been understood about its invasive mechanism and resistant mechanism to insecticide. In this paper, we studied the population dynamics, genetic diversity of invasive B. tabaci and its molecular mechanism of resistance to Thiamethoxam.From 2002-2004, the host plants of B. tabaci have been investigated and identified in Shanxi province, and all the host plants can be divided into 4 grades according to the damage degree caused by B. tabaci. There were 103 species of plant which belong to 27 families as hosts of B. tabaci, of them Cucurbitaceae. Solanaceae, Cruciferae and Leguminosae are the main families of host-plant, and the population density of B. tabaci was higher in south of Shanxi province than in north.The population dynamics of B. tabaci on 12 species of host-plants were studies from 2002 to 2004, which indicated that the infesting time and population densities of B. tabaci on different hosts were significantly different. the density of B. tabaci on different host plants was in the order of sunflower>pumpkin>cotton>soybean>cushaw>eggplant>hechima> tomato>chili>cucumber>kidney> bean>corn. In addition, spatial distributions of B. tabaci in cotton, sunflower and soybean fileds were studied, which indicated that they were collective distribution type.A amplified fragment length polymorphism (AFLP) molecular marker system on B. tabaci was funded by adjusting the DNA density, Mg~2+ density, dosage of dNTP and other reactions parameter etc.AFLP analysis showed that genetic differences exited among and within different host populations, but the difference among populations is bigger that that within populations.Analysis of the genetic diversity among 27 different geographical populations of B. tabaci and determination of biotypes of B. tabaci in China based on AFLP was conducted. The use of 5 primer combinations elected from 64 primer combinations allowed the identification of 229 polymorphic bands (97.03%) from 60 to 500 bp, suggesting abundant genetic diversity among different geographical populations of B. tabaci. Molecular phylogenetic tree based on AFLP analyse revealed the presence in China of at least four different genetic groups of B. tabaci. B biotype, Q biotype and two non B/Q biotype. B biotype was nationwide distributed. Q biotype was present only in local region of China including YunNan province and BeiJing city. Other two non B/Q biotype groups, one was found in ShanDong and HeBei provinces, and another in ZheJiang province.Genetic differences within different geographical populations of B. tabaci, including Zhejiang(ZJl-China),Shandong (SD2-China), Peking(BJ3-China),Shanghai (SH1-China), Shanxi(SX1-China)and Henan (HN-China) were studied using AFLP molecular marker. The results showed that geneticdifference and heterisity of population were lower. Heterisitys of several populations in order were:
Peking (BJ3- China) >Shandong (SD2-China)>Zhejiang (ZJ1-China) >Shanghai (SH1-China)>Shanxi (SX1-China)>Henan (HN-China).A 39.96 fold Thiamethoxam-resistant strain of B. tabaci was achieved by the selection with Thiamethoxam.Analysis of the genetic diversity between the resistant and susceptive strains of B. tabaci based on AFLP were conducted. The results showed that a significant genetical differentiation was found between the resistant and susceptive strains of B. tabaci. Additionally, peculiar amplified DNA fragments, the 150bp for resistant strain and the 315bp for susceptive strain were identified using the E-ACT/M-CTG primer, which may serve to
2002-2004年对山西省烟粉虱的寄主植物范围和危害情况调查结果:山西省现有烟粉虱寄主植物27科103种(变种),其中葫芦科、十字花科、茄科和豆科等危害较为严重,且以晋南地区受害最为严重,寄主种类较多。
通过对12种主要寄主植物上烟粉虱田间种群动态调查研究,得知不同寄主植物上烟粉虱种群数量达到高峰的时间有所差别,波动的幅度也不一致。烟粉虱在12种寄主植物上的种群数量大小比较依次为:油葵>西葫芦>棉花>大豆>南瓜>茄子>丝瓜>番茄>辣椒>黄瓜>菜豆>玉米,说明烟粉虱对其寄主植物具有一定的自然选择性。另外,采用扩散系数(C=S~2/(?)),对烟粉虱在棉花、油葵和大豆三种作物田的空间分布型进行了初步分析,结果表明均为聚集分布型。
经反复实验,探索了一套简便、可行、纯度高、带型完整的多头和单头烟粉虱DNA基因组的提取方法。该方法提取的DNA,适用于如AFLP等对模板纯度和质量要求较高的分子标记技术研究,同时也适用于其它小型昆虫DNA的提取。
通过DNA模板浓度、Mg~(2+)浓度、dNTP用量以及其它反应参数等的摸索、调整,建立了一套完善的烟粉虱AFLP分子标记技术体系。
采用AFLP分子标记体系,对同一地区不同寄主上烟粉虱种群的遗传分化研究结果表明:不同寄主烟粉虱种群之间已经存在一定的遗传分化,但分化的程度比较小;同一寄主烟粉虱种群内的遗传分化则很低。
采用AFLP分子标记体系,对来自美国、以色列、巴基斯坦、澳大利亚、肯尼亚、西班牙和中国等7个国家24个地区的27个烟粉虱种群进行了不同地理种群间遗传分化及系统发育地位研究。结果表明:烟粉虱不同地理种群间存在着丰富的遗传多样性。聚类分析结果显示,中国的烟粉虱种群可能存在多个来源,同时表明中国目前至少存在B型、Q型和非B/Q等4种生物型。采用AFLP分子标记体系,对来自我国浙江(ZJ1-China)、山东(SD2-China)、北京(BJ3一China)、上海(SH1-China)、山西(SX1-China)和河南(HN-China)等地区的烟粉虱种群内遗传分化研究结果表明,同一地区烟粉虱种群内的遗传分化和杂合度均较低,说明B型烟粉虱在中国的发展历史较短,还没有积累更多的遗传变异。另外,从测试结果看,几个种群的平均杂合度大小依次为:北京(BJ3-China)>山东(SD2-China)>浙江(ZJ1-China)>上海(SH1-China)>山西(SX1-China)>河南(HN-China)。该结果将在揭示烟粉虱的入侵路径方面起着一定作用。
用噻虫嗪对烟粉虱进行室内继代抗性汰选,并采用改进的琼脂保湿浸叶法进行生物测定,获得了抗性倍数为39.96倍的抗性品系和相对敏感品系。
采用AFLP分子标记技术,对噻虫嗪烟粉虱抗性品系的遗传分化进行了研究,在聚类树中首
Bemisia tabaci (Gennadius) is an important invasive species that has caused severe damage in the world. B. tabaci was recorded for the first time on cotton in China in 1949, but significant damages caused by this insect had not been noticed until 1990s. In the past several years, the importance of this whitefly in China has increased spectacularly, which has infested in more than 20 provinces in China on cotton, some ornamental plants and most of the vegetable crops, in most of these place, which has resulted in a significant decrease in yields. However, little have been understood about its invasive mechanism and resistant mechanism to insecticide. In this paper, we studied the population dynamics, genetic diversity of invasive B. tabaci and its molecular mechanism of resistance to Thiamethoxam.From 2002-2004, the host plants of B. tabaci have been investigated and identified in Shanxi province, and all the host plants can be divided into 4 grades according to the damage degree caused by B. tabaci. There were 103 species of plant which belong to 27 families as hosts of B. tabaci, of them Cucurbitaceae. Solanaceae, Cruciferae and Leguminosae are the main families of host-plant, and the population density of B. tabaci was higher in south of Shanxi province than in north.The population dynamics of B. tabaci on 12 species of host-plants were studies from 2002 to 2004, which indicated that the infesting time and population densities of B. tabaci on different hosts were significantly different. the density of B. tabaci on different host plants was in the order of sunflower>pumpkin>cotton>soybean>cushaw>eggplant>hechima> tomato>chili>cucumber>kidney> bean>corn. In addition, spatial distributions of B. tabaci in cotton, sunflower and soybean fileds were studied, which indicated that they were collective distribution type.A amplified fragment length polymorphism (AFLP) molecular marker system on B. tabaci was funded by adjusting the DNA density, Mg~2+ density, dosage of dNTP and other reactions parameter etc.AFLP analysis showed that genetic differences exited among and within different host populations, but the difference among populations is bigger that that within populations.Analysis of the genetic diversity among 27 different geographical populations of B. tabaci and determination of biotypes of B. tabaci in China based on AFLP was conducted. The use of 5 primer combinations elected from 64 primer combinations allowed the identification of 229 polymorphic bands (97.03%) from 60 to 500 bp, suggesting abundant genetic diversity among different geographical populations of B. tabaci. Molecular phylogenetic tree based on AFLP analyse revealed the presence in China of at least four different genetic groups of B. tabaci. B biotype, Q biotype and two non B/Q biotype. B biotype was nationwide distributed. Q biotype was present only in local region of China including YunNan province and BeiJing city. Other two non B/Q biotype groups, one was found in ShanDong and HeBei provinces, and another in ZheJiang province.Genetic differences within different geographical populations of B. tabaci, including Zhejiang(ZJl-China),Shandong (SD2-China), Peking(BJ3-China),Shanghai (SH1-China), Shanxi(SX1-China)and Henan (HN-China) were studied using AFLP molecular marker. The results showed that geneticdifference and heterisity of population were lower. Heterisitys of several populations in order were:
Peking (BJ3- China) >Shandong (SD2-China)>Zhejiang (ZJ1-China) >Shanghai (SH1-China)>Shanxi (SX1-China)>Henan (HN-China).A 39.96 fold Thiamethoxam-resistant strain of B. tabaci was achieved by the selection with Thiamethoxam.Analysis of the genetic diversity between the resistant and susceptive strains of B. tabaci based on AFLP were conducted. The results showed that a significant genetical differentiation was found between the resistant and susceptive strains of B. tabaci. Additionally, peculiar amplified DNA fragments, the 150bp for resistant strain and the 315bp for susceptive strain were identified using the E-ACT/M-CTG primer, which may serve to
引文
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