一株对TMV和PVY具有拮抗活性生防菌的筛选与鉴定  

Selection and Identification of a Biocontrol Bacteria Strain with Inhibitory Activity Against TMV and PVY

作　　者：翟熙伦[1] 杨金广[1] 申莉莉[1] 钱玉梅[1] 王盼[1] 孙丽萍[1] 王惠卿[2] 赵洪东[2] 王永[2] 王凤龙[1] 

机构地区：[1]中国农业科学院烟草研究所/烟草行业烟草病虫害监测与综合治理重点开放实验室,山东青岛266101 [2]山东中烟工业有限责任公司,济南250100

出　　处：《中国农业科学》2012年第11期2180-2188,共9页Scientia Agricultura Sinica

基　　金：国家烟草专卖局全国烟草有害生物调查研究(110200902065)

摘　　要：【目的】筛选对烟草花叶病毒(Tobacco mosaic virus,TMV)和马铃薯Y病毒(Potato virus Y,PVY)具有显著拮抗活性的菌株。【方法】从山东诸城等植烟区采集烟草病毒病高发区健康烟株中烟100(Nicotianatabacum var.Zhongyan 100)的根际土壤,分离抗TMV的活性菌株,通过生理生化测定和分子鉴定确定菌株。采用Real-time PCR技术定量检测该菌株对TMV和PVY的抑制作用进一步验证该菌株的抗病毒活性。田间试验进行3个处理,菌液与病毒混合,2 h后接种(I),喷菌液2 h后接种病毒汁液(II),接种病毒汁液2 h后喷施菌液(III),以8%宁南霉素和肥皂水分别与等体积病毒汁液混合为阳性对照,以LB培养基与病毒汁液混合为空白对照。利用菌株发酵液对剪刀上病毒的钝化作用进行验证试验。【结果】筛选到的菌株通过枯斑寄主半叶法显示其对TMV的抑制效果达98%以上,命名为4A1,分子鉴定结果显示该菌株16S rDNA序列与Pseudomonas monteilii的16s rDNA序列同源率达到99%;菌株4A1为革兰氏阴性,氧化酶、淀粉水解VP、吲哚等反应为阴性,明胶液化、葡萄糖反应为阳性,能运动,确定该菌株为蒙氏假单胞菌(P.monteilii)。Real-time PCR检测结果显示其发酵液对TMV、PVY具有显著的抑制活性。田间试验结果显示,处理I可有效降低大部分病毒侵染,TMV和PVY发病率分别为5.3%和7.7%,病情指数分别为0.8和2.9;处理II可显著减少TMV和PVY的侵染几率,发病率分别为33.3%和36.7%,病情指数分别为4.2和6.0;处理III烟株发病率为95.0%和92.0%,病情指数为24.5和20.1;CK组TMV、PVY发病率分别为99.3%和95.4%,病情指数为别为38.6和45.1。菌株对剪叶工具的处理,钝化病毒效果可达95%以上,防效显著高于其它处理。【结论】在烟叶生产上,该菌株可以作为生产工具的生物消毒剂和抗病毒剂。【Objective】The objective of this study is to screen a bacteria strain with significant anti-virus activity against TMV（Tobacco mosaic virus） and PVY（Potato virus Y）.【Method】The strain was isolated from the soil around the roots of healthy tobacco plants of Zhongyan100（Nicotiana tabacum var.Zhongyan 100） grown in the high incidence of virus disease in many regions plantation of Shandong Province.The anti-TMV strain was tested by half-leaf method on hypersensitive plants（N.tabacum var.Samsun）,and identified by the physiological and biochemical and molecular methods.Real-time quantitative PCR was used to test the inhibition of TMV and PVY for further verifying the anti-viral activity.Field trials were conducted with three treatments.The mixture was inoculated to tobacco NC89（N.tabacum var.NC89） after the virus juice mixed with fermentation for 2 h（I）.The fermentation was firstly sprayed on the tobacco plant,then inoculated the virus to NC89 2 h later（II）.The virus was inoculated to the tobacco plant,then the fermentation was inoculated to the tobacco plant 2 h later（III）.8% of ningnanmycin and soapy water mixed with virus respectively were chosen as the positive control,and LB culture was mixed with virus as the blank control.The text result was verified through the inactivation of fermentation to the virus on the scissors.【Result】The anti-virus capacity of the strain named as 4A1 was up to 98%.Sequence analysis results showed that the nucleotide sequence of the 16S rDNA of the strain had 99% identity with that of Pseudomonas monteiliis.The strain 4A1 was gram-negative.Enzyme,starch hydrolysis reaction such as VP and indole were negative,gelatin,liquefaction,glucose response were positive and could move.4A1 was identified as P.monteilii based on the above physiological,biochemical and molecular test results.Real-time quantitative PCR was used to test the inhibition of TMV and PVY for further verifying the anti-viral activity.And the results showed the fermentation of 4A1 h

关 键 词：烟草病毒病 生防菌 蒙氏假单胞菌 抗病毒活性 

分 类 号：S435.72[农业科学—农业昆虫与害虫防治]