Occurrence of Chrysanthemum chlorotic mottle viroid in Japan

Chrysanthemum chlorotic mottle viroid (CChMVd) was detected in Akita Prefecture, Japan, from chrysanthemums (Dendranthema grandiflorum) with distinct yellow leaf mottling and necrosis. The four clones are 398–399 nucleotides long and are thought to be the symptomatic type based on their UUUC sequence at positions 82–85 in the CChMVd tetraloop.The nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under accession numbers AB181857–AB181860     

Natural infection of Chrysanthemum stunt viroid in dahlia plants

For the first time, Chrysanthemum stunt viroid (CSVd) was detected in commercial dahlia bulbs in Japan. CSVd was found in 77.2% of the tested plants (Dahlia spp.). In nucleotide sequence analysis, a CSVd variant was detected consisting of 354 nucleotides, which differed slightly from previously reported CSVd variants. The nucleotide sequence data reported is available in the DDBJ/EMBL/GenBank databases under accession number AB255879.     

玉米褪绿斑驳病毒实时荧光RT-PCR检测方法研究

玉米褪绿斑驳病毒(Maize chlorotic mottle virus,MCMV)是我国对外公布的检疫性有害生物。本研究根据该病毒外壳蛋白基因的保守序列,设计得到特异性引物及Taqman荧光探针,建立了MCMV的实时荧光RT-PCR方法,并对其灵敏度与特异性进行了研究。该方法针对2个不同来源的毒株均能得到典型扩增曲线,而没有从小麦线条花叶病毒、玉米粗缩病毒和玉米矮花叶病毒的RNA得到扩增曲线,表明引物与荧光探针具有良好的特异性。针对玉米褪绿斑驳病毒RNA不同稀释度样品,实时荧光RT-PCR检测低限达到10-5稀释度,检测灵敏度要比普通RT-PCR高出100倍。因此,本研究建立的MCMV实时荧光方法具有特异性强、灵敏度高和快速有效的优点。     

玉米褪绿斑驳病毒3种PCR检测方法的建立与比较

 以带有玉米褪绿斑驳病毒(MCMV)的玉米叶片为材料,研究建立了MCMV 的普通RT-PCR、TaqMan 实时荧光RT-PCR 和SYBR Green Ⅰ实时荧光RT-PCR 检测方法,并比较了3种方法的灵敏度。结果表明:TaqMan 实时荧光RT-PCR 的检测灵敏度最高,最低检出量可达1.61 fg,SYBR Green Ⅰ实时荧光RT-PCR 略逊之,普通RT-PCR 的检测灵敏度则相对较低,与前两者相差10~100倍。     

基于MaxEnt模型分析气候变化下玉米褪绿斑驳病毒的潜在地理分布

为明确气候变化背景下玉米褪绿斑驳病毒 （maize chlorotic mottle virus, MCMV）的潜在分布范围,基于MCMV的全球分布数据,筛选影响其分布的关键环境变量,利用MaxEnt模型和ArcGIS软件预测MCMV在历史（1970—2000年）和未来（2021—2040年）气候条件下的潜在地理分布范围。结果表明, MaxEnt模型的受试者工作特征（receiver operating characteristic, ROC）曲线下面积（area under curve, AUC）均值为0.941,预测结果的准确性较高。历史气候条件下, MCMV在全球具有广泛的适生范围,在亚洲、美洲、非洲、欧洲和大洋洲均存在适生区;在我国其适生区主要集中于中部和南部地区,总适生区面积占我国陆地总面积的41.62%。未来气候情景下, MCMV在全球的适生性有所降低,在我国的总适生区面积有所减少,但适生范围仍较为广泛;未来气候变化虽不利于MCMV在全球的进一步扩张,但可能更利于其在欧洲生存。MCMV在我国的潜在地理分布范围广,特别是西南地区,在历史和未来气候条件下均存在高适生区,因此建议进境口岸检疫部门应加强检疫检测,严防MCMV传入;内检部门应加强检疫监测和防控,严防其扩散。     

一种高效的苹果褪绿叶斑病毒TaqMan探针实时荧光定量RT-PCR检测方法

为建立一种检测苹果褪绿叶斑病毒(Apple chlorotic leaf spot virus,ACLSV)的Taq Man探针实时荧光定量RT-PCR方法,根据ACLSV外壳蛋白基因(coat protein,cp)保守序列设计了特异性引物和Taq Man探针,以构建的ACLSV-cp重组质粒为阳性标准品绘制标准曲线,并对该方法的特异性、灵敏性、重复性进行检验。结果显示,以ACLSV-cp重组质粒为标准品建立的标准曲线相关系数达0.999,扩增效率为103.7%;建立的Taq Man探针实时荧光定量RT-PCR方法特异性好,与苹果茎沟病毒(Apple stem grooving virus,ASGV)、苹果茎痘病毒(Apple stem pitting virus,ASPV)、苹果锈果类病毒(Apple scar skin viroid,ASSVd)均无交叉反应;灵敏度为100拷贝/μL,比常规RT-PCR高100倍;批内和批间变异系数均小于0.84%。表明Taq Man探针实时荧光定量RT-PCR方法具有特异性强、灵敏性高、重复性好的优点,适用于实际样品中ACLSV的快速准确检测。     

Characterization of a new viroid strain from hops: evidence for viroid speciation by isolation in different host species

A new viroid was detected in hops cultivated in Akita Prefecture, Japan where it is prevalent in many hops fields. In a survey of hop samples collected during the 1986–2002 growing seasons, the new viroid was present in the major Japanese hop-cultivating areas as early as the 1980s. A single-stranded circular RNA of 368–372 nucleotides that assume a highly basepaired, stable, rod-like secondary structure, shares 93%–98% sequence homology with Apple fruit crinkle viroid (AFCVd) isolated from apple and 85%–87% with Australian grapevine viroid (AGVd) isolated from grapevine. Taking into account the present concept of viroid species, we conclude that the viroid is AFCVd. Circumstantial evidence suggests that AFCVd from apples and hops were endemic in Japan only where cultivation of the two host plants overlapped, thereby strongly supporting the possibility that AFCVd (or an ancestral viroid) was transmitted across the species barrier from apples to hops or hops to apples somewhere in the region. Phylogenetic analysis of AFCVd from hops, AFCVd from apples, and AGVd together with the other members of the genus Apscaviroid revealed that the Akita isolates of AFCVd from hops (AFCVd-hop) formed a cluster that is distinct from AFCVd-apple and AGVd. Accumulation of host-specific sequence variation following their isolation in different host species may be leading to the formation of two viroid species from a common ancestor.     

基于@RISK软件评估玉米褪绿斑驳病毒对我国玉米产业造成的潜在经济损失

为明确玉米褪绿斑驳病毒 （maize chlorotic mottle virus, MCMV）对我国玉米生产的经济损失,通过收集、整理玉米产量、种植面积、市场价格以及MCMV潜在地理分布、危害和防控等相关数据,基于随机模型利用@RISK软件分别预测MCMV在不防控和防控场景下对我国玉米产业造成的潜在经济损失。结果表明,在不防控场景下, MCMV对我国玉米产业造成的潜在经济损失总量的90%置信区间为329.44亿~508.96亿元;而在防控场景下, MCMV对我国玉米产业造成的潜在经济损失总量的90%置信区间为51.62亿~76.23亿元,可挽回潜在经济损失的90%置信区间为278.38亿~437.93亿元。说明MCMV严重威胁我国玉米生产,建议有关部门加强检疫阻截防控工作,保障我国玉米产业安全。     

First Report of <Emphasis Type="Italic">Pepper mottle virus</Emphasis> on <Emphasis Type="Italic">Capsicum annuum</Emphasis> in Japan

Pepper mottle virus, genus Potyvirus, was first identified in Japan based on particle morphology, host range, aphid transmission, and molecular classification using the nucleotide sequence of the coat protein gene and 3-untranslated region.     

Immunolocalization of Pepper mild mottle virus in Capsicum annuum seeds

To clarify the mechanism of seed transmission of Pepper mild mottle virus (PMMoV), the virus was immunolocalized in Capsicum annuum seeds using fluorescence microscopy. Two distinct patterns were observed: In the first, PMMoV was present in the epidermis and parenchyma but not in the endosperm or embryo; in the second, the virus was restricted to the surface of the epidermis and parenchyma. These findings shed light on the fundamental mechanisms of seed transmission of tobamoviruses and may aid in the design of new methods to prevent the spread of seedborne virus diseases.     

地被菊CVB病毒基因的RT-PCR检测

为建立可靠、灵敏且高效的地被菊中菊花B病毒(Chrysanthemum virus B,CVB)的检测方法,利用特异性CVB基因引物,通过RT-PCR技术进行了特异性、重复性和灵敏度测试,最终利用优化的RT-PCR体系对地被菊植株感染CVB病毒情况进行了检测。结果显示,以染病地被菊植株的总RNA为模板扩增出的特异性片段编码211个氨基酸,与基因数据库中其它来源的CVB基因外壳蛋白同源性为96%~99%,可确定为菊花B病毒外壳蛋白基因;重复性和灵敏度检测中均获得了清晰的目标条带,且1 ng的总RNA即可扩增出特异性片段;对7个地被菊品种共32个植株的检测中,CVB的检出率为56.2%,检测的准确率为68.75%。表明建立的地被菊CVB基因的RT-PCR检测体系可有效用于地被菊植株感染病毒情况的鉴定。     

Bionomics and importance of two species ofChaetocnema in rice yellow mottle virus transmission in lowland rice in Tanzania

Regular samplings were done of two important vectors in farmers’ fields during the 1999/2000 and 2000/01 rice seasons at crop stages susceptible to rice yellow mottle virus (RYMV) on a traditional rice variety (‘Supa’) under rainfed lowland conditions to provide information on the bionomics and importance of these vectors in the disease transmission. The population ofChaetocnema sp. (nr.varicornis Jacoby) (Coleoptera: Chrysomelidae) was significantly higher in hotspot than non-hotspot areas. However, there was no significant difference in theC. pulla Chapuis population between these two areas. In general, theChaetocnema sp. population was higher than that ofC. pulla, and both vectors reached the peak of their population at 63 days after planting. Early planting in the hotspot areas is suggested as a disease management strategy. Both vectors are naturally infective andChaetocnema sp. proved more efficient thanC. pulla in the transmission of RYMV.     

Construction of an infectious full-length cDNA clone of Chrysanthemum virus B

An infectious full-length cDNA clone of Chrysanthemum virus B (CVB, genus Carlavirus), was constructed. Four cDNA fragments covering the whole genome of CVB-S were cloned between the Cauliflower mosaic virus 35S promoter and the nopaline synthase (NOS) terminator. Chrysanthemum and garland chrysanthemum were inoculated with the constructed plasmid, named pCVB, using a gene gun system. As is the case in wild-type, CVB-infected plants, no visible symptoms were observed on plants inoculated with pCVB; however, western blotting and electron microscopy indicated the presence of the progeny virus of pCVB. pCVB could be a useful tool for analyzing the functions of carlaviral proteins.     

RT-PCR和实时荧光RT-PCR一步法检测大豆中菜豆荚斑驳病毒

针对进口大豆的菜豆荚斑驳病毒(Bean pod mottle virus,BPMV)检测,建立了一步法RT-PCR和一步法实时荧光RT-PCR检测方法.依据BPMV的外壳蛋白编码基因设计了特异引物和特异Taqman探针,特异引物的扩增片段约为500bp,阳性质粒的实时荧光PCR方法的检测下限为20fg/μL,是一步法RT-PCR方法的100倍,检测时间由约8 h缩短至4 h.种脐灰色斑驳的大豆种子BPMV检测呈阳性,种脐黑色斑驳的大豆种子BPMV检测呈阴性.     

病毒载体介导的基因沉默体系对朱顶红褪绿环斑病毒NSm基因的沉默效应

为探讨烟草脆裂病毒（tobacco rattle virus,TRV）载体介导的基因沉默技术对朱顶红褪绿环斑病毒（Hippeastrum chlorotic ringspot virus,HCRV）运动蛋白基因NSm的沉默效应,以本氏烟Nicotiana tabacum为材料,构建靶向HCRV NSm序列的基因沉默表达载体pTRV2-NSm,经农杆菌Agrobacterium介导侵染烟苗,检测其侵染效率,人工接种HCRV到沉默表达载体处理过的烟苗,通过观察发病症状、统计病情并应用实时荧光定量PCR(quantitative real-time PCR,qRT-PCR)技术检测TRV介导的基因沉默体系对HCRV侵染的沉默效应。结果表明,pTRV2-NSm沉默表达载体对本氏烟植株的侵染率达到95.00%,并且对本氏烟的生长无明显影响;与对照相比,经沉默载体处理的烟草植株接种HCRV后发病率显著降低,在接种后14 d发病率下降了90个百分点,病毒积累量明显下降,随着时间延长pTRV2-NSm对病毒的抑制效果持续升高,在接种后14 d对病毒病的防治效果最高,达93.13%;qRT-PCR检测发...     

High resistance to rice yellow mottle virus in two cultivated rice cultivars is correlated with failure of cell to cell movement

Rice yellow mottle virus (RYMV) accumulation in protoplasts and whole plants was investigated in two highly resistant cultivars, Tog5681 (Oryza glaberrima) and Gigante (Oryza sativa). Three susceptible cultivars, i.e. one O. glaberrima Tog5673 and two O. sativa (IR64, Ac. 2428), and a partially resistant cultivar (Azucena) were used as control. After inoculation, accumulation of coat protein (CP) and viral RNA were monitored on protoplasts, inoculated leaves, sheaths of inoculated leaves and newly infected leaves by serological and Northern blot analysis. Viral RNA accumulated to a similar extent in protoplasts from all cultivars studied. In contrast, three distinct in planta behaviors were noted. In susceptible plants (IR64, Tog5673 and Ac. 2428), there was high CP and RNA accumulation at 5 d.p.i. in whole plants, suggesting that cell to cell and vascular movements occurred before 5 d.p.i. in inoculated leaves. The second behavior concerned Azucena, which showed a delay (around 7 d.p.i.) of viral accumulation in inoculated leaves. The third behavior involved the highly resistant cultivars Tog5681 and Gigante. CP and viral RNA were not detected in these cultivars. The comparison of viral accumulation in protoplasts and plants suggested that resistance of the highly resistant cultivars Tog5681 (O. glaberrima) and Gigante (O. sativa) was not due to the inhibition of virus replication but rather to the failure of cell to cell movement.