【作者】 谢家建；

【导师】 彭于发； 王锡锋；

【作者基本信息】 中国农业科学院 ， 生物安全， 2004， 硕士

【摘要】 本研究初步建立了白叶枯病菌致病型的分子鉴定方法；在温室条件研究了3个水稻白叶枯病菌在转Xa21基因水稻、受体水稻皖A和汕优63叶面的侵染和定殖能力；以及田间种植转Xa21基因水稻、受体水稻皖A和汕优63的叶围细菌的时空动态变化。 比较了中国白叶枯病菌7个不同致病型菌株的内转录间隔区(Intergenic spacer region,ITS)序列、IS-PCR指纹图谱和全菌体蛋白SDS-PAGE电泳图谱的差异。7个菌株的ITS序列均为492bp(GenBank登陆号为AY250998-AY251004)，仅在4个位点上有差异。它们的全菌体蛋白SDS-PAGE电泳图谱相似性很高，仅C5菌株与其它6个菌株在谱带上有明显差异。IS-PCR指纹图谱可以将7个菌株分为3种不同谱型。测定了7个菌株IS-PCR均有的两条条带244bp和489bp的序列。根据489bp条带序列设计引物，能特异性在所有7个白叶枯菌株中扩增出355bp的条带。建立了白叶枯病菌快速鉴定的PCR方法和使用IS-PCR快速对不同白叶枯病菌进行分群的方法，为分析长期种植转Xa21基因水稻后对靶标白叶枯病菌的群落结构的变迁奠定了基础。 在温室研究了3个白叶枯致病型菌株在转Xa21基因水稻、受体水稻皖A和汕优63水稻叶面的侵染和定殖能力。在侵染力上，3种水稻侵染部位的病菌数量在初期都是迅速增殖。在达到约10~6CFU/leaf后，转Xa21基因水稻侵染部位的白叶枯病菌的数量开始下降，而受体水稻皖A、汕优63上白叶枯病菌的数量则继续上升2-3个数量级。在定殖能力上，白叶枯病菌在3种水稻健康的水稻叶片上都能存活较长的时间，在不同水稻品种上的动态变化是基本相似的，转Xa21基因水稻对白叶枯病菌的定殖能力没有明显的影响。 研究了田间转Xa21基因水稻、受体水稻皖A和汕优63水稻的细菌群落的时空变化。从细菌的总数量的空间分布来看，不同小区间有显著差异，但种植水稻品种间没有显著差异。细菌总数量随着时间的变化有着显著的变化，但同一时间不同的水稻品种间整体没有显著差异。测定从细菌群落中分离纯化的12个菌株的ITS序列(GenBank登陆号为AY485405-AY485416)，将其鉴定到8个属，占优势的为Clavibacter、Bacillus和Xanthomonas属。以属为分类单位计算叶围细菌群落的多样性指数。多样性指数随着时间的变化而有着显著变化，但同一时间的品种间的差异不显著。更多还原

【Abstract】 The molecular method to identify Xanthomonas oryzae pv.oryzae (Xoo) was built in this study. The dynamic changes of three Xoo strains in greenhouse and the phyllosphere bacterial communities in field trial of three rice varieties {Xa21 transgenic rice, unmodified control rice Wan A and conventional rice variety Shanyou 63) were studied.Seven Xoo strains were compared by their ITS (Intergenic spacer region) sequences, IS-PCR fingerprint and SDS-PAGE of total strains’ proteins. The Length of all the seven strains’ ITS sequences was 492 bp (Accession number AY250998-AY251004), only with four sites difference.The SDS-PAGE fingerprints of seven strains’ total bacterial proteins were very similar except strain C5. Seven strains were divided into three groups by their IS-PCR fingerprints. The two bands appeared in all the strains were sequenced to 244bp and 489bp, respectively. Depended on the sequence of the band of 489bp, a pair of prime was designed to amplify the specific band of 355bp for all the seven strains. Based on the primes and IS-PCR fingerprints, the community of Xoo in the Xa21 transgenic rice cultivated with a long time could be analyzed rapidly.The differences of Xoo strains’ abilities to infect and colonize in phyllosphere of three rice varieties were studied. After inoculation by clipping method, the numbers of Xoo increased rapidly in three rice varieties in the early time until it reached about 106CFU/leaf. The number of Xoo decreased in the Xa21 transgenic rice but increased 100-1000 fold in the other two rice varieties after that. When spraied in rice leaves, all the three Xoo strains could survive for a long time, but no significant differences among those three rice varieties were found.The phyllosphere bacterial communities of the rice varieties were studied in field trial. Significant differences were found among the total number of bacterial communities in different blocks at the same time, but no differences among three rice varieties were found. Significant differences were also found among the total number of phyllosphere bacterial communities of each rice variety in different time, but not among rice varieties at the each time. Twelve bacterial strains isolated from the rice phyllosphere were divided into eight genus by their ITS sequence (Accession number AY485405-AY485416).The predominance genus in phyllosphere of all three rice varieties were Clavibacter, Bacillus and Xanthomonas. Significant differences were found among the diversity indexes of bacterial communities of different time calculated based on genus, but no difference among the varieties at the each time were found.更多还原