Elimination of Sugarcane yellow leaf virus from infected sugarcane plants by meristem tip culture visualized by tissue blot immunoassay

Sugarcane yellow leaf virus (SCYLV), a member of the Luteoviridae , is implicated in the sugarcane disease known as yellow leaf syndrome (YLS), which is characterized by yellowing of the leaf midrib followed by leaf necrosis and possible growth suppression. YLS is distributed worldwide and susceptible cultivars are commonly infected with SCYLV. However, not all cultivars infected with SCYLV show symptoms of YLS and some cultivars that show symptoms do so sporadically. Since it is difficult to obtain virus-free plants of susceptible cultivars, it has not been possible to study the factors involved in SCYLV infection nor the effects of infection on plant growth and yield. A tissue blot immunoassay was used to visualize in vivo presence of the virus so that virus-infected and virus-free plants could be distinguished. Meristem tip cultures were used to produce virus-free plantings of six SCYLV-susceptible sugarcane cultivars. Nearly all of the regenerated sugarcane lines remained virus-free over a period of up to 4 years, whether grown in isolated fields or in the glasshouse. Experimental re-infection of the virus-free plants by viruliferous aphids demonstrated that meristem tip culture did not affect susceptibility of sugarcane to SCYLV. Improved diagnosis and production of virus-free plants of SCYLV-susceptible cultivars will facilitate research to quantify the effect of the virus on yield and to analyse the processes involved in disease development.     

甘蔗黄叶病毒复制酶基因的克隆及序列分析

 The gene of RNA-dependent RNA polymerase (RdRP) was cloned by RT-PCR from Sugarcane yellow leaf virus-Fuzhou isolate (CHN-FJ1) and then cloned into pMD18-T vector. The sequence showed that the fragment comprised 1 212 nucleotides including part gene of ORF1 and ORF2. The ORF2 was involved the RdRP gene consisted of 995 nucleotides and encoded putative protein of 331 amino acids. Compared the nucleo-tide sequence and encoded putative protein of CHN-FJ1 with the other isolates from different countries, they shared the homology above 92.0%. Phylogenetic tree suggested that the sixteen isolates were classified into four types according to the amino acid sequence of the RdRP. One of the groups contained CHN-FJ1 and the other isolates from China, American, Brazil, Australia and Colombia;however, there was the closest relation between CHN-FJ1 and BRA-YL1 isolate from Brazil.     

甘蔗黄叶病毒外壳蛋白基因克隆及其实时荧光RT-PCR检测

 甘蔗黄叶病毒（Sugarcane yellow leaf virus, SCYLV）引起的甘蔗黄叶病是一种新的全球性病毒病害。本文以YLSCPF1和YLSCPR591为引物,采用RT-PCR方法克隆了甘蔗黄叶病毒福建分离物（CHN-FJ1）外壳蛋白（CP）基因,编码196个氨基酸。分析不同地理来源的SCYLV病毒分离物cp基因核苷酸及其推导编码的氨基酸序列,同源性达95%以上。根据cp基因的保守序列,设计1对特异性引物和TaqMan探针,建立了SCYLV的TaqMan实时荧光RT-PCR方法。结果表明,检测下限为初始质粒模板DNA 1 000拷贝/μL（约3.61 fg/μL）,比常规PCR方法的灵敏度提高100倍。检测甘蔗花叶病毒、宿根矮化病菌和黑穗病菌,没有典型的扩增曲线和无Ct值。应用实时荧光RT-PCR、常规RT-PCR和组织印迹免疫杂交（TBIA）对田间甘蔗叶片样品进行检测,阳性检出率分别为100%、61.5%和69.2%,表明该方法比常规RT-PCR和TBIA具有更高的灵敏度,适合于对SCYLV的检测。     

Movement of aphid-transmitted Sugarcane yellow leaf virus (ScYLV) within and between sugarcane plants

Sugarcane yellow leaf virus (ScYLV) is distributed worldwide and has been shown to be the cause of the disease sugarcane yellow leaf syndrome (YLS). This study was an investigation of the transmission and spread of ScYLV in Hawaii. Several aphids are known to transmit the virus, but investigation of infestation and transmission efficiency showed Melanaphis sacchari to be the only vector important for field spread of the disease. The initial multiplication of ScYLV in a virus-free plant occurred exclusively in very young sink tissues. When a single leaf was inoculated on a plant, that leaf and all older leaves remained virus-free, based on tissue-blot immunoassay, whereas meristems and all subsequently formed new leaves became infected. Therefore, only after those leaves which had already developed before inoculation had been shed, did the complete plant contain ScYLV. Spread of the viral infection to neighbouring plants in the plantation fields via aphids was relatively slow and in the range of a few metres per year. No indication of long-distance transfer could be seen. This indicates that it may be possible to produce and use virus-free seed cane for planting of high-yielding but YLS-susceptible cultivars.     

北京地区番茄黄化曲叶病毒病的鉴定及防治对策

番茄黄化曲叶病毒病是一种由烟粉虱传播的病毒病,给番茄生产造成严重威胁。2009年在北京郊区调查时发现部分保护地种植的番茄植株表现典型黄化曲叶症状。通过提取典型症状样品总DNA利用粉虱传双生病毒检测简并引物PA/PB,进行PCR扩增到541bp的特异条带。通过测序和核苷酸序列比对表明该序列与番茄黄化曲叶病毒序列相似性最高为99%。分子检测结果表明北京郊区部分保护地种植的番茄已被烟粉虱传播的番茄黄化曲叶病毒侵染危害。     

河北省番茄黄化曲叶病毒病的分子鉴定初报

双生病毒是一类具有孪生颗粒形态的植物单链病毒,目前双生病毒病害已在多个国家和地区的作物上造成严重危害。近年来,我国多省报道作物上有这类病毒的发生,且有逐年加重和扩散的趋势。根据危害番茄的双生病毒DNA A序列保守区设计简并引物,对2009年4月采自河北省魏县表现叶片黄化、曲叶症状的4个番茄样品进行PCR检测,均为双生病毒阳性,对样品的扩增片断进行了克隆测序,经序列比对分析,与番茄黄化曲叶病毒（Tomato leaf curl virus,TYLCV）山东分离物（FJ646611.1）序列相似性为99.25%~99.55%,说明河北番茄黄化曲叶病由TYLCV引起。     

番茄黄化曲叶病毒与番茄褪绿病毒复合侵染对番茄黄化曲叶病毒传播的影响

番茄黄化曲叶病毒(tomato yellow leaf curl virus, TYLCV)是一种由烟粉虱传播的单链环状DNA病毒, 在田间可与多种病毒发生复合侵染, 如番茄褪绿病毒(tomato chlorosis virus, ToCV)等?本文对比了TYLCV单独侵染和TYLCV与ToCV复合侵染对烟粉虱获取和传播TYLCV的影响?结果表明, 与取食TYLCV单独侵染的番茄相比, 取食复合侵染番茄的烟粉虱对TYLCV的传毒率显著提高, 且番茄植株和烟粉虱体内TYLCV的病毒积累量也显著提高?试验结果说明复合侵染会提高烟粉虱的传毒率, 促进TYLCV的发生与流行?     

侵染广西番茄的番茄黄化曲叶病毒的分子鉴定

番茄黄化曲叶病毒病是番茄生产上的毁灭性病害,严重影响番茄产量及品质。2019年从广西百色市采集疑似番茄黄化曲叶病叶片样品,采用滚环扩增(RCA)及基因克隆等方法,获得了4个烟粉虱传双生病毒的全基因序列,4个分离物的全长序列分别为2 776、2 781、2 752、2 781 bp,均编码6个开放阅读框;核苷酸相似性比较发现,4个分离物彼此间的相似性均在90%以上,与已报道的番茄黄化曲叶病毒Tomato yellow leaf curl virus (TYLCV)各分离物间的相似性也在91%以上;系统进化分析表明,TYLCV广西分离物BS-2和BS-4与TYLCV-Hunan、BS-1和BS-3与TYLCV-YN6553等分离物处于独立的小分支,说明TYLCV广西分离物与TYLCV-Hunan、TYLCV-YN6553具有较近的亲缘关系。本研究首次报道TYLCV在广西发生。     

野茼蒿黄脉病毒的田间寄主范围及其基因多样性特征

为明确野茼蒿黄脉病毒(Crassocephalum yellow vein virus,CraYVV)的田间寄主范围及其群体基因结构特征,分别采用克隆和测序技术对采自云南省西双版纳傣族州、红河哈尼族彝族自治州的7种杂草和2种作物进行CraYVV的分离和鉴定,并通过生物信息学软件对分离到的CraYVV进行基因组结构、重组及基因遗传结构分析。结果显示,在赛葵、龙葵、臭牡丹、水茄、豨莶、野茼蒿和一点红7种杂草以及茄子和草莓2种作物中均分离到CraYVV,共获得20条CraYVV全长序列;CraYVV所有分离物分属2个株系,将其命名为YJ株系和JH株系;JH株系具有地理隔离特征,与YJ株系存在一定的遗传距离;重组分析显示,CraYVV是由烟草曲茎病毒(tobacco curly shoot virus,TbCSV)和云南烟草曲叶病毒(tobacco leaf curl Yunnan virus,TbLCYnV)重组产生;遗传结构分析显示,CraYVV中C1基因变异最显著,其次是C4基因和基因间隔区。表明CraYVV能侵染5科9种杂草和作物,具有较广的寄主范围,且菜豆金色花叶病毒属病毒能够侵染草莓、CraYVV能够侵染茄子,显示来源于杂草的菜豆金色花叶病毒属病毒在自然条件下能够侵染作物。     

番茄黄化曲叶病毒在湖北首次报道

2014年春季,在湖北省武汉市发现种植的番茄表现植株矮缩,叶片上卷,叶缘黄化等症状。PCR检测结果显示,所有采集的病样中均存在菜豆金色花叶病毒属病毒。进一步通过滚环扩增方法获得了该病毒的湖北分离物HB01的全基因组。基因克隆及序列分析结果表明,该病毒基因组全长为2 781nt,与已报道的番茄黄化曲叶病毒(TYLCV)各分离物同源性在89.0%以上,而与来自中国不同地区的TYLCV分离物的同源率均在97.0%以上。因此,HB01属于TYLCV的一个分离物。     

Phylogenetic and recombination analysis of sorghum isolates of Sugarcane yellow leaf virus

Recombination has played an important role in evolution and genetic diversity of Sugarcane yellow leaf virus (SCYLV) isolates sequenced to date. This study found that three newly sequenced SCYLV sorghum isolates from the USA underwent intraspecies recombination. No statistical significance on probable progeny–parent relationships involving SCYLV sorghum isolates were found in possible interspecies recombination with 18 members of the Luteoviridae family. Sorghum isolates deposited in the GenBank database under accession numbers KT960995, KT960996 and KT960997 were phylogenetically closely related to SCYLV genotypes IND, CUB and CHN1, all members of phylogroup II. Networked relationships among the sorghum isolates showed that numerous incompatibilities occurred in the sequences. These conflicting signals were probably due to recombination, especially in KT960997, which was heavily impacted by recombination. The KT960997 accession was positioned on a distinct branch compared to other members of phylogroup II, suggesting that it has probably emerged as a new genotype. Future studies on molecular evolution may reveal further insights into the adaptation capacity of these SCYLV lineages to new environments.     

甘蔗黄叶病及其病原分子生物学研究进展

甘蔗黄叶病是由甘蔗黄叶病毒(Sugarcane yellow leaf virus,SCYLV)引起的一种病毒病害,在全球主要甘蔗种植国家或地区普遍发生,危害日益严重。SCYLV经甘蔗蚜虫以持久性方式传播,在寄主植株内主要侵染韧皮部组织。该病毒起源于黄症病毒科属间基因重组,被国际病毒分类命名委员会确认为黄症病毒科马铃薯卷叶病毒属成员。文章综述了甘蔗黄叶病毒生物学特征、病害发生和危害、病原鉴定和检测、分子进化和遗传多样性、基因组结构和基因功能以及抗病转基因等方面研究进展,并对甘蔗黄叶病抗病育种和防治措施作了讨论。     

Impact of Sugarcane yellow leaf virus on Sugarcane Yield and Juice Quality in Réunion Island

Sugarcane yellow leaf virus (SCYLV) was first detected in sugarcane of Réunion Island in 1997. A field experiment was undertaken to assess the potential impact of this virus on sugarcane production. The agronomic characteristics of SCYLV-infected plants were compared to those of virus-free plants of three sugarcane cultivars (R570, R577 and R579) which occupy more than 90% of the cultivated sugarcane area on Réunion Island. In the plant crop, significant losses in stalk weight (28%) and in sugar content (11%) were detected for cultivar R577, but not for either of the two other cultivars. In the first ratoon crop, yield reduction was detected for cultivar R577 (37%), but also for cultivar R579 (19%). Cultivar R577 also showed significant losses in sugar content (12%) due to reduced amount and quality of extracted cane juice. No yield reduction was found for cultivar R570, although stalk height and diameter were reduced in SCYLV-infected canes of this cultivar in the first ratoon crop. Leaf yellowing was observed at harvest of plant and ratoon crops when sugarcane was no longer irrigated, and 10–59% of symptomatic stalks could be attributed to the presence of SCYLV. The most severe yellowing symptoms were related to infection of sugarcane by the virus.     

侵染甜椒的番茄黄化曲叶病毒的分子鉴定和全序列分析

<正>番茄黄化曲叶病毒(Tomato yellow leaf curl virus,TYLCV)在世界范围内可危害多种作物,造成植株矮化、叶片皱缩变形、局部黄化等症状。该病毒自1964年首次报道以来已蔓延至世界多地。在我国2006年上海首次报道该病毒[1],随后江苏、山东、安徽、北京、河北、天津等地相继报道,危害严重。TYLCV为双生病毒科(Geminiviridae)菜豆金色花叶病毒属(Begomovirus)成员,基因组为单组     

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?     

番茄黄化曲叶病毒V 2基因原核表达及多克隆抗体制备

 通过PCR的方法,从番茄黄化曲叶病毒江苏分离物DNA中扩增到V2基因,并克隆到pMD18-T载体,序列测定结果显示其与报道的XH2分离物序列完全一致,核苷酸序列全长351 bp,编码115个氨基酸,推测分子量约13 kD。将该V2基因亚克隆到原核表达载体PET32a中获得重组表达载体PET32a-V2,转化大肠杆菌BL21（DE3）,IPTG可诱导1个分子量约为32 kDa的融合蛋白（含His标签）表达,经Ni⁺ NTA亲和柱纯化,获得纯化的重组蛋白,免疫家兔制备TYLCV-V2蛋白的多克隆抗体Anti-V2,间接ELISA测定Anti-V2的效价达1∶655 360,Western blot及DIBA分析结果表明Anti-V2可与V2蛋白发生特异血清反应,可为进一步研究V2蛋白的功能提供参考。     

北京地区番茄黄化曲叶病病毒分离物测定及株系的初步鉴定

 番茄黄化曲叶病毒病是番茄生产中的一种毁灭性病毒病害,2009年传入北京。利用烟粉虱传双生病毒简并引物PA/PB对2010年~2011年采集自北京市5个区县的53个番茄样品进行检测,30个表现典型黄化曲叶病症状的样品均扩增得到约500 bp的特异条带,测定了其中7个样品的部分序列,经序列比对分析表明其为番茄黄化曲叶病毒（Tomato yellow leaf curl virus, TYLCV）。利用TYLCV特异引物TJ-F/TJ-R、TY-F/TY-R对样品BJDXXY、BJFS02、BJFS03、BJMY2231进行TYLCV基因组克隆和序列测定,经分析4个样品携带的TYLCV基因组长度均为2 781碱基,编码6个蛋白。基因组序列比较发现,这4个分离物与TYLCV-Israel株系同源性达到98%以上;通过建立系统发育树,发现BJDXXY、BJFS02、BJFS03与河北分离物（HBLF4）、山东分离物（SDSG）亲缘关系较近,BJMY2231与上海分离物（TYLCV-Israel）、江苏分离物（JSNJ1）亲缘关系较近。     

侵染新疆加工番茄的中国番茄黄化曲叶病毒 DNA-A的基因组特征

 从新疆加工番茄上分离到病毒分离物XJ26-4,对其基因组DNA-A 全序列测定表明,XJ26-4 DNA-A 全长2 737 个核苷酸(GenBank 登录号:FN985163),具有典型的双生病毒基因组特征。进一步序列比较发现,XJ26-4 DNA-A 与中国番茄黄化曲叶病毒(Tomato yellow leaf curl China virus, TYLCCNV)各分离物的同源性最高,达到91. 2% ~ 99. 5% ,而与其他双生病毒的序列相似性均在79. 5% 以下,表明XJ26-4 是TYLCCNV 的一个分离物。这是首次明确新疆加工番茄受到粉虱传双生病毒的侵染。     

河北省番茄黄化曲叶病毒和番茄褪绿病毒复合侵染及分布

 番茄黄化曲叶病毒(Tomato yellow leaf curl virus, TYLCV) 及番茄褪绿病毒(Tomato chlorosis virus, ToCV)是危害番茄的主要病毒。2016~2017年,在河北省18个番茄主产区调查番茄病毒病危害情况,采集262份植株矮化、叶片黄化、褪绿、卷曲的番茄样品,利用分子生物学技术对病原进行鉴定。结果表明:2016~2017年河北省番茄病毒病发生普遍,所检样品中TYLCV的侵染率为68.66%,其中与ToCV复合侵染率为19.5%;除栾城、滦南、乐亭、永年4地样品暂未检测到ToCV外,其他14个采样点的样品均检测到ToCV,侵染率为19.5%,且全部表现为与TYLCV复合侵染,河北省番茄主产区暂未发现ToCV单独侵染的样品。