本文選題：黃瓜細(xì)菌性流膠病　切入點(diǎn)：丁香假單胞流淚致病變種　出處：《中國(guó)農(nóng)業(yè)大學(xué)》2017年博士論文　論文類型：學(xué)位論文

【摘要】：近十年來(lái),我國(guó)北方多個(gè)省份發(fā)生了大面積的黃瓜細(xì)菌性流膠病,對(duì)當(dāng)?shù)氐霓r(nóng)民造成了嚴(yán)重的經(jīng)濟(jì)損失。本文對(duì)引起該病的病原菌進(jìn)行了鑒定,建立了兩套病原菌快速檢測(cè)體系,并應(yīng)用到土壤、水體和病殘?bào)w中病原菌的檢測(cè),主要的研究結(jié)果如下:(1)明確了引起我國(guó)黃瓜細(xì)菌性流膠病的病原菌。對(duì)山東、山西、河南、河北、遼寧、北京等6個(gè)省市的96個(gè)溫室進(jìn)行的病害調(diào)查,采集黃瓜細(xì)菌性流膠病樣本316份,通過(guò)分離、純化和柯赫氏法則驗(yàn)證,共獲得病原菌125株。經(jīng)過(guò)形態(tài)學(xué)、生理生化鑒定和分子生物學(xué)鑒定,明確了引起該病的病原菌分別為丁香假單胞流淚致病變種(Pseudomonas syringae pv.lachrymans,Psl)和胡蘿卜軟腐果膠桿菌巴西亞種(Pectobacterium carotovorum subsp.brasiliense,Pcb),其中Pcd引起的黃瓜細(xì)菌性莖軟腐病在中國(guó)屬于首次報(bào)道,而Psl引起的黃瓜細(xì)菌性角斑病是導(dǎo)致黃瓜流膠現(xiàn)象的主要病害,Psl占據(jù)全部病原菌的66%。(2)建立了基于疊氮溴化丙錠(propidium monoazide,PMA)結(jié)合熒光定量PCR(PMA-qPCR)的Psl活細(xì)胞定量檢測(cè)體系。通過(guò)處理?xiàng)l件的優(yōu)化,選擇終濃度為60 μmol/L的PMA進(jìn)行預(yù)處理。對(duì)菌懸液中活體Psl的最低檢測(cè)閾值為3.25×102 CFU/mL,而對(duì)于帶菌種子中病原菌的檢測(cè)閾值為9.5 CFU/g。對(duì)于田間采集的37個(gè)種子樣品,有9個(gè)樣品被檢測(cè)出攜帶有病原菌,并且?guī)Ь繛?03-104CFU/g。上述結(jié)果表明,PMA-qPCR是一種快速、可信的用于定量檢測(cè)菌懸液以及帶菌種子中病原菌活細(xì)胞的方法,對(duì)于提高該病害的防控措施具有潛在的應(yīng)用價(jià)值。(3)建立了基于環(huán)介導(dǎo)等溫?cái)U(kuò)增的Psl快速檢測(cè)體系�；趆rpZ基因的保守序列設(shè)計(jì)了 6條特異性引物用于LAMP擴(kuò)增,通過(guò)優(yōu)化反應(yīng)條件,LAMP擴(kuò)增的最適溫度設(shè)為67℃,LAMP結(jié)果通過(guò)鈣黃綠素實(shí)現(xiàn)自然光下可視化檢測(cè)。LAMP可以特異性的針對(duì)Psl進(jìn)行擴(kuò)增,而對(duì)非目的菌株的檢測(cè)結(jié)果為陰性。靈敏性檢測(cè)表明,LAMP對(duì)Psl菌懸液檢測(cè)的最低濃度為104 CFU/mL。該方法還被用于檢測(cè)受到感染的黃瓜葉片中,即使是發(fā)病初期的葉片也可以獲得檢測(cè)。此外,該方法適合于對(duì)田間的疑似受到Psl侵染的樣品直接進(jìn)行檢測(cè),不需要進(jìn)行菌株的富集和DNA提取,因此具有在田間進(jìn)行應(yīng)用的能力。(4)應(yīng)用PMA-qPCR研究了土壤、病殘?bào)w以及水體中Psl的動(dòng)態(tài)變化。Psl可以在病殘?bào)w中長(zhǎng)期存活,在保濕的土壤中以及保濕土埋藏的病殘?bào)w內(nèi)的Psl死亡率更高,并且可以在無(wú)營(yíng)養(yǎng)的水體中長(zhǎng)時(shí)間保持生物活性。因此,應(yīng)當(dāng)避免將病殘?bào)w長(zhǎng)期留存于溫室內(nèi),造成病害的二次傳播;土壤翻耕前進(jìn)行適當(dāng)灌水可以促使土壤中病殘?bào)w腐爛,有利于消除土壤中的Psl;溫室內(nèi)應(yīng)當(dāng)盡量保持較低的濕度,以切斷病原菌通過(guò)水進(jìn)行傳播的途徑。
[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，

本文編號(hào)：1590247