青枯菌LAMP检测方法的建立及植物精油抑菌活性评价

发布时间：2018-06-22 01:25

本文选题：青枯菌 + 演化型I型桑菌株　；参考：《西南大学》2017年硕士论文

【摘要】：植物细菌性青枯病(bacterial wilt of plants)是由茄科雷尔氏菌(Ralstonia solanacearum,简称青枯菌)引起的一种世界性重大病害。该病广泛分布于全球的热带、亚热带和温带地区,并逐渐向高纬度、高海拔冷凉地区扩散蔓延。青枯菌寄主范围广泛,可侵染包括马铃薯、香蕉、烟草、番茄、生姜和桑树等重要粮食与经济作物在内的54个科的450余种植物,于世界范围内造成巨大的经济损失。由演化型I型桑菌株引起的桑青枯病(bacterial wilt of mulberry)是对桑蚕生产具有毁灭性危害的主要桑树病害之一。鉴于其重要的经济与学术研究价值,2012年青枯菌被《Molecular Plant Pathology》杂志列为10大植物病原细菌之一。国内外围绕青枯菌基因组学、系统进化、致病机理、检测方法以及防控技术等诸多领域展开了深入研究,相继有GMI1000、Po82、PS107、UY031等13个青枯菌株完成了全基因组序列的测定,CMR15、FQY_4、B50等54个青枯菌株完成了基因组草图序列的测定。但是由于青枯菌群体复杂,具有广泛的生态适应性,栽培寄主抗病种质资源缺乏,尚无环保低毒、经济有效的化学杀菌剂,致使青枯病的防控成为难以突破的世界性瓶颈问题。建立精准、灵敏、高效的青枯菌种及种以下菌株水平上的分子检测技术,是有效阻断青枯病传播扩散,实现田间早期诊断并建立科学防控策略的基石;发掘植物源抑菌活性物质,研发新型生物农药不仅可为青枯病防控提供新的思路与技术储备,也有助于解决公众日益关切的食品安全、生态安全以及公共卫生安全问题。据此,本研究旨在:(1)建立青枯菌种及菌株分类单元水平上的特异性LAMP分子检测方法;(2)筛选对青枯菌具有抑制活性的植物精油,并评价抑菌效果,以期探寻新的青枯病防治方法。1.基于青枯菌特异性序列lpxC基因设计4条引物,建立青枯菌的LAMP快速检测方法,在1 h内获得可视化检测结果。通过单因素变化试验优化反应体系,确定反应体系中镁离子浓度为6 mmol/L,内外引物浓度比为8㑳1(1.6㑳0.2μmol/L),反应温度为63℃。在特异性测定试验中,24个青枯菌菌株反应液观察到荧光绿色的阳性反应,而对照菌株及阴性对照的反应液均保持橙色不变,表明该方法可特异性检测青枯菌。在灵敏度测定试验中,LAMP方法的检测灵敏度为1.42 pg,较常规PCR提高10倍。对人工接种和田间罹病样品进行检测,结果显示LAMP检测方法不受植物组织浸出液的干扰,可用于病株样品的检测。该方法特异性强、灵敏度高、操作简单、检测结果可用肉眼直接观察,为田间快速检测青枯菌提供新的方法。2.基于青枯菌演化型I型桑菌株的特异性核苷酸片段MG67设计了6条引物,建立了演化型I型桑菌株的LAMP快速分子检测方法,在20 min内获得可视化检测结果。通过ABI 7500Real Time PCR实时观测反应过程,确定最优反应温度为64℃,反应时间为20 min。在特异性测定试验中,23个青枯菌演化型I型桑菌株反应液观察到荧光绿色的阳性反应,23个非演化型I型桑菌株的青枯菌、7个对照菌株及阴性对照的反应液均保持橙色不变,表明该方法可特异性检测青枯菌演化型I型桑菌株。在灵敏度测定试验中,LAMP对基因组DNA的最低检测浓度为160 fg,较常规PCR提高10倍;LAMP对菌悬液的检测灵敏度为2.2×102 CFU/mL,且不受桑组织提取液的干扰,较常规PCR提高100倍。对田间收集桑植株样品进行检测,表明该方法可用于田间病株样品的检测,可为田间青枯菌演化型I型桑菌株的快速检测提供新的方法。3.采用抑菌圈及MIC值和MBC值的测定评价了4种植物精油对青枯菌的离体抑菌效果。抑菌圈测定结果显示,薄荷精油、草果精油、薰衣草精油和山苍子油对青枯菌Po82菌株均具有抑菌效果,其中山苍子油可完全抑菌;最小抑菌浓度MIC和最小杀菌浓度MBC测定结果显示,薄荷精油的MIC值和MBC值均为4μL/mL,草果精油的MIC值和MBC值分别为4μL/mL和8μL/mL,薰衣草精油的MIC值和MBC值均为4μL/mL,山苍子精油的MIC值和MBC值均为1μL/mL,山苍子油的抑菌效果最好。4种精油对青枯菌作用方式多样,精油挥发性气体抑菌效果测定结果显示,薄荷精油、草果精油、薰衣草精油和山苍子油的抑菌圈分别为7.80±0.037 mm、8.50±0.057 mm、13.10±0.122 mm和19.00±0.181 mm,对青枯菌Po82菌株均具有抑制作用。4.利用气相色谱-质谱联用技术初步鉴定出了所用山苍子油中的主要化学成分,并通过对含山苍子油的培养基中青枯菌生长情况及山苍子油熏蒸效果的测定,评价了山苍子油对青枯菌的抑菌效果及作用方式。气相色谱-质谱联用技术鉴定结果表明,所用山苍子油中有不同化学成分29种,其中烯烃类化合物是主要成分,约占总量的40%。生长曲线测定结果显示,不同浓度的山苍子油处理对青枯菌的生长均具有不同程度的抑制作用;随着精油浓度的升高,抑制作用增强;浓度为1.6μL/mL的山苍子油处理后,青枯菌的生长受到完全抑制。利用不同体积的山苍子油对200 mL三角瓶中的带菌培养基进行熏蒸处理,山苍子油的熏蒸浓度分别为1、0.75、0.5、0.25、0.125和0.0625μL/mL,培养40 h后A600值分别为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,与空白对照差异极显著,表明山苍子油通过熏蒸对青枯菌表现出明显的抑制作用。带菌土壤通过山苍子油熏蒸处理3 d后,可有效降低青枯病的发病指数,山苍子油浓度分别为0.10-0.50μL/mL时,移栽番茄苗8 d后病情指数降低36-78%。表明山苍子油可作为一种熏蒸剂,用于土壤中青枯菌的熏蒸处理。
[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.
【学位授予单位】：西南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S432.4

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