蔗糖水解酶注释基因XC0805与野油菜黄单胞菌8004菌株的蔗糖利用及致病相关

 野油菜黄单胞菌(Xanthomonas campestris pv.campestris,Xcc)是造成十字花科植物减产的重要病原菌,其通过水孔或伤口进入植物体内并在维管束中快速繁殖。光合产物(蔗糖)的运输主要依靠植物维管组织,同时蔗糖也是维管组织重要的能量来源。本研究利用生物信息学和遗传学等方法对Xcc中参与蔗糖利用的基因进行了分析,并研究了这些基因与其致病力的关系。Xcc 8004菌株中注释的参与蔗糖分解蛋白的编码基因有XC1002、XC1642、 XC1645和XC0805。 XC1002和XC1642的编码产物属于糖苷水解酶GH97蛋白超家族,而XC1645和XC0805的编码产物则属于GH13家族。突变分析发现除XC0805外,其他3个都不影响Xcc的蔗糖利用能力。接种实验表明,XC0805参与Xcc的侵染或在植物体内的生长,其他3个不参与致病过程。另外,Xcc 8004中预测参与蔗糖转运的基因(XC0806和XC0807)突变并不影响细菌的蔗糖利用和致病力,表明其可能存在其他的蔗糖转运通路。上述结果说明XC0805可能是Xcc 8004主要的蔗糖水解酶基因,且在致病中起重要作用。     

玫烟色棒束孢耐热菌株的筛选及对麦长管蚜的致病力测定

采用48℃恒温水浴热胁迫法对玫烟色棒束孢7个菌株进行了耐热性测定。结果表明,热胁迫45 min后,不同菌株的残存活孢率差异显著,其中IF-502、IF-904和IF-9606菌株耐热性较强,残存活孢率均达50%以上;IF-7606菌株耐热性最差,残存活孢率仅为28.70%;IF-14、IF-511和IF-609菌株耐热性居中,残存活孢率34.72%～36.00%。对耐热性强的IF-502、IF-904和IF-9606三个菌株对麦长管蚜若蚜的致病力测定表明, IF-904菌株在1×10~7孢子/mL时对麦长管蚜的致病力最高,校正死亡率达88.20%,致死中时最短,为2.67 d。     

安徽省猪伪狂犬病毒的分离鉴定及其主要毒力基因分子特征

猪伪狂犬病是由猪伪狂犬病毒(porcine pseudorabies virus,PRV)引起的一种急性、热性传染病。自2011年底以来,PRV发生了变异,传统的PRV弱毒疫苗已不能对PRV变异株提供完全保护,这给我国PR防控带来了巨大挑战。为了解安徽省PRV流行特征及其主要毒力基因遗传变异情况。利用PCR技术、细胞接种试验、电子显微镜观察、间接免疫荧光试验及兔体接种试验等方法,对安徽省临诊病例中疑似PRV感染的病猪进行病原检测及PRV分离鉴定,并通过设计6对特异性引物对PRV分离株主要毒力基因(gE、gI、TK、gB、gC、gD)进行克隆及测序分析。2016—2018年安徽省临诊病例中共分离鉴定15株PRV;PRV分离株主要毒力基因序列均与2011年后国内PRV变异株同源性较高;与2011年前国内PRV经典株序列比对,PRV分离株gE、gB、gC及gD基因存在多个位点的一致性替换、插入或缺失,且gE、gC基因多位点突变位于其重要的抗原表位区。本研究分离的15株PRV均为变异株,变异株已成为安徽省主要的流行毒株。15株PRV分离株的gI、TK基因序列较为保守,而gE、gC蛋白抗原表位区域氨基酸的突变可能导致其毒力及抗原性发生改变。部分PRV分离株与邻近地区PRV序列同源性均为100%,可能与频繁跨省调运生猪、跨区引种等原因有关。     

不同新型作用机制杀菌剂对梨树主要病菌毒力及防治流程应用

 为明确多种新型作用机制杀菌剂与引起梨树褐斑病、黑星病、白粉病等病原菌的有效对靶关系及制定梨树主要病害防治流程,采用菌丝生长速率法、离体叶片法与田间药效方法研究新型杀菌剂对靶标病原菌的毒力、对靶标病害的防效及其田间有效应用,建立替代梨树主要病害传统化学杀菌剂的防治流程技术。结果显示,双胍三辛烷基苯磺酸盐在离体叶片法下对褐斑病的防效大于85%,田间药效验证3次用药后7 d防效大于85%、30 d防效大于80%、90 d防效仍大于60%,兼治轮纹病菌其毒力EC₅₀值均小于1 μg·mL^-1。双胍三辛烷基苯磺酸盐、辛菌胺醋酸盐、吩嗪α-2羧酸在离体叶片法下对黑星病防效大于90%且毒力EC₅₀值小于1 μg·mL^-1;田间药效验证1次用药后7 d防效大于80%、30 d防效仍大于75%。噻肟菌酯、硝苯菌酯、丙硫菌唑在离体叶片法下对白粉病的防效大于85%;田间药效验证3次用药后7 d铲除效果大于70%、30 d仍大于60%。丙硫菌唑对黑斑、轮纹病菌毒力EC₅₀值均小于1 μg·mL^-1;吩嗪α-2羧酸同时对褐斑、黑斑、轮纹病菌毒力EC₅₀值小于1 μg·mL^-1。针对梨树主要病害发生期,选用新型杀菌剂替代传统杀菌剂制定防治技术流程,其综合防效达到88.94%。不同新型作用机制杀菌剂在防治梨树主要病害上的应用,能够降低抗药性产生,同时达到有效防治的目的。     

A novel glycoside hydrolase 74 xyloglucanase CvGH74A is a virulence factor in Coniella vitis

Grape white rot is a destructive fungal disease occurring worldwide. Recently, Coniella vitis was identified as the predominant pathogen causing this disease in China. As the periderms of grape shoots are severely degraded by C. vitis, it was speculated that cell wall-degrading enzymes (CWDEs) might play a key role in the pathogenesis of this disease. Therefore, this study aimed to examine the hydrolytic activity of the CWDEs of C. vitis. The results showed that xylanase (Xy) and xyloglucanase (XEG) had high levels of hydrolytic activity both in vitro and in vivo. Furthermore, a high-virulence fungal strain exhibited higher levels of Xy and XEG activities compared with a low-virulence strain. The genome of the fungus was found to harbor two XEG-coding genes CvGH74A and CvGH74B, which belonged to the glycoside hydrolase (GH)74 family. The expression level of CvGH74A was found to be high during pathogen infection. CvGH74A gene deletion mutants were generated using the split-marker method. The deletion of CvGH74A decreased both the hydrolytic activities of XEG and Xy and also the ability of the fungus to infect the grape leaves. No differences in the hyphal growth, morphology of colonies, or conidiation were found between the ΔCvGH74A mutant strains and the wild-type strain. Together, these results suggested that CvGH74A acted as an important virulence factor, and its enzymatic activity might regulate the virulence of the pathogen. This study was novel in reporting that GH74 XEG acted as a virulence factor in C. vitis.     

不同培养基继代培养蜡蚧轮枝菌对产孢量和烟粉虱毒力的影响

为了探究不同培养基继代培养的蜡蚧轮枝菌对烟粉虱的作用效果,通过继代培养方法测定JMC-01、JMC-02菌株的产孢量、对烟粉虱的毒力及其几丁质酶活性。结果表明,JMC-01、JMC-02菌株在添加蝉蜕和烟粉虱的PDA培养基中连续培养5代的产孢量、对烟粉虱的毒力、几丁质酶活性均高于无添加的PDA培养基,且JMC-01菌株的各项参数均高于JMC-02菌株。JMC-01菌株在添加蝉蜕的PDA培养基中T_1和T_3代的产孢量最大,均为1.279×10~7个/mL,T_3代对烟粉虱的毒力最大,为88.33%,T_5代的几丁质酶活最高,为0.268 3 U/mL;在添加烟粉虱的PDA培养基中,JMC-01菌株的最大产孢量、对烟粉虱的最高毒力和最高几丁质酶活分别出现在S_2、S_4和S_5代,分别为1.270×10~7个/mL、88.33%和0.325 4 U/mL。回归分析结果显示,JMC-01、JMC-02菌株几丁质酶活性与烟粉虱校正死亡率呈显著的正相关,且拟合优度较好。本试验表明JMC-01、JMC-02菌株均对烟粉虱具有较强的毒力,添加蝉蜕和烟粉虱的培养基可以维持菌株生长特性、增加产孢量、延缓菌株退化、增强菌株的几丁质酶活性,为烟粉虱的生物防治提供了新的菌种资源。     

Genome-wide comparison of Carnobacterium maltaromaticum derived from diseased fish harbouring important virulence-related genes

Although Carnobacterium maltaromaticum has been used as a probiotic in fish, it was reported to cause disease for the first time in Korea. The objective of this study was to understand the differences between pathogenic and non-pathogenic strains. Pathogenicity was tested by challenging rainbow trout with C. maltaromaticum ATCC35586 and 18ISCm isolated from diseased fish, and DSM20342 isolated from a dairy product. We also compared 24 genomes of C. maltaromaticum strains plus the genome of our isolate 18ISCm sequenced in this study. Only the strains from diseased fish caused high mortality with severe histopathological changes. Although all strains shared more than 90% of Ko_id, wecC and xtmA were found only in strains from diseased fish. Interestingly, only strains from diseased fish harboured two wecC paralogs involved in the production of D-mannosaminuronic acid which is a major component of a well-known virulence factor, teichuronic acid. Two wecC paralogs of 18ISCm were increased when they were co-cultured with trout blood cells, suggesting that wecC genes might play a role in virulence. The results of this study show that strains isolated from diseased fish are different from strains derived from food in terms of pathogenicity to fish and the presence of virulence-related genes.     

Piscine orthoreovirus: Biology and distribution in farmed and wild fish

Piscine orthoreovirus (PRV) is a common and widely distributed virus of salmonids. Since its discovery in 2010, the virus has been detected in wild and farmed stocks from North America, South America, Europe and East Asia in both fresh and salt water environments. Phylogenetic analysis suggests three distinct genogroups of PRV with generally discrete host tropisms and/or regional patterns. PRV-1 is found mainly in Atlantic (Salmo salar), Chinook (Oncorhynchus tshawytscha) and Coho (Oncorhynchus kisutch) Salmon of Europe and the Americas; PRV-2 has only been detected in Coho Salmon of Japan; and PRV-3 has been reported primarily in Rainbow Trout (Oncorhynchus mykiss) in Europe. All three genotypes can establish high-load systemic infections by targeting red blood cells for principal replication. Each genotype has also demonstrated potential to cause circulatory disease. At the same time, high-load PRV infections occur in non-diseased salmon and trout, indicating a complexity for defining PRV's role in disease aetiology. Here, we summarize the current body of knowledge regarding PRV following 10 years of study.     

Characterization and growth conditions of Vibrio parahaemolyticus strains with different virulence degrees that cause acute hepatopancreatic necrosis disease in Litopenaeus vannamei

The phenotypic characteristics and growth kinetics at several temperatures, salinities, and pH values of three Vibrio parahaemolyticus (Vp) strains with different virulence and one nonpathogenic strain were evaluated. Independent of the virulence of the strain, a high metabolic diversity was found, which yielded different colored phenotypes on the CHROMagar? Vibrio. All strains were resistant to ampicillin and carbenicillin, and Vp AHPND+ organisms were the most sensitive to enrofloxacin. The exponential growth of Vp strains started at 1–2 hr of incubation, although no relationship was observed between the bacterial density and degree of virulence. Moreover, the growth of the most virulent strain was independent of the nutrients in the incubation media during the initial hour postinoculation. No strain grew at 4°C in 0% NaCl and pH 4, but only Vp AHPND+ grew at 44°C. For all strains, the lag phase was proportional to the NaCl concentration, and the growth was better at pH 8–9. However, the Vp AHPND? strain displayed a greater variability, was more sensitive to extreme conditions, and showed a lag phase of 9 hr independent of the pH.     

白叶枯病菌效应子XopN在拥有OsSWEET11同源基因的水稻品种上发挥毒性作用

【目的】黄单胞菌细胞外泌蛋白质(Xanthomonas outer proteins, Xops)是植物病原黄单胞菌高度保守的毒性效应子或毒性辅助组分,通过细菌Ⅲ型分泌系统分泌到细菌细胞外部,然后转入植物细胞而发挥病理作用。水稻黄单胞菌水稻致病变种即水稻白叶枯病菌的标准菌株PXO99^A分泌的XopN是一种毒性效应子,通过影响寄主免疫反应而使水稻发病。但是,XopN对病菌毒性的影响是否因水稻品种的不同而异,还有待研究。【方法】利用同源双交换技术,先敲除了PXO99^A的XopN基因,获得了∆XopN突变体,又经过遗传互补,得到了回补菌株∆XopN/XopN。通过营养肉汤液体培养,测定了XopN对病菌繁殖能力的影响;根据文献选用14个水稻品种,通过接种实验,测定了PXO99^A对这些品种的毒性与XopN敲除或回补的影响;在XopN发挥毒性作用的水稻品种上,测定了隐性抗病基因OsSWEET11/xa13与显性感病基因OsSWEET11/Xa13受病菌侵染而表达的情况,分析了XopN敲除或回补的影响。【结果】在营养肉汤液体培养过程中,病菌突变体菌株∆XopN的繁殖速度明显低于野生型PXO99^A。PXO99^A、∆XopN和∆XopN/XopN接种水稻后,根据其在水稻叶片组织内的繁殖量及随后产生的白叶枯病症状的严重程度,将供试的14个水稻品种分为两种情况。一是感病程度与病菌XopN是否敲除或回补无关,这有10个水稻品种（IRBB1、IRBB3、IRBB8、IRBB10、IRBB14、IR24、IRBB203、IRBB204、IRBB205和IRBB211）,PXO99^A、∆XopN和∆XopN/XopN对它们的毒性无明显差别。二是XopN对病菌毒性发挥作用的水稻品种,包括高度感病的3个品种（IRBB208、Asominori和日本晴）和低感品种IRBB13。与PXO99^A或∆XopN/XopN相比,∆XopN对这4个水稻品种的毒性大为降低。在IRBB13上,病菌侵染对隐性抗病基因OsSWEET11/xa13在叶片内的表达发生抑制作用,这一效应与XopN的毒性功能相关。相反,日本晴显性感病基因OsSWEET11/Xa13却受病菌侵染的诱导,在叶片内的表达水平大幅度提高。IRBB208和Asominori携带OsSWEET11/Xa13同源基因,该同源基因在叶片内的表达水平也因病菌侵染而大幅提高。在这4个水稻品种上,PXO99^A和∆XopN/XopN能够诱导OsSWEET11/Xa13或其同源基因表达,但∆XopN无此作用。另外,XopN对病菌在非寄主植物烟草上诱发过敏反应有量变贡献,相比PXO99^A或∆XopN/XopN,∆XopN引起过敏反应的程度有所降低。【结论】XopN是一个有限广谱性效应子,在拥有OsSWEET11同源基因的水稻品种上发挥毒性作用。XopN也是病菌繁殖所需要的,对病菌在非寄主植物上诱导过敏反应有一定贡献。