青枯病菌噬菌体P3株系的生物学特性及其应用研究

作物青枯病是一种土传细菌性维管束病害,由于作物病害的发生和严重度与土壤中的病原菌数量呈正相关,因而快速准确测定土壤中的病原菌数量是病害预测及有效防控的前提。本文对筛选获得的一株具有生防潜力的噬菌体P3株系进行了生物学特性测定,并探讨其在检测烟草根际土壤中青枯病菌数量的可行性,为烟草根际土壤青枯病菌的检测提供一种新方法。研究结果表明,噬菌体P3株系由一个二十面体的头部和非常短的尾部构成;裂解谱广,可裂解烟草、番茄、辣椒、马铃薯和甘薯的青枯病菌;最佳感染复数为0.0001;潜伏期60 min,裂解期80 min,裂解量179;对1%氯仿不敏感;55℃以下及pH 4~12的环境中保持稳定;对紫外线敏感,照射18 min失活;20℃~28℃存放一个月效价稳定。同等条件下,检测烟草根际土壤中青枯病菌数量,平板检测法的实测值仅为1.79×10⁴CFU/g,而噬菌体检测法实测值为2.83×10⁵CFU/g,说明噬菌体检测法的灵敏度较平板检测法高10倍。本研究通过噬菌体检测法检测了烟草根际土壤的青枯病菌数量,可为今后烟草青枯病的预测预报提供技术支持。     

青枯雷尔氏菌噬菌体的研究与应用进展

青枯雷尔氏菌(Ralstonia solanacearum)引起的烟草等茄科作物青枯病,是一种世界性重大细菌病害,危害严重且防治困难。利用“噬菌体疗法”防治细菌性病害是近年来生物防治研究的热点。由于近年来抗药性和超级细菌的出现,噬菌体作为病害控制的新方法重新受到重视。综述了噬菌体的发现历程、青枯雷尔氏菌噬菌体发现与分类、挖掘利用,并讨论了噬菌体应用中的问题及发展方向。     

繁殖青枯菌噬菌体无毒菌株的筛选及应用

利用噬菌体防治植物细菌性病害,关键技术是噬菌体的扩大繁殖,利用宿主菌繁殖,应用时存在一定的风险。本研究通过继代培养筛选致病性丧失的青枯菌菌株作为青枯菌噬菌体扩繁的宿主,结果表明,通过对野生青枯菌株RSsw326连续超过20代的培养,获得了一株菌落圆形、游动性弱的变异菌株;再通过刺叶、注射和伤根等方法接种烟苗,30 d后无萎蔫症状出现,将该菌株命名为RSsw326-2;测试表明,该菌株对青枯病菌没有拮抗作用,可被从福建不同烟区分离纯化的8株噬菌体裂解。利用菌株RSsw326-2作为宿主,进行青枯菌噬菌体的扩大繁殖,培养36 h,效价可达10¹⁰ PFU/mL。将无毒菌株RSsw326-2+噬菌体的共培养液刺叶、伤根和不伤根接种烟苗,35 d后均无症状出现,而将毒性菌株RSsw326+噬菌体的共培养液刺叶接种后14 d、伤根接种后28 d发病率都为100%,不伤根接种烟苗35 d,发病率为66.7%。无毒菌株RSsw326-2+噬菌体的共培养液对盆栽烟苗防病试验结果表明,发病时间推迟8 d,并且在接种后21 d时对烟草青枯病的防效达74.1%,显著高于对照组。本研究采用继代培养筛选获得的无毒菌株作为噬菌体扩大繁殖的宿主,为今后研制噬菌体制剂的生产提供了参考。     

无菌水常温保存番茄青枯菌的效果检测

不同 pH的灭菌水常温保存不同浓度的番茄青枯菌 ,6年后检测其存活情况。结果表明 ,灭菌水常温保存番茄青枯菌效果与初始菌浓度和无菌水 pH有关。试验表明此种方法保存番茄青枯菌是一种保存时间长 ,保存性状稳定的方法。     

中生菌素对马铃薯青枯病菌的溶菌作用

马铃薯青枯病菌［Ｐｓｅｕｄｏｍｏｎａｓｓｏｌａｎａｃｅａｒｕｍ（Ｓｍｉｔｈ）Ｄｏｗｓｏｎ］是一种寄主范围非常广泛的植物病原细菌,可以侵染所有茄科和茄科以外一百多种植物。中生菌素（农抗７５１）是中国农科院生防所研制成功的一种新型农用抗生素,属于Ｎ－糖苷...     

梨火疫病菌噬菌体的初步研究

为找到梨火疫病菌适度专化性的噬菌体，用于该病的检测和鉴定，从英国病山楂枝条上分离到一株噬菌体，测定了其寄主范围、潜育期、繁殖量等，还观察了其形态。分离到的噬菌体为高度专化性噬菌体，不能单位用于梨火疫病菌的检测和鉴定，可与其他梨火疫病菌噬菌体株系配合使用。     

双重PCR检测马铃薯晚疫病菌和青枯病菌方法的建立及应用

 利用真菌通用引物ITS1和ITS4扩增马铃薯晚疫病菌转录间隔区并进行序列测定,通过序列比较,设计了1对马铃薯晚疫病菌的特异引物INF1/INF2,并对15种不同真菌、细菌和7种疫霉属和腐霉属卵菌基因组DNA进行PCR扩增,结果只有不同来源的马铃薯晚疫病菌株可获得324 bp的特异带。将引物INF1/INF2与卵菌通用引物进行巢式PCR扩增后,其检测灵敏度在DNA水平上可达30 fg。运用设计的引物与马铃薯青枯病菌特异引物结合建立了双重PCR体系,能从马铃薯晚疫病菌和马铃薯青枯病菌总基因组DNA以及人工接种和自然发病的马铃薯植株中分别或同时扩增到324 bp和281 bp的特异片段。实现了同时对马铃薯晚疫病菌和马铃薯青枯病菌的快速可靠检测。     

黄瓜霜霉病菌保存方法

 Pseudoperonospora cubensis(Berk. & M. A. Curtis) Rostovzev, the causal agent of cucumber downy mildew, is an obligate parasitic fungus. Up to now it can not be preserved on culture medium. In this study, the sporangia of P. cubensis were preserved in protective substances of 10% dimethyl sulfoxide plus 5% skim milk, 10% dimethyl sulfoxide plus 10% skim milk, sterilized water and leaves in vitro, and stored at -20℃, -70℃, and preliminary freezing at -20℃ for 24 h prior to -70℃ respectively. The results showed that the sporangia preserved in 10% dimethyl sulfoxide plus 5% skim milk, preliminary freezing at -20℃ for 24 h prior to -70℃ were still highly pathogenic after 12 months preservation. The percentage of germination of sporangia, incidence and disease index were 46%, 50.0% and 40.0 respectively 9 d after inoculation. This method solved the problem of P. cubensis preservation.     

PMA-qPCR定量检测青枯菌活菌方法的建立

本文将叠氮溴化丙锭(PMA)与荧光实时定量PCR技术相结合,建立了一种适于青枯菌不同小种菌株活细胞精准、快速检测的PMA-qPCR方法。通过单因素变化试验对PMA预处理反应体系中的各参数进行优化,确立了PMA终浓度为15 ng/μL,黑暗孵育时间为10 min,曝光时间为5 min的PMA预处理体系。试验结果表明,当活菌比例大于10%,PMA-qPCR的测定结果均在理论活菌数相对应的95%置信区间内。检测灵敏度测试结果显示,该方法适用于活菌数在5.0×10~2～5.0×10~8 cfu/mL范围内菌悬液的检测。本文建立的PMA-qPCR方法可在一定范围内有效去除青枯菌死菌的干扰,定量检测出活菌数量,研究结果可为植物细菌性青枯病的流行规律研究提供新的技术支撑。     

假茄科雷尔氏菌引起向日葵青枯病在中国的首次报道

茄科雷尔氏菌复合种侵染引起的青枯病是众多作物上的毁灭性病害。2020年笔者首次在广东省东莞市发现向日葵青枯病,并对其病原菌进行了鉴定。室内人工接种试验、16S rDNA序列比对和演化型鉴定结果表明,引起向日葵青枯病的病原菌为假茄科雷尔氏菌Ralstonia pseudosolanacearum。生理生化特性和致病性鉴定结果表明,分离自向日葵的15株假茄科雷尔氏菌为1号生理小种和生化变种3。egl基因部分序列系统进化分析表明,15株假茄科雷尔氏菌分属4个序列变种,其中8株菌株为序列变种17,5株菌株为序列变种13,其余2株菌株分别为序列变种14和序列变种54。本文是我国首次报道假茄科雷尔氏菌侵染引起向日葵青枯病。     

Genetic diversity of Ralstonia solanacearum species complex strains obtained from Guangxi,China and their pathogenicity on plants in the Cucurbitaceae family and other botanical families

Bacterial wilt, caused by the Ralstonia solanacearum species complex (RSSC), is a destructive plant disease in Guangxi, China. However, the diversity of RSSC populations in the area is unknown. To this end, we performed an extensive bacterial wilt survey from 2015 to 2018. Using phylotype-specific multiplex PCR (Pmx-PCR) and an egl-based tree, 189 strains collected from 20 plant species were identified as R. pseudosolanacearum phylotype I, which included 14 sequevars (12, 13, 14, 15, 16, 17, 18, 30, 34, 44, 48, 54, 70, and 71); two strains isolated from potato plants belonged to R. solanacearum phylotype II, sequevar 1. Sequevars 13, 17, and 44 were prevalent in Guangxi, and sequevar 13 dominated the RSSC sequevars of four Cucurbitaceae plants. The susceptibility of different Cucurbitaceae species to bacterial wilt and the host range of 16 representative strains were further tested. Members of the Cucurbita, Momordica, and Luffa genera were susceptible to bacterial wilt, with wilt incidence ranging from 73% to 100%. Most strains were pathogenic to solanaceous plants, mulberry, and ginger plants but not to melon crops; however, the strains from kidney bean, pepper, and Cucurbitaceae plants were highly virulent to melon crops. This is the first comprehensive report on the genetic and host range diversity of the RSSC in Guangxi and the susceptibility of different Cucurbitaceae species to bacterial wilt, which can provide valuable information for the development of bacterial wilt control strategies.     

Survival of Ralstonia solanacearum and Ralstonia pseudosolanacearum in drain water

The survival in drain water of two strains of Ralstonia solanacearum and three strains of Ralstonia pseudosolanacearum, including two strains able to cause wilt in roses, was determined. Water draining from drip‐irrigated rock wool mats on which roses were grown was supplemented with the pathogen and survival was monitored at 4, 12, 20 and 28°C for up to 112 days. All strains were able to survive for at least 112 days in drain water at 12 and 20°C, but at 4°C maximum survival was 56 days. At 28°C, the survival period was strain dependent, but was at least 56 days. Populations declined gradually in non‐sterile drain water to a low level (maximum 100 cfu mL^?1 after 112 days). In sterile drain water (autoclaved prior to addition of populations), no or only a limited decline in populations was found at 112 days, dependent on strain and temperature. Drain water that tested negative for Ralstonia in the dilution plating assay was tested for the presence of cells in a viable but non‐culturable state (VBNCs). Tomato plants were inoculated, but no symptoms developed, and plants sampled 22 days post‐inoculation were negative in a plating assay. Therefore, no indications were found that VBNCs were present.     

星油藤青枯病病原菌鉴定

 星油藤(Plukenetia volubilis L.) 是一种重要的藤本油料植物,在我国华南地区广泛种植。青枯病是近两年在海南星油藤种植区发生的新病害,为探究星油藤青枯病菌的基本特性及种下分化情况,本研究对分离的6株代表菌株进行了相关分析。细菌学鉴定及致病性测定结果表明,该病害是由类茄科雷尔氏菌(Ralstonia pseudosolanacearum)侵染引起。同时,从传统分类及分子生物学不同层面分析了星油藤青枯病菌的遗传分化情况。生理小种及生化变种的测试结果表明,星油藤青枯病菌属于1号生理小种和生化变种Ⅲ;16S rDNA和egl基因部分序列聚类分析显示,星油藤青枯病菌属类茄科雷尔氏菌演化型Ⅰ即亚洲分支菌株,序列变种34。     

Pathogenicity evaluation of Ralstonia pseudosolanacearum race 4 on ginger by leaf-clipping inoculation

Journal of General Plant Pathology - An easy, rapid method has been needed to test the pathogenicity of strains of Ralstonia pseudosolanacearum, the cause of serious bacterial wilt on ginger...     

星油藤青枯病病原菌鉴定

 星油藤(Plukenetia volubilis L.) 是一种重要的藤本油料植物,在我国华南地区广泛种植。青枯病是近两年在海南星油藤种植区发生的新病害,为探究星油藤青枯病菌的基本特性及种下分化情况,本研究对分离的6株代表菌株进行了相关分析。细菌学鉴定及致病性测定结果表明,该病害是由类茄科雷尔氏菌(Ralstonia pseudosolanacearum)侵染引起。同时,从传统分类及分子生物学不同层面分析了星油藤青枯病菌的遗传分化情况。生理小种及生化变种的测试结果表明,星油藤青枯病菌属于1号生理小种和生化变种Ⅲ;16S rDNA和egl基因部分序列聚类分析显示,星油藤青枯病菌属类茄科雷尔氏菌演化型Ⅰ即亚洲分支菌株,序列变种34。     

The long-term preservation of potato virus Y and watermelon mosaic virus in liquid nitrogen in comparison to other preservation methods

Samples of watermelon mosaic virus (WMMV), consisting of clarified sap of diseased squash plants, were found to be very infective after more than 4 years of storage if the samples had been deep frozen at –18°C, and to have lost most of their infectivity within 4 years of preservation by freeze drying and subsequent storage of the samples at 4°C.Potato virus Y (PVY) inocula, consisting of samples of clarified sap of diseased red peppers, lost their infectivity in less than 4 years if the samples had been deep frozen at –18°C but retained their infectivity for more than 4 years if the samples had been freeze dried and stored at 4°C.A decrease in infectivity of both viruses in the deep frozen at –18°C and freeze dried inocula could be observed even during the first months of storage. However, samples of clarified sap stored in or over liquid nitrogen maintained their activity for at least 22 months for WMMV and 32 months for PVY with no indication of a decrease in infectivity of the viruses. Storage in or over liquid nitrogen seems therefore a very promising long-term preservation method for plant viruses.Samenvatting Watermeloenmozaïekvirus inocula, bestaande uit helder gecentrifugeerd sap van zieke squashplanten, bleken hun infectievermogen zeer goed te hebben behouden na meer dan vier jaar bewaring bij –18°C, maar binnen vier jaar grotendeels te hebben verloren na droogvriezen en bewaren bij 4°C.Overeenkomstig bereide aardappelvirus Y inocula uit zieke paprikaplanten hadden hun infectievermogen binnen vier jaar verloren na bewaring bij –18°C, maar konden hun infectievermogen grotendeels behouden gedurende meer dan vier jaar na droogvriezen en bewaring bij 4°C.Binnen enkele maanden na het begin der bewaringsperiode kon al een afname van het infectievermogen van beide virussen in de diepgevroren (–18°C) en drooggevroren inocula worden gevonden. Helder gecentrifugeerde sapmonsters die in of boven vloeibare stikstof bewaard waren, hadden gedurende tenminste 22 maanden voor het watermeloenmozaïekvirus en 32 maanden voor het aardappelvirus Y hun volle infectievermogen behouden. Bewaring in of boven vloeibare stikstof is daarom de beste van de drie getoetste bewaarmethodes voor opslag op lange termijn van beide virussen. Deze methode lijkt ook veelbelovend voor een groot aantal andere virussen.     

Factors influencing Ralstonia pseudosolanacearum infection incidence and disease development in rose plants

Glasshouse experiments were conducted to study infection and disease development in rockwool-grown rose plants inoculated with Ralstonia pseudosolanacearum. A R. pseudosolanacearum strain isolated from rose plants was more aggressive than strains from anthurium or curcuma. The three rose cultivars tested, Avalanche, Red Naomi, and Armando, differed in susceptibility. At 20°C, the rose strain caused hardly any symptoms over a 6-week period, whereas at 28°C typical wilt symptoms were observed within 2 weeks after stem inoculation of Armando, the most susceptible cultivar. Inoculating roots with the rose strain resulted only in weak atypical symptoms. Nevertheless, inoculating roots of cv. Armando at a relatively low inoculum dose of 10⁴ cfu/ml led to high densities in the base of stems in one out of two experiments. R. pseudosolanacearum occasionally spread from stem inoculated plants with symptoms in rockwool slabs. This limited spread resulted in a low infection incidence, and only of plants directly adjacent to the plants with symptoms.     

烟草感染青枯菌后的组织病理变化

病症观察表明,烟草感染青枯菌后168h叶片全部萎蔫下垂、褐变。茎部切片表明,青枯菌处理72h,个别导管内出现了染色较深的物质,髓部和皮层的部分薄壁细胞出现破损。120h后导管内染色较深的物质增多,导管堵塞程度加大。168h后局部区域的木质部和韧皮部分离。对感病植株的叶进行青枯病菌分离,结果表明:从叶中可分离出致病青枯菌,并可以导致复感植株得病继而死亡。