进境美国小麦中小麦线条花叶病毒检疫鉴定

小麦线条花叶病毒(WSMV)是我国进境植物检疫性有害生物。2016年11月天津出入境检验检疫局通过反转录PCR(RT-PCR)和双抗体夹心酶联免疫吸附(DAS-ELISA)方法,从美国小麦中检出WSMV。利用实时荧光PCR(Realtime RT-PCR)、免疫捕获RT-PCR(IC-RT-PCR)和序列比对分析等方法进行了验证。这是我国在进境小麦中截获WSMV的首次报道。     

小麦线条花叶病毒检测及病毒在燕麦种子内的分布研究

小麦线条花叶病毒(wheat streak mosaic virus,WSMV)是禾本科粮食作物上重要的检疫性有害生物,能引起严重花叶、矮化和坏死。本文报道从美国进境的种用燕麦种子中检出WSMV,并研究了该病毒在种子内部的分布。挑选表面带有花纹的种子,利用两对不同的引物进行RT-PCR检测,结果均从样品中扩增出特异性条带。PCR产物测序分析结果表明为WSMV多聚蛋白部分序列。通过对幼苗及种子不同部位的检测,发现WSMV主要存在于种子稃皮及种皮内,而胚及胚乳中则未检出病毒,这一结果有助于提高WSMV的检出效率。     

宁波口岸首次从进境乌克兰玉米中截获检疫性有害生物——小麦线条花叶病毒

正2016年5月,宁波局对一批来自乌克兰的玉米进行抽样与检疫,发现该批玉米中部分玉米粒具有红色线状斑点,经分子生物学与血清学方法检测,证实携带小麦线条花叶病毒,这是全国首次从进境乌克兰玉米中截获小麦线条花叶病毒。此病毒属于进境植物检疫性有害生物,对粮食产量影响大,造成的经济损失严重;一旦传入我国,可对我国的农业与生态环境造成较大危害。小麦线条花叶病毒(wheat streak mosaic virus,WSMV)是一种重要的麦类作物病毒,在分类地位     

美国进口大豆中菜豆荚斑驳病毒和玉米褪绿斑驳病毒的检疫鉴定

菜豆荚斑驳病毒(Bean pod mottle virus,BPMV)和玉米褪绿斑驳病毒(Maize chlorotic mottle virus,MCMV)是我国进境植物检疫性有害生物。2018年6月秦皇岛海关通过反转录PCR(RT-PCR)和双抗体夹心酶联免疫吸附(DAS-ELISA)方法,从一批美国进口大豆以及杂质中检出BPMV和MCMV;同时利用实时荧光PCR(Real time RT-PCR)和序列比对分析方法进行验证。这是河北口岸首次在进境大豆中截获BPMV和MCMV。     

重组酶聚合酶扩增技术(RPA)快速检测玉米褪绿斑驳病毒

玉米褪绿斑驳病毒(maize chlorotic mottle virus,MCMV)是我国颁布的一种禁止进境的检疫性有害生物,自然寄主有玉米、小麦、黍、大麦等。自然情况下,MCMV常与甘蔗花叶病毒(sugarcane mosaic virus,SCMV)、玉米矮花叶病毒(maize dwarf mosaic virus,MDMV)、小麦线条花叶病毒(wheat streak mosaic virus,WSMV)共同侵染玉米,可造成玉米产量损失高达10%~90%(Hilker et al.,2017)。     

从进境印度尼西亚辣椒种子中截获番茄环斑病毒

本文利用DAS-ELISA方法从印度尼西亚进境的辣椒种子中初步筛选出疑似感染有番茄环斑病毒(ToRSV)的样品,利用IC-RT-PCR(immune capture RT-PCR)扩增特异性片段,进而对扩增的PCR产物进行了测序和序列比对,确认我们在进境的辣椒种子中检出了我国关注的检疫性有害生物——番茄环斑病毒。此前该病毒未有在印度尼西亚辣椒上危害的报道,是系统内首次在蔬菜种子上截获该病毒。     

论进境植物种苗检疫技术法规体系的构建

简要分析了我国进境植物种苗检疫管理状况,比较分析新西兰、澳大利亚和加拿大等国家在进境植物种苗检疫管理的经验和我国进境种苗管理存在的主要问题,提出了我国进境种苗检疫技术法规体系构建建议。     

进境菠菜种子中藜草花叶病毒的免疫捕获RT-PCR检测

本研究基于藜草花叶病毒(Sowbane mosaic virus,SoMV)外壳蛋白基因的保守区域设计特异性引物,建立了SoMV的免疫捕获反转录PCR(IC-RT-PCR)方法。应用建立的IC-RT-PCR方法,从来自丹麦和荷兰的进境菠菜种子中检测到SoMV。这是我国进境种子中截获SoMV的首次报道。     

美国进境樱桃果实中梨火疫病菌的检测

为了筛选快速、灵敏的梨火疫病菌检测方法,利用常规PCR、套式PCR和实时荧光PCR方法分别对美国进境的326批樱桃果实中梨火疫病菌进行检测。结果显示,3种PCR方法的检出率不同,不同引物或探针的检出率也存在差异。在常规PCR中,引物Ams3/Ams4c、P29A/P29B和PEANT1/PEANT2的检出率分别为35.28%、24.85%和16.87%;单管套式PCR和套式PCR的检出率分别为23.01%和50.61%;4种实时荧光PCR的检出率分别为17.48%(探针PA)、32.21%(探针Ams)、29.14%(探针ITS)和23.93%(SYBR GreenⅠ)。在所有试验方法中由引物P29A/P29B和PEANT1/PEANT2组成的套式PCR的检出率最高。检测结果证实了进境樱桃果实中存在梨火疫病菌DNA,套式PCR和常规PCR(引物Ams3/4c)可用于进境樱桃样品中梨火疫病菌的常规检测。     

豌豆种传花叶病毒检测鉴定

豌豆种传花叶病毒(pea seed-borne mosaic virus,PSbMV)世界广泛分布,在国外已造成豌豆大量减产,国内有仅小范围低密度分布的报道。加拿大豌豆种子中挑拣皱缩、黑斑等症状的豌豆,先采用双抗体夹心酶联免疫吸附(DAS-ELISA)方法检出PSbMV阳性,再利用反转录PCR(RT-PCR)、序列比对分析和构建系统进化树对阳性样本进行验证鉴定,结果显示与PSbMV序列相似度为99.25%,综上确定从进境豌豆种子中检出豌豆种传花叶病毒。     

An isolate of Wheat streak mosaic virus from foxtail overcomes Wsm2 resistance in wheat

Wheat streak mosaic virus (WSMV) is an economically important pathogen of wheat (Triticum aestivum) causing major yield losses in regions where severe infection occurs. To detect the presence of any new virus or new WSMV isolates, green foxtail (Setaria viridis) plants exhibiting virus-like symptoms were sampled in a summer-fallowed wheat field at the Agricultural Research Center-Hays, Kansas State University, Hays, Kansas. These plants were tested serologically for four wheat viruses: WSMV, Triticum mosaic virus (TriMV), High Plains wheat mosaic virus (HPWMoV) and Foxtail mosaic virus (FoMV). Among 38 plant samples exhibiting virus-like symptoms, 29 contained WSMV as indicated by ELISA. Four isolates from samples with relatively strong reactions were transferred to healthy wheat seedlings by mechanical inoculation in a growth chamber for pathogenicity testing. Three isolates were avirulent to a wheat variety RonL, which contains Wsm2, a gene providing temperature-sensitive resistance to currently prevalent isolates of WSMV. However, one isolate, KSH294, was able to infect RonL and showed more virulence on two other varieties/lines containing Wsm2. Further sequence and phylogenetic analysis of KSH294 confirmed that this isolate displays a sequence homology with WSMV, but has sequence differences making it distinct from previously identified WSMV isolates included in the phylogenetic analysis.     

Phylogenetic relationships within the family potyviridae: wheat streak mosaic virus and brome streak mosaic virus are not members of the genus rymovirus

ABSTRACT The complete nucleotide sequence of wheat streak mosaic virus (WSMV) has been determined based on complementary DNA clones derived from the 9,384-nucleotide (nt) RNA of the virus. The genome of WSMV has a 130-nt 5' leader and 149-nt 3'-untranslated region and is polyadenylated at the 3' end. WSMV RNA encodes a single polyprotein of 3,035 amino acid residues and has a deduced genome organization typical for a member of the family Potyviridae (5'-P1/HC-Pro/P3/6K1/CI/6K2/VPg-NIa/NIb/CP-3'). Because WSMV shares with ryegrass mosaic virus (RGMV) the biological property of transmission by eriophyid mites, WSMV has been assigned to the genus Rymovirus, of which RGMV is the type species. Phylogenetic analyses were conducted with complete polyprotein or NIb protein sequences of 11 members of the family Potyviridae, including viruses of monocots or dicots and viruses transmitted by aphids, whiteflies, and mites. WSMV and the monocot-infecting, mite-transmitted brome streak mosaic virus (BrSMV) are sister taxa and share a most recent common ancestor with the whitefly-transmitted sweet potato mild mottle virus, the type species of the proposed genus "Ipomovirus." In contrast, RGMV shares a most recent common ancestor with aphid-transmitted species of the genus Potyvirus. These results indicate that WSMV and BrSMV should be classified within a new genus of the family Potyviridae and should not be considered species of the genus Rymovirus.     

Comparison of ambisense m RNA of watermelon silver mottle virus with other tospoviruses

ABSTRACT Double-stranded genomic RNAs (dsRNAs) extracted from Chenopodium quinoa infected with watermelon silver mottle virus (WSMV) were similar to those of tomato spotted wilt virus (TSWV, serogroup I) and impatiens necrotic spot virus (INSV, serogroup III), except that the S dsRNA of WSMV is 0.75 and 0.6 kbp longer than those of TSWV and INSV, respectively. The complete nucleotide sequence of the genomic M RNA of WSMV was determined from cDNA clones generated from separated M dsRNA. The M RNA is 4,880 nucleotides in length with two open reading frames (ORFs) in an ambisense organization. The M RNA-encoded nonstructural (NSm) ORF located on the viral strand encodes a protein of 312 amino acids (35 kDa), and the G1/G2 ORF located on the viral complementary strand encodes a protein of 1,121 amino acids (127.6 kDa). The RNA probe corresponding to the NSm or G1/G2 ORF of WSMV failed to hybridize with the M dsRNAs of TSWV and INSV. Comparison of M and S RNAs of WSMV, TSWV, INSV, and peanut bud necrosis virus (PBNV, serogroup IV) revealed a consensus sequence of eight nucleotides of 5'-AGAGCAAU...-3' at their 5' ends and 5'-...AUUGCUCU-3' at their 3' ends. The low overall nucleotide identities (56.4 to 56.9%) of the M RNA and the low amino acid identities of the NSm and G1/G2 proteins (30.5 to 40.9%) with those of TSWV and INSV indicate that WSMV belongs to the Tospovirus genus but is phylogenetically distinct from viruses in serogroups I and III. The M RNA of WSMV shares a nucleotide identity of 79.6% with that of PBNV, and the two viruses share 83.4 and 88.7% amino acid identities for their NSm and G1/G2 proteins, respectively. It is concluded that they are two related but distinct species of serogroup IV. In addition to the viral or viral complementary full-length M RNA, two putative RNA messages for the NSm gene and the G1/G2 gene, 1.0 and 3.4 kb, respectively, were detected from the total RNA extracted from WSMV-infected tissue of Nicotiana benthamiana. The 1.0- and 3.4-kb RNAs were also detected in the viral RNAs extracted from purified nucleocapsids, suggesting that the putative messages of the M RNA of WSMV can also be encapsidated by the nucleocapsid protein.     

2009年－2019年全国口岸进口大麦中检疫性有害生物截获情况分析

为给进口大麦中检疫性有害生物截获策略提供参考,本研究统计了全国口岸2009年-2019年大麦进口基本情况,比较并分析了不同来源国(或地区)大麦中截获杂草、软体动物、真菌、昆虫、病毒等检疫性有害生物的种类数和种次数。结果表明,近11年来我国大麦进口量呈波动上升的趋势,其中2015年大麦进口量最大。截至2019年,大麦中截获检疫性有害生物共计70种,包括杂草53种、软体动物2种、真菌5种、昆虫8种、病毒2种,其中疫情最突出的是杂草。从来源国来看,澳大利亚、法国、加拿大、乌克兰是截获检疫性有害生物种类及种次数最多的国家。其中,澳大利亚、法国大麦中主要截获杂草、软体动物、真菌等有害生物,加拿大大麦中主要截获杂草、真菌及病毒,乌克兰大麦则主要截获杂草、软体动物及昆虫。本研究汇总了2009年-2019年不同来源国(或地区)进口大麦中截获检疫性有害生物的名单,为进口大麦检疫工作提供参考。     

Serological and Molecular Characterization of a High Temperature-recovered Virus Belonging to Tospovirus Serogroup IV

A serologically and cytologically distinct gloxinia tospovirus (HT-1) previously isolated from a gloxinia plant infected with Impatiens necrotic spot virus (INSV) when propagated in a high-temperature environment was characterized. Rabbit antisera produced for INSV and Tomato spotted wilt virus (TSWV) nucleocapsids (N) failed to react with HT-1 proteins in western blot analysis. The HT-1 antibodies reacted strongly with homologous antigen but failed to react with INSV and TSWV. However, the HT-1 antiserum reacted in ELISA with Watermelon silver mottle virus (WSMV) from Taiwan and in western blot analysis with the WSMV N protein. A reciprocal test showed that the antiserum prepared against the N protein of WSMV also reacted with the HT-1 N protein in both ELISA and western blot analysis. DNA probes derived from the N gene of HT-1 or WSMV hybridized to RNAs prepared from plants infected with either virus. Stronger signals were obtained with homologous than with heterologous reactions. Neither probe detected INSV or TSWV. The M and S RNAs of HT-1 were sequenced. The M RNA contains two open reading frames (ORF) ; one in the sense orientation encoding a nonstructural (NSm) protein of 308-amino-acids (aa) and the other in the ambisense orientation, a 1122-aa precursor of Gl and G2 glycoproteins. The S RNA also contains two ORFs ; one in the sense orientation encoding a nonstructural (NSs) protein of 439 aa and the other in the ambisense orientation, an N protein of 277 aa. HT-1 is distantly related to INSV and TSWV as shown by low nucleotide (40–52%) and amino acid (28–48%) similarities in the four ORF sequences. The HT-1 virus shares high nucleotide (76–81%) and amino acid (85–92%) similarities with WSMV and peanut bud necrosis virus (PBNV). Based on the serological properties and sequence data, we propose that HT-1 is a distinct species of serogroup IV in the genus Tospovirus. This is the first time that a tospovirus similar to those found in the Far East and in Southeast Asia has been identified in the US. Received 16 October 1999/ Accepted in revised form 20 December 1999     

新疆小麦线条花叶病毒(WSMV)的研究

 在新疆五家渠分离到一株病毒,鉴定为小麦线条花叶病毒WSMV,致死温度55~60℃,体外存活期3~7天,稀释限点500~1000倍。病毒颗粒长度700×18毫微米,在感病的小麦叶细胞质中观察到风轮状包含体。其病原由螨(Aceria tulipae)传播,人工接种可侵染大麦(Hordeum vulgare L.)小麦、(Triticum acestivum L.)、黑麦(Secale Cereale L.)、燕麦(Avenae sativa L.)、高粱(Sorghum valgare L.)、黍(Panicum miliaceum L.)、苋色藜(Chenopodium amaranitcolor Cost et Reyn)表现系统花叶病状,可通过介体感染玉米。
该病毒与玉米矮花叶病毒,大麦条纹花叶病毒,马铃薯Y病毒的血清反应为阴性。该病毒在我区的发生量约占小麦上总病毒数量的80%以上。     

乳状耳形螺的检疫鉴定与风险管理措施

本文采用贝壳形态和软体解剖相结合的方法对广东出入境检验检疫局从西班牙进境集装箱中截获的一头活体蜗牛标本进行鉴定,结果确认为乳状耳形螺Otala lactea(Müller,1774),为国内首次截获。该螺原产于地中海东部沿岸地区,是当地蔬菜、瓜果、花卉最重要的有害生物之一,被美国、加拿大、澳大利亚等国家及有关国际和地区植物保护组织列为检疫性有害生物。该螺在中国无分布,本文在风险评估的基础上,提出了相应的风险管理措施。     

Resistance to Wheat streak mosaic virus– a survey of resources and development of molecular markers

Wheat streak mosaic virus (WSMV) has been newly documented in Australia. The vulnerability of contemporary Australian elite wheat germplasm prompted a survey for effective resistance against an Australian isolate, WSMV‐ACT. This study confirms the effectiveness of previously reported sources of resistance and shows that new sources of resistance also confer protection. The resistance derived from Thinopyrum intermedium (Wsm1) as a 4D translocation and a new 4A translocation, and two bread wheat resistances, Wsm2 and the new source c2652, were effective against WSMV‐ACT in glasshouse experiments. Wsm1 was effective at lower temperatures but ineffective above 20°C, a temperature sensitivity shared with many of the derivatives of Wsm2 except for one new selection which was effective at 26°C. True wheats c2652 and Wsm2 selection CA745, and amphiploids Zhong1, Zhong2, Zhong4, Zhong5, TAF46, Summer1, Ot38 and OK7211542 were uniformly resistant at 20, 25 and 28°C. New sources of resistance were identified in a Th. scirpeum‐wheat amphiploid, B84‐994, and in chromosome addition lines Z2, Z6 and TAi27, derived from wheat‐Th. intermedium partial amphiploids. Several new, tightly linked SSR, RAPD and EST‐ILP PCR markers were developed for tracking the various Th. intermedium translocations associated with Wsm1, including the smaller translocations on wheat chromosome 4AS and 4DS. Three markers for the 4A‐Wsm1 translocation were validated on a segregating breeding population.