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。     

Identification of Pathogenicity Defective Mutants from a T-DNA Insertion Mutant Library of Verticillium dahliae

[Objective] Our research aimed to identify pathogenicity defective mutants from a T-DNA insertion mutant library of Verticillium dahliae strain Vd991 and to analyze pathogenicity-related genes. [Method] In total, 294 T-DNA insertion mutants of V. dahliae were tested for their virulence using a cotton infection assay. Southern blot assays were performed to identify the T-DNA insertion copy number of each pathogenicity defective mutant. DNA sequences flanking the T-DNA insertional sites of each mutant were analyzed by high-efficiency thermal asymmetric interlaced PCR. [Result] Based on the virulence assay, the disease indices of cotton plants inoculated with each mutant decreased very significantly in comparison with the index of those inoculated with Vd991. The Southern blot assay revealed that only one mutant contained two T-DNA insertions, while the remaining eight mutants harbored a single T-DNA insert. An analysis of biological characteristics found that the growth and conidial production of these mutants were impaired by the T-DNA insertions compared with the wild type Vd991. The T-DNAs' insertion position and distribution in each mutant were identified by comparison with genome sequences of strain VdLs.17. Furthermore, the pathogenicity-related genes were cloned from strain Vd991. [Conclusion] The screening and identification of T-DNA insertion mutants is an effective method to identify the pathogenicity-related genes of V. dahliae on a genome-wide scale. This laid a foundation for the further breeding of disease-resistant cotton varieties and will promote the study of the pathogenic molecular mechanisms of V. dahliae.     

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。     

广西卵形鲳鲹海豚链球菌基因分型、耐药谱型以及毒力基因检测

为了解近年来广西卵形鲳鲹海豚链球菌流行菌株的基因型信息以及菌株间的差异,对2015—2016年从广西地区7个养殖场的患病卵形鲳鲹体内分离得到的17株海豚链球菌菌株分别进行了基因型分析、耐药谱测定以及毒力基因检测。采用随机扩增多态性DNA标记技术(RAPD)和基因组重复序列PCR(rep-PCR)分析其基因型。结果显示,RAPD和rep-PCR指纹图谱结果一致,17株海豚链球菌可分为2种基因型。对海豚链球菌7种主要的毒力相关基因特异PCR检测,所有菌株均为sim A+scp I+pdi+sag A+cps D+pgm A+cfi+毒力基因型,表明这2种基因型的海豚链球菌似乎均为毒力较强的菌株。采用K-B法进行了20种抗生素敏感实验分析其耐药谱,结果表明归于基因1型的菌株耐药谱为AZT,基因2型菌株则出现3种相似的耐药谱,分别为SIZ/T/S/PEN/AZT/SPE/CAZ/PB、SIZ/T/S/PEN/AZT/SPE/CAZ/CRO和SIZ/T/S/PEN/AZT/SPE/CAZ/PB/RIF,证实基因型相同的菌株耐药谱型也相似,2种基因型的菌株耐药谱型差异显著,因此基因型和耐药谱型存在相关性。此结果为卵形鲳鲹海豚链球菌病流行病学研究、疫苗研制以及疫病监测提供理论依据。     

Highly specific assays to detect isolates of Pseudomonas syringae pv. actinidiae biovar 3 and Pseudomonas syringae pv. actinidifoliorum directly from plant material

Pseudomonas syringae pv. actinidiae (Psa) is responsible for bacterial canker of kiwifruit. Biovar 3 of Psa (Psa3) has been causing widespread damage to yellow‐ and green‐fleshed kiwifruit (Actinidia spp.) cultivars in all the major kiwifruit‐producing countries in the world. In some areas, including New Zealand, P. syringae pv. actinidifoliorum (Pfm), another bacterial pathogen of kiwifruit, was initially classified as a low virulence biovar of Psa. Ability to rapidly distinguish between these pathovars is vital to the management of bacterial canker. Whole genome sequencing (WGS) data were used to develop PCR assays to specifically detect Psa3 and Pfm from field‐collected material without the need to culture bacteria. Genomic data from 36 strains of Psa, Pfm or related isolates enabled identification of areas of genomic variation suitable for primer design. The developed assays were tested on 147 non‐target bacterial species including strains likely to be found in kiwifruit orchards. A number of assays did not proceed because although they were able to discriminate between the different Psa biovars and Pfm, they also produced amplicons from other unrelated bacteria. This could have resulted in false positives from environmental samples, and demonstrates the care that is required when applying assays devised for pure cultures to field‐collected samples. The strategy described here for developing assays for distinguishing strains of closely related pathogens could be applied to other diseases with characteristics similar to Psa.     

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.