【作者】 王运生；

【导师】 戴良英； 高必达； 荒井啓；

【作者基本信息】 湖南农业大学 ， 植物病理学， 2004， 硕士

【摘要】 柑桔黄龙病是由韧皮部杆菌Candidatus Liberibacter引起的一种柑桔毁灭性病害，在多个国家与地区均有发生。该病在日本1988年于冲绳县西表岛初次发现，随后河野于1994年在冲绳本岛发现该病并确认，近年来在日本有向北扩展的趋势，最近在鹿儿岛县奄美大岛也有发现。 自1970年Bove等用电镜观察到柑桔黄龙病病原以来，电镜观察一直是作为一种重要的鉴定和诊断手段，后来基于扩增16S rDNA特异性片断的PCR检测方法成了柑桔黄龙病检测的主要方法。 由于柑桔黄龙病病原只存在于韧皮部筛管细胞中，而对柑桔叶片维管组织固定比较困难。本研究通过对材料细切，小心去掉木质部部分以便于固定。由于植物组织中都含有一定的空气，致使树脂不能顺利浸润细胞器，这就给材料包埋带来困难，这可以通过在预包埋过程中处以真空处理以抽出植物组织中的空气，以便更好包埋。本研究对日本柑桔体内的柑桔黄龙病原菌的形态结构进行了电镜观察，并与发生在其它地区的柑桔黄龙病的病原进行了比较，结果表明柑桔黄龙病病原体存在于柑桔韧皮部的筛管细胞中，有时可见寄主细胞膜内陷成小泡状，柑桔黄龙病病菌日本株系绝大多数是球形和短椭圆形，极少观察到长条形病原，直径为100-500nm，外被一层胶状物质包围，厚约10-50nm，这与其它地区发现的柑桔黄龙病病原体形态不同。本研究还对植物总DNA的提取过程和PCR扩增程序进行了优化，利用Ready-To-Go^TM PCR bead法可以快速、准确检测到柑桔黄龙病病原体。更多还原

【Abstract】 Citrus Huanglongbing caused by Candidatus Liberibacter is one of the most severe citrus diseases, which has been reported in many countries and areas. In Japan, citrus Huanglongbing was observed in the island of Iriomote in Okinawa and in the main island of Okinawa in 1988 and in 1994 respectively. The disease was also observed in kagoshima island recently. It is intend to spread to the north area.The prokaryote associated with citrus Huanglongbing was first observed in 1970 by Lafleche and Bove in the phloem of infected sweet orange leaves. The electron microscope is one of the most important direction methods to study the morphology and to detect the pathogen associated with citrus Huanglongbing. According to the sequence of 16S RNA, a fast and accurate method based on PCR has been developed to detect the pathogen and now it is the most frequently used method.Because the pathogen of citrus huanglongbing is restricted in the sieve tubes in phloem which is surrounded by xylem tissue, it is difficult to fix the vascular tissue. In order to get good fixation, we wiped off the xylem tissue and cut the slices into 1mm cubes. Application of a few drops of Fixative No.1(2% glutaraldehyde in 0.1M cacodylate buffer at pH7.2) to the tissue before cutting into slices can prevent sticking and drying and initiate the fixing process. The air in the plant tissues prevent the resin to reach the organelles, which makes the fixation difficult. During the pre-embedding process, application of vacuum will make a rather good embedding. The pathogen of citrus huanglongbing found in Japan was detected in ultrathin section of infected citrus leaves and a comparation with the pathogen found in other areas was also carried out. It has show that the pathogen associated with Japanese citrus huanglongbing differ from others in some characters as the pathogen is mostly in round or oval forms, rarely in filamentous forms, diameter in 100-500nm with a 10-50nm envelope. A rapid and accurate detection method, with an improved DNA extraction process and Ready-To-GoTM PCR procedure was also developed.更多还原