發(fā)布時間：2018-06-22 01:25

  本文選題：青枯菌 + 演化型I型桑菌株　； 參考：《西南大學(xué)》2017年碩士論文

【摘要】：植物細菌性青枯病(bacterial wilt of plants)是由茄科雷爾氏菌(Ralstonia solanacearum,簡稱青枯菌)引起的一種世界性重大病害。該病廣泛分布于全球的熱帶、亞熱帶和溫帶地區(qū),并逐漸向高緯度、高海拔冷涼地區(qū)擴散蔓延。青枯菌寄主范圍廣泛,可侵染包括馬鈴薯、香蕉、煙草、番茄、生姜和桑樹等重要糧食與經(jīng)濟作物在內(nèi)的54個科的450余種植物,于世界范圍內(nèi)造成巨大的經(jīng)濟損失。由演化型I型桑菌株引起的桑青枯病(bacterial wilt of mulberry)是對桑蠶生產(chǎn)具有毀滅性危害的主要桑樹病害之一。鑒于其重要的經(jīng)濟與學(xué)術(shù)研究價值,2012年青枯菌被《Molecular Plant Pathology》雜志列為10大植物病原細菌之一。國內(nèi)外圍繞青枯菌基因組學(xué)、系統(tǒng)進化、致病機理、檢測方法以及防控技術(shù)等諸多領(lǐng)域展開了深入研究,相繼有GMI1000、Po82、PS107、UY031等13個青枯菌株完成了全基因組序列的測定,CMR15、FQY_4、B50等54個青枯菌株完成了基因組草圖序列的測定。但是由于青枯菌群體復(fù)雜,具有廣泛的生態(tài)適應(yīng)性,栽培寄主抗病種質(zhì)資源缺乏,尚無環(huán)保低毒、經(jīng)濟有效的化學(xué)殺菌劑,致使青枯病的防控成為難以突破的世界性瓶頸問題。建立精準(zhǔn)、靈敏、高效的青枯菌種及種以下菌株水平上的分子檢測技術(shù),是有效阻斷青枯病傳播擴散,實現(xiàn)田間早期診斷并建立科學(xué)防控策略的基石;發(fā)掘植物源抑菌活性物質(zhì),研發(fā)新型生物農(nóng)藥不僅可為青枯病防控提供新的思路與技術(shù)儲備,也有助于解決公眾日益關(guān)切的食品安全、生態(tài)安全以及公共衛(wèi)生安全問題。據(jù)此,本研究旨在:(1)建立青枯菌種及菌株分類單元水平上的特異性LAMP分子檢測方法;(2)篩選對青枯菌具有抑制活性的植物精油,并評價抑菌效果,以期探尋新的青枯病防治方法。1.基于青枯菌特異性序列l(wèi)pxC基因設(shè)計4條引物,建立青枯菌的LAMP快速檢測方法,在1 h內(nèi)獲得可視化檢測結(jié)果。通過單因素變化試驗優(yōu)化反應(yīng)體系,確定反應(yīng)體系中鎂離子濃度為6 mmol/L,內(nèi)外引物濃度比為8㑳1(1.6㑳0.2μmol/L),反應(yīng)溫度為63℃。在特異性測定試驗中,24個青枯菌菌株反應(yīng)液觀察到熒光綠色的陽性反應(yīng),而對照菌株及陰性對照的反應(yīng)液均保持橙色不變,表明該方法可特異性檢測青枯菌。在靈敏度測定試驗中,LAMP方法的檢測靈敏度為1.42 pg,較常規(guī)PCR提高10倍。對人工接種和田間罹病樣品進行檢測,結(jié)果顯示LAMP檢測方法不受植物組織浸出液的干擾,可用于病株樣品的檢測。該方法特異性強、靈敏度高、操作簡單、檢測結(jié)果可用肉眼直接觀察,為田間快速檢測青枯菌提供新的方法。2.基于青枯菌演化型I型桑菌株的特異性核苷酸片段MG67設(shè)計了6條引物,建立了演化型I型桑菌株的LAMP快速分子檢測方法,在20 min內(nèi)獲得可視化檢測結(jié)果。通過ABI 7500Real Time PCR實時觀測反應(yīng)過程,確定最優(yōu)反應(yīng)溫度為64℃,反應(yīng)時間為20 min。在特異性測定試驗中,23個青枯菌演化型I型桑菌株反應(yīng)液觀察到熒光綠色的陽性反應(yīng),23個非演化型I型桑菌株的青枯菌、7個對照菌株及陰性對照的反應(yīng)液均保持橙色不變,表明該方法可特異性檢測青枯菌演化型I型桑菌株。在靈敏度測定試驗中,LAMP對基因組DNA的最低檢測濃度為160 fg,較常規(guī)PCR提高10倍;LAMP對菌懸液的檢測靈敏度為2.2×102 CFU/mL,且不受桑組織提取液的干擾,較常規(guī)PCR提高100倍。對田間收集桑植株樣品進行檢測,表明該方法可用于田間病株樣品的檢測,可為田間青枯菌演化型I型桑菌株的快速檢測提供新的方法。3.采用抑菌圈及MIC值和MBC值的測定評價了4種植物精油對青枯菌的離體抑菌效果。抑菌圈測定結(jié)果顯示,薄荷精油、草果精油、薰衣草精油和山蒼子油對青枯菌Po82菌株均具有抑菌效果,其中山蒼子油可完全抑菌;最小抑菌濃度MIC和最小殺菌濃度MBC測定結(jié)果顯示,薄荷精油的MIC值和MBC值均為4μL/mL,草果精油的MIC值和MBC值分別為4μL/mL和8μL/mL,薰衣草精油的MIC值和MBC值均為4μL/mL,山蒼子精油的MIC值和MBC值均為1μL/mL,山蒼子油的抑菌效果最好。4種精油對青枯菌作用方式多樣,精油揮發(fā)性氣體抑菌效果測定結(jié)果顯示,薄荷精油、草果精油、薰衣草精油和山蒼子油的抑菌圈分別為7.80±0.037 mm、8.50±0.057 mm、13.10±0.122 mm和19.00±0.181 mm,對青枯菌Po82菌株均具有抑制作用。4.利用氣相色譜-質(zhì)譜聯(lián)用技術(shù)初步鑒定出了所用山蒼子油中的主要化學(xué)成分,并通過對含山蒼子油的培養(yǎng)基中青枯菌生長情況及山蒼子油熏蒸效果的測定,評價了山蒼子油對青枯菌的抑菌效果及作用方式。氣相色譜-質(zhì)譜聯(lián)用技術(shù)鑒定結(jié)果表明,所用山蒼子油中有不同化學(xué)成分29種,其中烯烴類化合物是主要成分,約占總量的40%。生長曲線測定結(jié)果顯示,不同濃度的山蒼子油處理對青枯菌的生長均具有不同程度的抑制作用;隨著精油濃度的升高,抑制作用增強;濃度為1.6μL/mL的山蒼子油處理后,青枯菌的生長受到完全抑制。利用不同體積的山蒼子油對200 mL三角瓶中的帶菌培養(yǎng)基進行熏蒸處理,山蒼子油的熏蒸濃度分別為1、0.75、0.5、0.25、0.125和0.0625μL/mL,培養(yǎng)40 h后A600值分別為0.0332±0.0069、0.0540±0.0183、0.0619±0.0069、0.0981±0.0746、1.3744±0.0164和1.7317±0.0466,與空白對照差異極顯著,表明山蒼子油通過熏蒸對青枯菌表現(xiàn)出明顯的抑制作用。帶菌土壤通過山蒼子油熏蒸處理3 d后,可有效降低青枯病的發(fā)病指數(shù),山蒼子油濃度分別為0.10-0.50μL/mL時,移栽番茄苗8 d后病情指數(shù)降低36-78%。表明山蒼子油可作為一種熏蒸劑,用于土壤中青枯菌的熏蒸處理。
[Abstract]:Bacterial wilt disease (bacterial wilt of plants) is a major worldwide disease caused by Ralstonia solanacearum (Ralstonia solanacearum for short). The disease is widely distributed in the tropical, subtropical and temperate regions of the world and gradually spread to high latitudes and high altitude cold regions. It can infect more than 450 species of 54 families, including potatoes, bananas, tobacco, tomatoes, ginger and mulberry trees and other important grain and economic crops, which cause huge economic losses worldwide. The mulberry blight (bacterial wilt of mulberry) caused by the evolutionary I type mulberry strain is the main mulberry which has devastating harm to the production of silkworm. One of the tree diseases. In view of its important economic and academic research value, 2012 young blight bacteria have been listed as one of the top 10 plant pathogens in magazine. The research on genomics, phylogenetic evolution, pathogenic mechanism, detection methods and prevention and control techniques of Rhizoctonia blight has been studied in many fields, including GMI1000, Po. 82, PS107, UY031 and other 13 Blight Strains completed the whole genome sequence determination, CMR15, FQY_4, B50 and other 54 Blight Strains completed the genome sequence analysis. But because of the complex population of the bacterial blight, it has extensive ecological adaptability, the germplasm resources of culture host are lack, and there is no environmental low toxicity and economic and effective chemical fungicide. The prevention and control of bacterial wilt has become a world bottleneck problem that is difficult to break through. The establishment of a precise, sensitive and efficient molecular detection technique on the level of bacterial and bacterial strains is the cornerstone of effectively blocking the spread of bacterial wilt, realizing early diagnosis in the field and establishing a scientific prevention and control strategy. The biological pesticides can not only provide new ideas and technical reserves for the prevention and control of bacterial wilt disease, but also help to solve the public concern of food safety, ecological security and public health safety. Based on this, this study aims at: (1) the specific LAMP molecular detection methods on the level of the Jian Liqing bacterial species and the strain classification unit; (2) screening for the blight of green blight. The bacteria have the inhibitory activity of plant essential oil and evaluate the effect of bacteriostasis in order to explore the new method of prevention and control of bacterial wilt disease,.1., based on the design of 4 primers, based on the lpxC gene of the specific sequence of the bacterial blight, the rapid detection method for the LAMP of the bacterial blight bacteria was established, and the visual detection results were obtained in the 1 h. The concentration of magnesium ion in the system was 6 mmol/L, the concentration ratio of the internal and external primers was 8? 1 (1.6? 0.2 mu mol/L) and the reaction temperature was 63. In the specific test, the positive reaction of the fluorescent green was observed in the reaction solution of 24 bacterial strains, while the control strain and the negative control reacted to the orange color, indicating that the method can be specifically tested for the bacterial blight. In the sensitivity test, the sensitivity of the LAMP method is 1.42 PG, which is 10 times higher than that of the conventional PCR. The results show that the LAMP detection method is not disturbed by the plant tissue leaching solution, and can be used for the detection of the samples of the plant. The method is specific, sensitive, simple and easy to operate, and the results can be detected. Direct observation by the naked eye to provide a new method for rapid detection of the bacterial wilt bacteria in the field, 6 primers were designed based on the specific nucleotide fragment MG67 of the evolutionary I mulberry strain of the bacterial blight bacteria. A rapid LAMP molecular detection method for the evolutionary I mulberry strain was established, and the visual detection results were obtained in the 20 min. ABI 7500Real Time PCR was used in real time. The optimal reaction temperature was determined to be 64 C and the reaction time was 20 min. in the specific test. The positive reaction was observed in the reaction solution of the 23 strains of the I type mulberry strain of the bacterial wilt bacteria. The 23 non evolutionary I mulberry strains, the 7 control strains and the negative control reacted to the orange. This method can be used to detect the evolution type I mulberry strain of bacterial blight. In sensitivity test, the minimum detection concentration of LAMP to genomic DNA is 160 FG, which is 10 times higher than that of conventional PCR; the sensitivity of LAMP to bacterial suspension is 2.2 x 102 CFU/mL, and is not affected by the dry disturbance of Mulberry Tissue Extract, and is 100 times higher than that of conventional PCR. In the field, Sangzhi is collected. The test results showed that the method could be used to detect the samples of field plant strains, which could provide a new method for the rapid detection of I type mulberry strain in the field. The bacteriostatic ring and the MIC value and the MBC value were used to evaluate the bacteriostasis effect of 4 kinds of plant essential oils. The results of bacteriostasis test showed that the peppermint essential oil and grass were found. Fruit essential oil, lavender essential oil and Litsea cubeba oil have bacteriostasis effect on Po82 strains of Rhizoctonia blight, and the Zhongshan Xanthium oil can be completely bacteriostasis. The minimum inhibitory concentration MIC and minimum bactericidal concentration MBC results show that the MIC value and MBC value of the mint essential oil are 4 u L/mL, the MIC value and MBC value of the fruit essential oil are 4 u L/mL and 8 micron L/mL, lavender essential oil, respectively. The MIC value and MBC value are 4 L/mL, the MIC value and MBC value of Litsea cubeba essential oil are 1 L/mL. The bacteriostasis effect of Litsea oil is better than that of.4 seed oil, and the bacteriostatic effect of volatile oil in essential oil, volatile oil, lavender essential oil and Litsea cubeba oil are 7.80 + 0.037 mm, 8, respectively. .50 + 0.057 mm, 13.10 + 0.122 mm and 19 + 0.181 mm, which had inhibitory effect on all Po82 strains of bacterial blight bacteria, and identified the main chemical components in the oil of Litsea cubeba by gas chromatography-mass spectrometry, and evaluated the growth and fumigation effect of Litsea cubeba oil in the medium of Rhizoctonia Xanthium oil. The bacteriostasis effect and action mode of Litsea cubeba oil to Rhizoctonia blight. The results of gas chromatography-mass spectrometry identification showed that there were 29 different chemical components in Litsea cubeba oil, of which olefin compounds were the main components. The results of 40%. growth curve of the total amount showed that different concentrations of Litsea oil were treated for the growth of bacterial blight. With the increase of essential oil concentration, the inhibitory effect increased with the increase of essential oil concentration. The growth of Rhizoctonia blight was completely suppressed after the treatment of Litsea oil with a concentration of 1.6 L/mL. The fumigation concentration of Litsea cubeba oil in 200 mL trigonometric bottles was fumigated with different volumes of Litsea oil, and the fumigation concentration of Litsea cubeba oil was 1,0.7, respectively 5,0.5,0.25,0.125 and 0.0625 mu L/mL, after culture 40 h, A600 values were 0.0332 + 0.0069,0.0540 + 0.0183,0.0619 + 0.0746,1.3744 + 0.0164 and 1.7317 + 0.0466 respectively. The difference was very significant with the blank control. After 3 D, the disease index of bacterial wilt could be effectively reduced. When the concentration of Litsea cubeba oil was 0.10-0.50 L/mL, the disease index of transplanted tomato seedlings decreased after 8 D, and the 36-78%. table of xanshan oil could be used as a fumigant to fumigate the Rhizoctonia blight in soil.
【學(xué)位授予單位】：西南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S432.4

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