广西柑橘衰退病和黄脉病调查及其病原病毒的遗传多样性分析

为明确柑橘衰退病毒(citrus tristeza virus, CTV)和柑橘黄脉病毒(citrus yellow vein clearing virus, CYVCV)在广西柑橘上的发生?分布及其遗传变异情况, 于2020年至2021年对百色?北海?崇左?贵港?桂林?河池?贺州?来宾?柳州?南宁?梧州和玉林等12个柑橘产区进行了病毒病调查?采用RT-PCR对采集样品进行了病毒检测, 并基于病毒分离物外壳蛋白(coat protein, CP)基因的核苷酸序列进行比对分析, 构建系统发育树?结果表明:采集的737份柑橘样品中, CTV的检出率为20.62%, CYVCV检出率为18.32%, CTV的检出率略高于CYVCV?病毒复合侵染的现象在采集的柑橘样品中普遍存在, CTV和CYVCV复合侵染率高达34.50%?对RT-PCR产物测序共获得12个CTV分离物和6个CYVCV分离物的CP基因序列?遗传多样性分析发现, CTV和CYVCV的CP基因序列都较保守, CTV分离物的遗传进化与地理来源?寄主来源均没有明显相关性, 但CYVCV分离物的遗传进化与地理位置具有相关性, 而与寄主来源无明显相关性?上述研究结果可为深入了解CTV和CYVCV在广西的流行情况以及柑橘病毒病的检疫和防控提供参考?     

烟草蚀纹病毒山东分离物全基因组序列的克隆和保守性分析

为利用RNA介导的病毒抗性策略,培育抗性稳定或抗多烟草蚀纹病毒（Tobacco etch virus,TEV）株系的转基因植株,采用RT-PCR及5＇-RACE方法克隆了烟草蚀纹病毒山东分离物TEV-SD1的全基因组序列。TEV-SD1全基因组核苷酸序列长度为9494 bp,包含1个9165 bp的开放阅读框架（open reading frame,ORF）,编码3054个氨基酸。将TEV-SD1基因组序列与GenBank中已公布的4个TEV全基因组序列和11个外壳蛋白（coat protein,CP）基因序列比对分析发现,各分离物CP基因间的核苷酸和氨基酸序列平均相似性分别为96.65%和98.31%,高于其它功能基因间的相似性;各分离物CP基因3＇端核苷酸序列相似性平均为96.55%,高于5＇端序列。聚类分析发现TEV在自然界中的分子变异与其寄主关系密切。     

6种柑橘类植物对柑橘衰退病毒分离株TR-L514变异的影响

 柑橘衰退病毒(Citrus tristeza virus,CTV)存在复杂的株系分化现象,运用弱毒株交叉保护防治柑橘衰退病时需了解不同类型柑橘对CTV构成的影响。作者将CTV分离株TR-L514嫁接接种到6类柑橘植物上获得了18个亚分离株,并对这18个亚分离株和TR-L514进行了指示植物鉴定,p25基因的限制性片段长度多态性(RFLP)和单链构象多态性(SSCP)分析,p23基因的序列比较。结果表明,CTV株系对不同柑橘类植株的适应性存在差异。TR-L514嫁接到不同柑橘类植株其构成会发生变化,在甜橙上可以同时检测出p25//HinfⅠ RFLP第1和6组群,并具有比在其它4类柑橘上更为复杂的SSCP谱型构成,因此甜橙可能更适宜于CTV的增殖。TR-L514及18个亚分离株的p23基因序列差异可能与不同类柑橘植株的适应性有关。     

建兰花叶病毒外壳蛋白基因和3′UTR序列测定与分析

采用单链构象多态性分析从建兰花叶病毒(Cymbidium mosaic virus,CyMV)的8个分离物筛选得到5个存在遗传变异的分离物,并测定了外壳蛋白(coat protein,CP)基因和3'非编码区(3'UTR)序列,经序列分析表明5个分离物与其他分离物间的CP核苷酸和氨基酸序列相似性分别为87,1％～99.9％和91.1％～99.6％,属于亚组A成员.CP基因不同片段受到正选择压力的作用不一样,C端受到负选择压力的作用不易发生变异,而N端受到正选择压力的作用容易发生变异.通过RNAsharpes软件分析3 'UTR序列发现各分离物均形成带有Potexvirus属保守核苷酸序列“AC(C/T) TAA”的三叶草茎环结构,这种结构的功能可能与病毒RNA复制有关.     

4种柑橘衰退病毒源单蚜传毒分离株CP基因的分子特征

 对4种柑橘衰退病毒源及其31个单蚜传毒分离株的CP基因做了限制性片段长度多态性(RFLP)和单链构象多态性(SSCP)分析,并对其中8个单蚜传毒分离株的CP基因进行了序列分析。实验明确了所研究柑橘衰退病毒(Citrus tristeza virus,CTV)的CPG/Hinf I RFLP组群和CPG/SSCP模式,二者能较好地对应并有效验证了单蚜传毒对CTV毒源的分离纯化作用;CP基因序列分析结果表明,相同CPG/Hinf I RFLP组群的单蚜传毒分离株间具有高度的序列相似性,而不同CPG/Hinf I RFLP组群单蚜传毒分离株间则存在较大差异;通过与已知生物学特性CTV分离株比较,初步建立了上述CP基因分子特征与病毒生物学特性之间的联系。     

甘薯病毒G CH株系和CH2株系中国分离物基因组全序列克隆及结构特征分析

为明确甘薯病毒G(sweet potato virus G,SPVG)CH株系中国分离物SPVG-CH-Ch1和CH2株系中国分离物SPVG-CH2-Ch1的基因组结构特征及遗传变异情况,利用RT-PCR和RACE方法克隆获得分离物SPVG-CH-Ch1和SPVG-CH2-Ch1的基因组全序列,应用DNAMAN软件对基因组全序列及不同编码区序列进行分子变异分析,并基于多聚蛋白基因序列利用MEGA 7软件进行系统进化分析。结果表明,分离物SPVG-CH-Ch1和SPVG-CH2-Ch1的基因组分别包含10 813个和10 834个核苷酸,均包含1个开放阅读框,由10 467个核苷酸组成,编码含有3 488个氨基酸残基的多聚蛋白。分离物SPVG-CH-Ch1与SPVG-CH2-Ch1之间的基因组全序列核苷酸一致性为78.6%,二者与GenBank中登录的其它SPVG分离物基因组全序列核苷酸一致性分别为78.6%～99.1%和77.9%～98.6%,其中SPVG-CH-Ch1与IS103分离物(KM014815)的核苷酸一致性最高,为99.1%,与WT325分离物(KF790759)的核苷酸一致性最低,为78.6%;SPVG-CH2-Ch1与WT325分离物的核苷酸一致性最高,为98.6%,与66Al分离(KX279878)的核苷酸一致性最低,为77.9%。系统进化树显示,SPVG-CH-Ch1与CH株系的7个分离物形成1个分支,SPVG-CH2-Ch1与CH2株系的WT325分离物形成1个分支。表明SPVG的CH株系和CH2株系之间的变异较大,株系内较保守。     

利用小RNA深度测序技术鉴定上海地区‘红美人’柑橘病毒种类

为明确上海地区‘红美人’柑橘的病毒种类, 2020年4月在崇明、金山、奉贤、嘉定、闵行和浦东采集疑似病毒感染的叶片样品11份, 采用小RNA深度测序技术结合RT-PCR对不同来源的混合样品进行病毒种类鉴定。结果表明, 样品中含有柑橘衰退病毒Citrus tristeza virus (CTV)、柑橘黄化脉明病毒Citrus yellow vein clearing virus(CYVCV)、柑橘树皮裂纹类病毒Citrus bark cracking viroid(CBCVd)和柑橘类病毒Ⅴ Citrus viroid V(CVd-Ⅴ)。同时发现, 上海地区‘红美人’柑橘病毒复合侵染现象普遍。基于CTV和CYVCV的CP全长基因及CBCVd和CVd-Ⅴ全基因组序列系统进化分析结果表明:除了CYVCV的分离物与地理来源具有明显的相关性外, 其余病毒及类病毒均没有严格的地域相关性。研究结果为开展‘红美人’柑橘种苗病毒检测及病毒病的防治奠定基础。     

菜豆普通花叶病毒RT-PCR承检测及3'末端序列分析

根据哈尔滨地区豇豆感病植株的症状,初步鉴定感染豇豆的病毒为菜豆普通花叶病毒(Bean common mosaic virus,BCMV).利用马铃薯Y病毒属通用引物扩增出病毒基因组3'末端序列,经BLAST检索表明该病毒为BCMV,将该序列与GenBank上的21个BCMV分离物的3'末端序列进行比较,显示其核苷酸序列与其他分离物的序列同源性为91.7%～97.3%.系统进化分析显示不同分离物可聚为5个类群,并显示出一定的寄主相关性.哈尔滨分离物BCMV-X与2个浙江分离物、1个澳大利亚分离物聚为一支,且该4株分离物的寄主均为豇豆.RNA二级结构分析显示BCMV基因组3'末端非编码区形成4个茎环结构,不同分离物的序列变化并未引起茎环结构的明显变化.     

吉林番茄黄化曲叶病毒分离物的检测及其DNA-A全基因组序列分析

从吉林省松原市田间表现黄化曲叶症状的番茄上分离到病毒分离物JLSY,基因组全序列测定结果表明,基因组全长2781 bp,共编码6个ORF。序列比对表明,JLSY基因组与番茄黄化曲叶病毒（TYLCV）安徽分离物（FN650807）相似性最高,为99.5%,而与其他双生病毒的序列相似性低于89%。经系统进化分析,该病毒分离物属于TYLCV以色列株系（TYLCV-IL）。这是首次在我国吉林省检测到番茄黄化曲叶病毒。     

Geographical cline of DNA variation within the F intersterility group of Heterobasidion annosum in Italy

Eighty-six Heterobasidion annosum isolates, mainly belonging to the F intersterility group and obtained from 32 different geographical localities in Italy, were subjected to genetic analysis by the Random Amplified Polymorphic DNA (RAPD) markers. The similarity between F and S groups was higher than that between F and P. In UPGMA Cluster Analysis, the F isolates originating from the same locality usually grouped in the same cluster. The isolates also showed a tendency to group at the level of larger geographical areas. Within the F group, isolates from the south of the Italian peninsula showed the highest genetic variation and northern isolates from the Alpine regions showed the lowest. This indicates a gradual cline along the peninsula. The genetic variability in the Italian F group is discussed in relation to the past and present distribution of the host species in Italy and Europe.     

Geographic distribution and genetic diversity of Fusarium graminearum and F. asiaticum on wheat spikes throughout China

A large number of Fusarium graminearum and F. asiaticum isolates were collected from wheat spikes from all regions in China with a history of fusarium head blight (FHB) epidemics. Isolates were analysed to investigate their genetic diversity and geographic distribution. Sequence characterized amplified region (SCAR) analyses of 437 isolates resolved both species, with 21% being F. graminearum (SCAR type 1) and 79% being F. asiaticum (SCAR type 5). AFLP profiles clearly resolved two groups, A and B, that were completely congruent with both species. However, more diversity was detected by AFLP, revealing several subgroups within each group. In many cases, even for isolates from the same district, AFLP haplotypes differed markedly. Phylogenetic analyses of multilocus DNA sequence data indicated that all isolates of SCAR type 1, AFLP group A were F. graminearum , whilst isolates of SCAR type 5, AFLP group B were F. asiaticum , demonstrating that it is an efficient method for differentiating these two species. Both species seem to have different geographic distributions within China. Fusarium graminearum was mainly obtained from wheat growing in the cooler regions where the annual average temperature was 15°C or lower. In contrast, the vast majority of F. asiaticum isolates were collected from wheat growing in the warmer regions where the annual average temperature is above 15°C and where FHB epidemics occur most frequently. This is the first report of the distribution of, and genetic diversity within, F. graminearum and F. asiaticum on wheat spikes throughout China.     

2010-2011年小麦条锈菌群体的温度敏感性与毒性及遗传多样性的关系

为了明确小麦条锈菌毒性、遗传多样性以及温度敏感性三者之间的关系,本研究利用21个已知抗条锈病基因近等基因品系对2010-2011年生长季采自6个省市78株已知温度敏感性(ET50)的小麦条锈菌群体进行毒性鉴定,并利用AFLP技术对其进行遗传多样性分析。苗期毒性鉴定结果表明,不同省市小麦条锈菌群体的毒性基因多样性存在一定差异,甘肃菌株群体毒性多样性指数H值最高,为0.269 3,云南菌株群体最低,为0.150 4。遗传多样性结果显示,6省市小麦条锈菌群体遗传多样性指数H值范围在0.125 5~0.165 3之间,遗传一致度GI为0.964 7~0.987 2,遗传距离GD为0.012 9~0.036 0。三者的相关性分析表明,条锈菌群体毒性多样性与平均ET50显著负相关,与ET50变异系数正相关,而与遗传多样性没有显著的相关关系。     

甘肃省大麦条纹病病原菌致病力分化、rDNA-ITS及遗传多样性分析

为明确甘肃省大麦条纹病病原菌麦类核腔菌Pyrenophora garminea的致病力分化、r DNA-ITS序列特征及变异,采用"三明治"法测定甘肃省大麦条纹菌的致病力,通过r DNA-ITS序列分析菌株间变异类型,并对菌株进行ISSR遗传多样性分析。结果表明,共筛选到43株大麦条纹病菌,生长7 d后的菌落直径为2.60~7.93 cm,其中菌株TB生长速度最快,菌株JT生长速度最慢,强、中等和弱致病力菌株分别为2、21和20株;43株菌的核糖体DNA-ITS序列与麦类核腔菌菌株SMCD2015同源性在99%以上;在9个菌株中检测到15个变异位点,共17种变异类型;8个ISSR标记从43个菌株中扩增出41条带,平均每个标记5.13条带,90.24%片段具有多态性,遗传相似系数为0.44~1.00,平均值为0.71,当遗传相似系数为0.68时,可将供试菌株划分为4个类群。表明甘肃省大麦条纹病病原菌存在致病力分化,菌株核糖体DNA-ITS序列变异丰富,且菌株间遗传结构复杂。     

Genetic variation within and between southern Ontario populations of Sclerotinia homoeocarpa

Restriction fragment length polymorphisms (RFLP) of the intergenic spacer region (IGS) of rDNA and random amplified polymorphic DNA (RAPD) markers were used to survey genetic variability among 181 isolates of Sclerotinia homoeocarpa from Ontario and 10 isolates from Japan. RAPD and IGS-RFLP analyses revealed polymorphisms within and between populations of S . homoeocarpa , distinguishing 151 genotypes. Both types of markers gave similar results in phenetic analysis of genetic distances between populations. Cluster analysis showed that Japanese isolates of S. homoeocarpa were genetically distinct from Ontario isolates, demonstrating significant intraspecific differentiation. An average genetic similarity of 0.66 was found between Japanese isolates. Among Ontario isolates, average genetic similarity was 0.86, and genotypic diversity analysis showed that 49.3% of the total genetic variation observed within Ontario populations occurred among individuals within populations compared to 50.7% between populations. Gametic linkage disequilibrium analysis within Ontario populations revealed an average 15.6% significant nonrandom associations between putative RAPD loci, and that half of the populations showed signs of significant linkage disequilibrium. These results suggest that both clonal propagation and recombination events occurred in local populations of S. homoeocarpa . The high level of genetic similarity between populations and the low levels of intraspecific genetic variation may reflect a small founding population for southern Ontario isolates of S. homoeocarpa .     

甜菜西方黄化病毒两个山东辣椒分离物全基因组扩增及序列分析

 通过RACE和RT-PCR技术相结合的方法,在山东省潍坊市和济宁市辣椒上克隆出2个甜菜西方黄化病毒(beet western yellows virus,BWYV)的全基因组序列,基因组全长均为5 699 nt。与GenBank比对发现,2个分离物的5′UTR(Untranslated region,UTR)变异程度小、相对比较保守,而3′UTR变异程度大、为突变热点区域。潍坊、济宁两地分离物与GenBank中该病毒的全基因组序列相似度平均值分别为89.89%和89.72%。系统进化树分析发现BWYV亚洲地区的分离物聚类在3个不同的组别,本研究克隆的2个山东分离物和3个日本分离物(LC428355、LC428356、LC428357)、2个韩国分离物(LC198684、KM076647)聚在一组,而美国分离物与法国分离物聚在其他分支,表明该病毒的进化存在地理相关性。这是首次对BWYV中国辣椒分离物的全基因组序列克隆,丰富了BWYV的序列信息,有助于进一步了解该病毒种群的遗传进化关系。     

甜菜西方黄化病毒两个山东辣椒分离物全基因组扩增及序列分析

 通过RACE和RT-PCR技术相结合的方法,在山东省潍坊市和济宁市辣椒上克隆出2个甜菜西方黄化病毒(beet western yellows virus,BWYV)的全基因组序列,基因组全长均为5 699 nt。与GenBank比对发现,2个分离物的5′UTR(Untranslated region,UTR)变异程度小、相对比较保守,而3′UTR变异程度大、为突变热点区域。潍坊、济宁两地分离物与GenBank中该病毒的全基因组序列相似度平均值分别为89.89%和89.72%。系统进化树分析发现BWYV亚洲地区的分离物聚类在3个不同的组别,本研究克隆的2个山东分离物和3个日本分离物(LC428355、LC428356、LC428357)、2个韩国分离物(LC198684、KM076647)聚在一组,而美国分离物与法国分离物聚在其他分支,表明该病毒的进化存在地理相关性。这是首次对BWYV中国辣椒分离物的全基因组序列克隆,丰富了BWYV的序列信息,有助于进一步了解该病毒种群的遗传进化关系。     

瓜类褪绿黄化病毒山东分离物全基因组序列扩增及分析

为明确分离自山东省寿光市甜瓜上的瓜类褪绿黄化病毒(cucurbit chlorotic yellows virus,CCYV)分离物的全基因组序列信息和遗传变异情况,利用毛形病毒属Crinivirus简并引物进行RTPCR检测,利用RACE技术结合RT-PCR方法克隆CCYV山东分离物2条RNA链的全基因组序列,通过与GenBank中其它地区CCYV分离物的全长序列进行比对分析其同源性,并基于CP基因序列构建系统进化树分析其遗传变异情况。结果表明,山东分离物经RT-PCR检测和测序后确定为CCYV。CCYV山东分离物与其它CCYV分离物的RNA1链和RNA2链的全基因组序列一致性的平均值分别为99.82%和99.88%,且2条链的5′末端均比较保守,没有碱基突变的情况发生;RNA1链3′末端存在2个碱基变异,RNA2链3′末端存在1个碱基变异。CCYV不同地区分离物主要分为3个簇群,其中山东分离物和中国其它地区分离物、日本分离物、苏丹分离物、黎巴嫩分离物和塞浦路斯分离物聚类在一起。研究表明CCYV基因组序列比较保守,该病毒的分化可能与地理来源存在一定的相关性。     

甘薯病毒2中国分离物全基因组序列克隆及遗传进化分析

正甘薯病毒2(Sweet potato virus 2,SPV2)是马铃薯Y病毒科(Potyviridae)马铃薯Y病毒属(Potyvirus)成员。SPV2也称为甘薯脉花叶病毒(ipomoea vein mosaic virus,IVMV)和甘薯Y病毒(sweet potato virus Y,SPVY)~[1],是甘薯上常见的病毒之一。SPV2病毒粒体为线条状,长度为850 nm,在细胞质中形成风轮状或卷轴状内含体~[2]。SPV2可由桃     

Molecular variability of the 5'- and 3'-terminal regions of citrus tristeza virus RNA

ABSTRACT Isolates of citrus tristeza virus (CTV) differ widely in their biological properties. These properties may depend on the structure of viral RNA populations comprising the different isolates. As a first approach to study the molecular basis of the biological variability, we have compared the sequences of multiple cDNA clones of the two terminal regions of the RNA from different CTV isolates. The polymorphism of the 5' untranslated region (UTR) allowed the classification of the sequences into three groups, with intragroup sequence identity higher than 88% and intergroup sequence identity as low as 44%. The variability of an open reading frame (ORF) 1a segment adjacent to the 5' UTR supports the same grouping. Some CTV isolates contained sequences of more than one group. Most sequences from Spanish isolates belonged to group III, whereas a Japanese isolate was composed mostly of sequences of groups I and II. The mildest isolates contained only sequences of group III, whereas the most severe isolates also contained sequences of groups I, II, or both. The most stable secondary structure predicted for the 5' UTR was composed of two stem-loops and remained essentially unchanged as a result of compensatory mutations in the stems and accommodation of most of the variability in the loops. In contrast to the 5'-terminal region, the variability of the 3'-terminal region of CTV RNA was very much restricted, with nucleotide identity values higher than 90%. The presence of a conserved putative "zinc-finger" domain adjacent to a basic region in p23, the predicted product of ORF 11, suggests that this protein might act as a regulatory factor during virus replication.