葡萄根瘤蚜的熒光定量PCR檢測(cè)及間種植物防控研究

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

作者：郭　慶　　導(dǎo)師：王忠躍

中國(guó)農(nóng)業(yè)科學(xué)院　　昆蟲(chóng)生理及分子生物學(xué)2011屆碩士

摘　要

葡萄根瘤蚜Daktulosphaira vitifoliae(Fitch),屬于同翅目（Homoptera），根瘤蚜科（Phylloxeridae）,原產(chǎn)于北美洲落基山脈東部，是葡萄上的毀滅性害蟲(chóng)，為專食性害蟲(chóng)，只危害vitis屬植物，是我國(guó)植物檢疫性有害生物。1935年在我國(guó)首次發(fā)現(xiàn)葡萄根瘤蚜后，通過(guò)采取砍伐葡萄，使葡萄根瘤蚜銷聲匿跡。2005年在我國(guó)再次發(fā)現(xiàn)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，沒(méi)有其他有效防治方法。由于我國(guó)葡萄大多以扦插方式栽培，嫁接抗性砧木面積較少，葡萄根瘤蚜對(duì)我國(guó)迅速發(fā)展的葡萄產(chǎn)業(yè)構(gòu)成嚴(yán)重威脅。同時(shí)，目前主要依靠常規(guī)土壤及根系調(diào)查葡萄根瘤蚜，但常規(guī)地面方法費(fèi)時(shí)費(fèi)力。因此，為了給葡萄根瘤蚜監(jiān)測(cè)提供快速、準(zhǔn)確、敏感性高的分子生物學(xué)調(diào)查方法，以及為葡萄根瘤蚜防控探索有效的方法，本試驗(yàn)對(duì)實(shí)時(shí)定量PCR方法檢測(cè)葡萄根瘤蚜，以及在葡萄園間作植物防控葡萄根瘤蚜進(jìn)行了研究。主要結(jié)果總結(jié)如下：

1.　參考Karen Herbert等的方法，根據(jù)葡萄根瘤蚜ITS2中的保守序列設(shè)計(jì)的特異性引物與TaqMan-MGB熒光探針，通過(guò)試驗(yàn)驗(yàn)證上述引物和探針適用于我國(guó)葡萄根瘤蚜的檢測(cè)，并且成功構(gòu)建了標(biāo)準(zhǔn)質(zhì)粒，制作出以標(biāo)準(zhǔn)陽(yáng)性質(zhì)粒作為標(biāo)準(zhǔn)品的標(biāo)準(zhǔn)曲線，經(jīng)優(yōu)化反應(yīng)條件，建立了一種絕對(duì)定量檢測(cè)葡萄根瘤蚜的實(shí)時(shí)熒光PCR方法。并對(duì)該方法進(jìn)行敏感性和重復(fù)性試驗(yàn)，結(jié)果顯示，該方法的靈敏度可達(dá)1.625 copies/ul。3次重復(fù)檢測(cè)的變異系數(shù)均小于5%。提取0.25g含有10頭葡萄根瘤蚜蠕蟲(chóng)土壤的DNA，并將其梯度稀釋，用建立的實(shí)時(shí)熒光定量PCR方法進(jìn)行檢測(cè)，結(jié)果表明，將DNA稀釋10倍后，仍能檢測(cè)出陽(yáng)性結(jié)果，同時(shí)，對(duì)受害葡萄根際土壤檢測(cè)結(jié)果也為陽(yáng)性，葡萄根瘤蚜TaqMan-MGB探針實(shí)時(shí)熒光PCR檢測(cè)技術(shù)具有特異性強(qiáng)、敏感度高、易操作等優(yōu)點(diǎn)，有很好的應(yīng)用前景和研究?jī)r(jià)值。

2.　按照殺蟲(chóng)活性、經(jīng)濟(jì)合理性、耐陰性、植株高度等篩選原則，選取裂葉荊芥、黃芩和黃芪三種植物作為防控葡萄根瘤蚜的間作植物，并且在果實(shí)成熟期對(duì)葡萄樹(shù)勢(shì)進(jìn)行調(diào)查，在葡萄根系第二次生長(zhǎng)高峰時(shí)和越冬時(shí)，對(duì)0～15cm土壤層的葡萄根瘤蚜種群數(shù)量進(jìn)行調(diào)查。研究結(jié)果表明，間作裂葉荊芥、黃芩和黃芪三種植物對(duì)葡萄根瘤蚜種群的控制均有顯著效果，其中間作荊芥在葡萄樹(shù)勢(shì)恢復(fù)方面也有較明顯的作用。另外，在成熟期根系調(diào)查中發(fā)現(xiàn)裂葉黃芪有促進(jìn)葡萄根系生長(zhǎng)的作用，所以采用葡萄園間作植物的方法對(duì)防控葡萄根瘤蚜及樹(shù)勢(shì)的恢復(fù)均可達(dá)到較好的效果，可以作為未來(lái)葡萄根瘤蚜防治的研究方向。

關(guān)鍵詞　葡萄根瘤蚜　實(shí)時(shí)熒光定量PCR　TaqMan-MGB　間作植物

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