葡萄根瘤蚜的荧光定量检测及间种植物防控研究

葡萄根瘤蚜的荧光定量PCR检测及间种植物防控研究

USING THE INTERCROP TO CONTROL AND REAL-TIME PCR DETECTION OF GRAPE PHYLLOXERA

作者：郭　庆　　导师：王忠跃

中国农业科学院　　昆虫生理及分子生物学2011届硕士

摘　要

葡萄根瘤蚜Daktulosphaira vitifoliae(Fitch),属于同翅目（Homoptera），根瘤蚜科（Phylloxeridae）,原产于北美洲落基山脉东部，是葡萄上的毁灭性害虫，为专食性害虫，只危害vitis属植物，是我国植物检疫性有害生物。1935年在我国首次发现葡萄根瘤蚜后，通过采取砍伐葡萄，使葡萄根瘤蚜销声匿迹。2005年在我国再次发现葡萄根瘤蚜危害。目前世界上主要利用嫁接抗性砧木防治葡萄根瘤蚜，没有其他有效防治方法。由于我国葡萄大多以扦插方式栽培，嫁接抗性砧木面积较少，葡萄根瘤蚜对我国迅速发展的葡萄产业构成严重威胁。同时，目前主要依靠常规土壤及根系调查葡萄根瘤蚜，但常规地面方法费时费力。因此，为了给葡萄根瘤蚜监测提供快速、准确、敏感性高的分子生物学调查方法，以及为葡萄根瘤蚜防控探索有效的方法，本试验对实时定量PCR方法检测葡萄根瘤蚜，以及在葡萄园间作植物防控葡萄根瘤蚜进行了研究。主要结果总结如下：

1.　参考Karen Herbert等的方法，根据葡萄根瘤蚜ITS2中的保守序列设计的特异性引物与TaqMan-MGB荧光探针，通过试验验证上述引物和探针适用于我国葡萄根瘤蚜的检测，并且成功构建了标准质粒，制作出以标准阳性质粒作为标准品的标准曲线，经优化反应条件，建立了一种绝对定量检测葡萄根瘤蚜的实时荧光PCR方法。并对该方法进行敏感性和重复性试验，结果显示，该方法的灵敏度可达1.625 copies/ul。3次重复检测的变异系数均小于5%。提取0.25g含有10头葡萄根瘤蚜蠕虫土壤的DNA，并将其梯度稀释，用建立的实时荧光定量PCR方法进行检测，结果表明，将DNA稀释10倍后，仍能检测出阳性结果，同时，对受害葡萄根际土壤检测结果也为阳性，葡萄根瘤蚜TaqMan-MGB探针实时荧光PCR检测技术具有特异性强、敏感度高、易操作等优点，有很好的应用前景和研究价值。

2.　按照杀虫活性、经济合理性、耐阴性、植株高度等筛选原则，选取裂叶荆芥、黄芩和黄芪三种植物作为防控葡萄根瘤蚜的间作植物，并且在果实成熟期对葡萄树势进行调查，在葡萄根系第二次生长高峰时和越冬时，对0～15cm土壤层的葡萄根瘤蚜种群数量进行调查。研究结果表明，间作裂叶荆芥、黄芩和黄芪三种植物对葡萄根瘤蚜种群的控制均有显著效果，其中间作荆芥在葡萄树势恢复方面也有较明显的作用。另外，在成熟期根系调查中发现裂叶黄芪有促进葡萄根系生长的作用，所以采用葡萄园间作植物的方法对防控葡萄根瘤蚜及树势的恢复均可达到较好的效果，可以作为未来葡萄根瘤蚜防治的研究方向。

关键词　葡萄根瘤蚜　实时荧光定量PCR　TaqMan-MGB　间作植物

Abstract

Grape phylloxera,daktulosphaira vitifoliae (Honoptera:phylloxeridae),originated from the east part of Rocky Mountains in North America ,is a kind of devastating grape insect. Found on vitis vine,grape phylloxera do harm to vitis grape only. Grape phylloxera,was found in 1935 in China,supposed to be introduced when importing grape seedings around 1900 and has been a quarantine pest since 1957 when it was identified to be it in1954,but vanished(could not to be found)though cut measures according to law or political reasons during 1965～1978,but it has been rediscovered again at suburb vineyards of Shanghai city in 2005.By grafting resistance rootstocks,the pest has been under control for more than 100 years in the word and there is no other equally effective method for substitution or rotating to avoid resistance. Even worse situation for grape industry in China,most of the grapes are cultivated by cottage,and resistance rootstocks are rarely used,so the risk of spreading of the pest poses a serious threat to the industry. Routine ground survey is still the main detecting method for grape phylloxera,but this method is time consuming and ineffective. In order to provide a quick,accurate and sensitive method to detect and explore grape phylloxera and population dynamics,a real-time PCR method had been studied,and a new method for control the pest,intercropping management ,was preliminarily taken for. The results were as follows.

1.　A real-time PCR method for detection of grape phylloxera,according to specific primers and TaqMan-MGB probe established by Karen Hebert et al,had been designed based on the internal transcribed space region 2(ITS2).Primers and probe could be used to detect grape phylloxera in vineyard through the experiment ,the recombinant plasmid containing the ITS2 sequence was constructed as a standard control,then an absolute quantitative method of FQ-PCR was developed grape phylloxera in vineyard through the experiment,the recombinant plasmid containing the ITS2 sequence was constructed as a standard control,then an absolute quantitative method of FQ-PCR was developed by optimizing reaction conditions,and sensitivity and reproducibility were carried out. The sensitivity of this method was proved to be 1,625 copies/ul；the coefficient of variation value was less than 5%. The DNA,that is distilling from 0.25g soil which contain 10 grape phylloxera crawlers,can be detected efficiently,even as few as diluted a thousand times,and rhizosphere soil was detected qualitatively. The TaqMan-MGB probe-based real-time fluorescent PCR assay served as a promising approach for easy,rapid and highly specific and sensitive detection of grape phylloxera.

2.　Under the selection criteria of having insecticidal activity,economic rationality,shade tolerance,and dearf plant but more roots,schizone peta tenuifolia (Benth.) Briq,Astragalusmenbtanaceus (Fisch) Bunge and Scutellaria baicalensis Georgi were chosen as intercrops to control grape phylloxera. Field trials had been taken for two years. Vine vigour was investigated in maturity stage,and the grape phylloxera population was investigated in 0～15cm soil layer both in the second peak of root growth stage and overwintering period. The results showed that intercropping of Schezone peta tenuifolia(Benth.) Briq had effect on tree vigor recovery. In addition,the maturation roots. In conclusion ,the intercropping could be an effective method for grape phylloxera controlling,and further research need to be taken to check and explore the mechanism for intercropping.

Key words　Grape phylloxera　FQ-PCR　TaqMan-MGB　Intercrop