黄瓜细菌性流胶病的病原鉴定、检测技术及应用

发布时间：2018-03-09 21:04

  本文选题：黄瓜细菌性流胶病　切入点：丁香假单胞流泪致病变种　出处：《中国农业大学》2017年博士论文　论文类型：学位论文

【摘要】：近十年来,我国北方多个省份发生了大面积的黄瓜细菌性流胶病,对当地的农民造成了严重的经济损失。本文对引起该病的病原菌进行了鉴定,建立了两套病原菌快速检测体系,并应用到土壤、水体和病残体中病原菌的检测,主要的研究结果如下:(1)明确了引起我国黄瓜细菌性流胶病的病原菌。对山东、山西、河南、河北、辽宁、北京等6个省市的96个温室进行的病害调查,采集黄瓜细菌性流胶病样本316份,通过分离、纯化和柯赫氏法则验证,共获得病原菌125株。经过形态学、生理生化鉴定和分子生物学鉴定,明确了引起该病的病原菌分别为丁香假单胞流泪致病变种(Pseudomonas syringae pv.lachrymans,Psl)和胡萝卜软腐果胶杆菌巴西亚种(Pectobacterium carotovorum subsp.brasiliense,Pcb),其中Pcd引起的黄瓜细菌性茎软腐病在中国属于首次报道,而Psl引起的黄瓜细菌性角斑病是导致黄瓜流胶现象的主要病害,Psl占据全部病原菌的66%。(2)建立了基于叠氮溴化丙锭(propidium monoazide,PMA)结合荧光定量PCR(PMA-qPCR)的Psl活细胞定量检测体系。通过处理条件的优化,选择终浓度为60 μmol/L的PMA进行预处理。对菌悬液中活体Psl的最低检测阈值为3.25×102 CFU/mL,而对于带菌种子中病原菌的检测阈值为9.5 CFU/g。对于田间采集的37个种子样品,有9个样品被检测出携带有病原菌,并且带菌量为103-104CFU/g。上述结果表明,PMA-qPCR是一种快速、可信的用于定量检测菌悬液以及带菌种子中病原菌活细胞的方法,对于提高该病害的防控措施具有潜在的应用价值。(3)建立了基于环介导等温扩增的Psl快速检测体系。基于hrpZ基因的保守序列设计了 6条特异性引物用于LAMP扩增,通过优化反应条件,LAMP扩增的最适温度设为67℃,LAMP结果通过钙黄绿素实现自然光下可视化检测。LAMP可以特异性的针对Psl进行扩增,而对非目的菌株的检测结果为阴性。灵敏性检测表明,LAMP对Psl菌悬液检测的最低浓度为104 CFU/mL。该方法还被用于检测受到感染的黄瓜叶片中,即使是发病初期的叶片也可以获得检测。此外,该方法适合于对田间的疑似受到Psl侵染的样品直接进行检测,不需要进行菌株的富集和DNA提取,因此具有在田间进行应用的能力。(4)应用PMA-qPCR研究了土壤、病残体以及水体中Psl的动态变化。Psl可以在病残体中长期存活,在保湿的土壤中以及保湿土埋藏的病残体内的Psl死亡率更高,并且可以在无营养的水体中长时间保持生物活性。因此,应当避免将病残体长期留存于温室内,造成病害的二次传播;土壤翻耕前进行适当灌水可以促使土壤中病残体腐烂,有利于消除土壤中的Psl;温室内应当尽量保持较低的湿度,以切断病原菌通过水进行传播的途径。
[Abstract]:In recent ten years, a large area of cucumber bacterial fluid gum disease has occurred in many provinces of northern China, which has caused serious economic losses to local farmers. The pathogenic bacteria causing the disease have been identified in this paper. Two sets of rapid detection systems of pathogenic bacteria were established and applied to the detection of pathogens in soil, water and diseased bodies. The main results are as follows: 1) the main results are as follows: (1) the pathogens causing bacterial fluid gum disease of cucumber in China are identified. The disease investigation was carried out in 96 greenhouses in 6 provinces and cities of Hebei, Liaoning and Beijing. 316 samples of cucumber bacterial fluid gum disease were collected. 125 strains of pathogenic bacteria were obtained by isolation, purification and verification of Koch's rule. Physiological and biochemical identification and molecular biological identification, The pathogens of the disease were Pseudomonas syringae pv.lachrymansspsls) and Pectobacterium carotovorum subsp. brasilienseus Pcbb, respectively. The bacterial stem rot caused by Pcd was reported for the first time in China. The bacterial keratoplakia of cucumber caused by Psl was the main disease causing cucumber gelatin phenomenon. Psl occupied 66% of all the pathogenic bacteria. A quantitative detection system of Psl living cells based on propidium monoazide PMAs and fluorescence quantitative PCRG PMA-qPCRs was established. Optimization of overprocessing conditions, PMA with final concentration of 60 渭 mol/L was selected for pretreatment. The minimum detection threshold for live Psl in bacterial suspension was 3.25 脳 10 ~ 2 CFU / mL, while for pathogen in bacterial seed was 9.5 CFU / g. For 37 seed samples collected in the field, the lowest detection threshold was 3.25 脳 10 ~ (2) CFU 路mL ~ (-1) 路mL ~ (-1). Nine samples were detected to carry pathogenic bacteria, and the amount of bacteria was 103-104 CFU / g. The results showed that PMA-qPCR was a rapid and reliable method for quantitative detection of live cells of pathogenic bacteria in bacterial suspension and seeds. The rapid detection system of Psl based on cyclization isothermal amplification was established. Six specific primers were designed for LAMP amplification based on the conserved sequence of hrpZ gene. By optimizing the reaction conditions, the optimum temperature for the amplification of lamp was 67 鈩，

本文编号：1590247