高温胁迫诱导水稻白叶枯病菌在病叶中进入存活但不可培养状态的研究  

Study on high temperature stress inducing Xanthomonas oryzae pv.oryzae to enter the viable but non-culturable state in diseased leaf of rice bacterial leaf blight

作　　者：张含 王冰倩 杜逸晨 宋康丽 李佳杨 聂婧源 宋从凤 ZHANG Han;WANG Bingqian;DU Yichen;SONG Kangli;LI Jiayang;NIE Jingyuan;SONG Congfeng(College of Plant Protection/Key Laboratory of Integrated Management of Crop Disease and Pests,Ministry of Education,Nanjing Agricultural University,Nanjing 210095,China)

机构地区：[1]南京农业大学植物保护学院/农作物生物灾害综合治理教育部重点实验室,江苏南京210095

出　　处：《南京农业大学学报》2022年第3期521-528,共8页Journal of Nanjing Agricultural University

基　　金：国家自然科学基金项目(1471744)。

摘　　要：[目的]探究水稻白叶枯病菌是否存在存活但不可培养状态(viable but non-culturable,VBNC),将有助于更好地了解水稻白叶枯病的病害循环,为有效防治水稻白叶枯病提供理论基础。[方法]对分离采集自田间、保存在室温和4℃下14年的水稻白叶枯病叶,采用划线稀释分离法进行病原菌的平板分离,探索不同温度处理下病叶中白叶枯病菌的可培养情况。将带利福平抗性的白叶枯病菌PXO99^(A)菌株剪叶接种感病水稻品种‘深两优871’,病叶剪成小段分别置于4、28和37℃下保存,每隔30 d取病叶小段,通过平板分离进行病原菌含量的测定。利用染色全细胞染料Microlight^(TM) Green JJ98 and JJ99以及染死细胞染料碘化丙啶(PI)对喷菌液中细胞的存活程度进行测定。[结果]采集自田间、保存在室温和4℃下的病叶都有明显的喷菌现象;只有从4℃保存(甚至保存4年)的样品上可以分离到病原菌,而保存在室温的样品上都不能分离到白叶枯病菌。从人工接种的保存在3个温度下的病叶上分离的病原菌,随着保存时间的增加,可培养的PXO99^(A)菌量呈现下降趋势,其中37℃保存60 d后未分离到病原菌。荧光染色结果显示,保存60 d后,4、28和37℃条件下保存的样本中活细胞的比例分别为52.52%、32.17%和24.02%。以上结果说明,37℃病叶中的病原菌部分进入了VBNC状态。[结论]高温诱导了水稻白叶枯病样中的病原菌进入了VBNC状态。这为研究水稻白叶枯病菌的侵染生物学特征和制定有效的防治策略提供了科学依据。[Objectives]The paper aimed to explore if the pathogen of rice bacterial leaf blight(BLB),Xanthomonas oryzae pv.oryzae(Xoo),entered the viable but non-culturable(VBNC)state under the stress of high temperature.The study will help to better understand the disease cycle of rice BLB and provide a theoretical basis for effective control of the disease.[Methods]BLB samples collected in the field were stored at either room temperature or 4℃for about 1-4 years.Xoo was isolated by streaking based dilution separation method to explore the culturability of the pathogen in diseased rice leaves under different temperatures.The diseased rice variety‘Shenliangyou 871’leaves which were artificially inoculated with rifampicin resistant Xoo strain PXO99^(A) were cut into segments and stored at 4,28 and 37℃,respectively.Every 30 days,leaf segments were taken out to determine the bacterial population by counting bacteria colonies grown on medium plate and cell viability by dye staining using Microlight^(TM) Green JJ98 and JJ99 which stained both dead and living cells and propodium iodide(PI)specific for staining dead cells.[Results]All of collected field BLB samples stored either at room temperature or 4℃had bacterial exudation without obvious difference.The Xoo bacteria could be isolated only from the samples stored at 4℃even up to four years,but not from the samples stored at room temperature at least for two years.Further,the artificially inoculated BLB leaves were stored at three different temperatures of 4,28 and 37℃,and the samples were collected every 30 days to isolate the bacteria on NA plates.The results showed that with the increase of days,the amount of PXO99^(A) that could be cultured from the BLB leaves stored at three temperatures showed a downward trend.Xoo could not be isolated from the samples stored at 37℃for 60 days.The results of fluorescence staining showed that after 60 days of storage,the proportions of living cells in the samples stored at 4,28 and 37℃were 52.52%,32.17%and 24.02%,respectiv

关 键 词：水稻白叶枯病菌 VBNC 荧光染料染色 温度胁迫 

分 类 号：S432.1[农业科学—植物病理学]