【作者】 王笑；

【导师】 谢关林；

【作者基本信息】 浙江大学 ， 植物病理学， 2007， 硕士

【摘要】 西瓜细菌性果斑病(Bacterial Fruit Blotch)是西瓜、甜瓜上的一种毁灭性病害，由噬酸菌属燕麦种西瓜亚种(Acidovorax avenae subsp. citrulli)引起，是我国对内和对外的重要植物检疫性有害生物。该病主要通过种子带菌传播。随着我国农业科技和商业贸易的发展，种质资源的交换和种子的远程运输十分频繁和活跃，但由此引起的病害蔓延已对我国的西瓜和甜瓜产业构成严重威胁。本研究旨在明确我国西瓜、甜瓜的种植分布及西瓜果斑病的发生情况；建立准确、快速、灵敏的西瓜果斑病检测方法，并在实际的种子带菌检测中完善该技术，以有效监控和治理该病害。本文通过收集资料得出，中国西瓜甜瓜的种植面积及总产量均居世界首位，该产业已成为我国一种具有国际竞争力和较大经济增长空间的重要园艺产业，但是，据统计我国目前已有十个省份的局部地区有细菌性果斑病的发生报道，其中新疆、内蒙古、福建、台湾等地发病较严重，对当地造成了很大的经济损失。另据调查表明，我国种类繁多的西瓜甜瓜品种对该病的抗性存在差异，但尚未发现免疫品种。从调查不同年份西瓜果斑病发生情况看，该病的发生受气候因子的影响很大，西瓜生产期雨水较多、气温较高的年份发病尤为严重。本实验室于2006年建立了免疫捕捉PCR法检测西瓜果斑病菌，其检测灵敏度达50-100cfu／ml，比直接PCR的灵敏度高出100倍左右。本研究在该方法的基础上，优化了种子带菌检测的条件，采用MOPS缓冲液在28℃、220r／min摇培4h所得到的病菌要显著多于其他缓冲液处理和其他温度处理，因此该浸提条件更易于带菌量较少的种子检测。本研究采用不同缓冲液浸提模拟病种进行免疫捕捉PCR检测，结果发现20粒种子在2ml缓冲液种浸提均能检测出特异性片断，但是用1ml不同缓冲液浸提1粒模拟病种时，只有MOPS缓冲液的处理可以检测出带菌，证明了MOPS缓冲液可以从种子上洗脱出更多的病菌，从而可以提高检测的灵敏度。病菌在种子上的分布研究结果显示，果斑病以附着在种皮上为主，种仁上未检测到该病菌。对杭州市售种子进行带菌检测，结果发现产地为新疆的浙蜜4号、早佳84—24和和产地为台湾的台湾寿山王三个品种的种子有出现阳性结果，可见其对浙江省西瓜和甜瓜产业存在潜在危险性。更多还原

【Abstract】 Bacterial Fruit Blotch(BFB) caused by Acidovorax avenae subsp. citrulli (Aac) which is an important national plant quarantine disease in China is a destructive disease on watermelon and melon. The disease is seed-borne and seed transmitted. As the rapid development of science and technology in agriculture and international trade in recent years, the exchange and the long-distance transportation of melon germ plasm resources become more frequent, for what the disease has seriously threatened the development of watermelon and melon production in China. The study undertaken has following objectives: to know the growing areas of the melons and the occurrence situation of BFB in China; to develop an accurate, rapid and sensitive method to detect the pathogen in the seed quarantine for effective management of the disease.At present study, a great deal of information was collected and concluded that either the planting area or the yield of watermelon and melon that has become the important international competitive horticulture industry with great economical rise in China was the largest in the world. It is reported that the watermelons in 10 provinces in China have suffered from BFB in recent years, and especially in Xinjiang, Inner-Mongolia, Fujian and Taiwan province, the disease caused significant economic losses for farmers. The investigation also showed that there’s no immunity cultivar against BFB, although the resistance varies with varieties of the melons. The outbreak of BFB was affected greatly by the weather. The more rainfall and the higher temperature during the melon growth period, the more serious the disease was.An immuno-capture polymerase chain reaction (IC-PCR) method for detection of Acidovorax avenae subsp. citrulli has been established in 2006 in our lab. The minimum detection concentration for the IC-PCR was about 50-100cfu/ml, which is 100 times higher than that of direct PCR method. The present study found that MOPS buffer is more effective for extraction compared with another buffer when the seeds were incubated at 28℃(220rpm) for 4 hours. IC-PCR detection was performed by immersing diseased seeds in different buffer. When 20 seeds were added into 2ml buffer, Aac could be detected in different buffer; however, when 1 of the seeds were added into 1ml buffer, Aac could be detected only in MOPS buffer. Therefore, it seems that the sensitive of detection could be improved when immersing the seeds in MOPS buffer. The distribution of Aac on the seed was determined and most of them were colonized on seed coat and no bacteria were detected on seed kernel. The detection of melon seeds from market of Hangzhou were performed and the result showed Aac was present on cultivar Zhemi-4 and Zaojia 84-24 from Xinjiang and cultivar Shoushanwang from Taiwan, therefore, it should be pay attention to the potential risk of Aac on production of melon and watermelon in Zhejiang.更多还原