【作者】 金伟；

【导师】 王相晶；

【作者基本信息】 东北农业大学 ， 生物化学与分子生物学， 2012， 硕士

【摘要】 甜瓜细菌性果斑病(Acidovorax avenae subsp.citrulli)和火鹤细菌性叶枯病(Xanthomonas campestris pv.dieffenbachiae)是世界性分布的毁灭病害,给瓜类产业和火鹤产业带来了巨大的经济损失。本研究主要从病害的传播途径和检测技术方面进行了深入研究,以期为病害的综合防治提供一定的参考和依据。1.明确了带菌种子、花粉管通道、带菌土壤及迸溅的水滴是甜瓜细菌性果斑病的传播途径。采用浸种法、点滴接种法、拌土法及水滴进溅法接种细菌性果斑病菌于健康的甜瓜种子、雌花柱头、土壤及幼苗上,对细菌性果斑病的传播方式进行了研究。结果表明,浸种及点滴接种雌花柱头获得的带菌种子均可传病；菌土中的幼苗在出苗后13d就有病症出现,且随着生长期的延长,病情逐渐加重,发病最重幼苗的病叶率和病指分别为69.13%和25.72；病原菌随进溅的水滴传播时,离菌源越近的幼苗发病情况越为严重,病叶率和病指分别为63.34%和47.12。2.明确了带菌土壤及迸溅的水滴是火鹤细菌性叶枯病的传播途径。采用拌土法、水滴迸溅法接种细菌性叶枯病菌于健康的火鹤植株上,对细菌性叶枯病的传播方式进行了研究。结果表明,菌土中的火鹤植株在移栽后14d有发病症状出现,后随着移栽天数的增加,病情逐渐加重,发病最重幼苗的病叶率和病指分别为84.74%和72.31；病原菌随进溅的水滴传播时,离菌源越近发病越为严重,病叶率和病指分别为75.00%和55.54。3.建立了选择性培养基和Nested-PCR方法检测甜瓜带菌种子。采用选择性培养基(ASCM)对甜瓜带菌种子上果斑菌进行分离及富集,引用特异性引物,对ASCM培养液进行Nested-PCR扩增。结果表明,选择性培养基(ASCM),可特异性对甜瓜带菌种子上的果斑病菌进行分离、培养和富集；引物对SEQ10/SEQ12、SEQ11/SEQ12和Nested-PCR引物只在细菌性果斑病菌中分别扩增出262bp、241bp和241bp的特异片段,在其他菌种中无特异性扩增片段。进行常规PCR扩增,检测细菌性果斑病菌的灵敏度为1.92x10-4mg/L和1.92x10-3mg/L,而Nested-PCR的灵敏度可达到1.92×10-6mg/L。Nested-PCR方法检测ASCM培养液的最低浓度为2×105cfu/ml。4.建立了Nested-PCR方法检测火鹤细菌性叶枯病的病株。引用特异性引物,对土传试验中火鹤发病植株的根、茎、叶进行Nested-PCR扩增。结果表明,引物对PXadU/PXadL. NXadU/NXadL和Nested-PCR引物只在细菌性叶枯病菌中分别扩增出1570bp、785bp和785bp的特异片段,在健康植株和其他菌种中均无特异性扩增片段。用两对引物进行常规PCR扩增,检测叶枯菌的灵敏度为1.23×10-5mg/L和1.23×10-3mg/L,而Nested-PCR的灵敏度可达到1,23×10-7mg/L。Nested-PCR方法对发病植株的检测灵敏度可达到病情指数高于16.6的样品。本研究首次明确了甜瓜细菌性果斑病和火鹤细菌性叶枯病的传播途径；建立了选择性培养基及Nested-PCR方法检测甜瓜带菌种子上的果斑病菌；建立了Nested-PCR方法检测火鹤病株中的叶枯病菌。对甜瓜细菌性果斑病和火鹤细菌性叶枯病的防控具有重要的意义。更多还原

【Abstract】 Melon Bacterial Fruit Blotch (Acidovorax avenae subsp.citrulli) and Anthurium Bacterial Leaf Blight (Xanthomonas axonopodis pv.dieffenbachiae) are worldwide destructive disease that cause significant economic losses for melons industry and anthurium industry. In order to provide a basis for integrated control,the channel of disease spread,and detection technologies were studied in this paper.1.It has been clarified that bacteria-contaminated seeds and soil, pollen tube and splashing water drops are the major source of bacterial fruit blotch. Seed soaking、drip inoculation、mixing bacterial fruit blotch pathogens with soil and splashing water drops were used to inoculated bacterial fruit blotch pathogen in healthy melon seeds, stigma, soil and seedlings, the transmission of bacterial fruit blotch was studied.The results indicated that bacterial fruit blotch can transmison by seed soaking and pollen tube;Melon seedlings planted in contaminated soil demonstrated symptoms after emergence13d,With the growth of seedlings, the situation would get worse, the morbidity and disease index of the worst seedlings were69.13%and25.72;bacterial fruit blotch pathogens can spread by water splashing, the closer the more serious, the morbidity and disease index were63.34%and47.12.2.We are clear about infected soil, and splashing water drops are the main transmissions of bacterial leaf blight. Inoculate bacterial leaf blight pathogens on sterilized soil and splashing water drops were used to study the transmission of bacterial leaf blight.The results showed that Anthurium plant in contaminated soil displayed symptoms after transplanted14d, with the increase of transplant days, the incidence would getting more serious, the morbidity and disease index of the worst seedlings were84.74%and72.31;bacterial leaf blight pathogens can spread by water splashing, the more recent onset,the worse, the morbidity and disease index were75.00%and55.54. 3.We have created selective media and nested-PCR to identify seeds infected by bacterial fruit blotch pathogens. Selective medium (ASCM) was used to separation and detection pathogens on the contaminated seed, two primer pairs were referenced to amplify bacterial fruit blotch pathogen from ASCM liquid by nested-PCR. The results indicated that selective medium can specificity separation, training and enrichment bacterial fruit blotch pathogen; SEQ10/SEQ12, SEQ11/SEQ12and nested-PCR could amolify262bp,241bp and241bp fragment only from Acidovorax avenae. In conventional PCR,the sensitivity of detection was1.92×10-4mg/L and1.92×10-3mg/L for both primes,in the nested-PCR the detection limit was1.92×10-6mg/L. The lowest ASCM liquid concentration that can be identified by nested-PCR was2×105cfu/ml.4. A nested-PCR detection technology was established to identify plants infected by bacterial leaf blight. Two primer pairs were referenced to amplify bacterial leaf blotch pathogen from the plant roots, stems, leaves by nested-PCR. The results showed that PXadU/PXadL,NXadU/NXadL and nested-PCR could amolify1570bp,785bp and785bp fragment only from Xanthomonas axonopodis, respectively, no amplification product was observed from healthy plants. In conventional PCR,the sensitivity of detection was1.23×10-5mg/L and1.23×10-3mg/L for both primes, in the nested-PCR the detection limit was1.23×10-1mg/L.The sensitivity of detection of infected plants DSI reached the samples with DSI of16.6.This is the first clarifying the main transmission of bacterial fruit blotch and bacterial leaf blight. Established selective media and nested-PCR to identify seeds infected by bacterial fruit blotch pathogens,A nested-PCR to detection anthurium plants infected with bacterial leaf blight was created,which is contribute to the rapid disease prediction and early prevention.更多还原