稻曲病菌薄壁分生孢子制备的影响因素分析

本文研究了培养基、温度、振荡速度等因素对稻曲病菌Ustilaginoidea virens薄壁分生孢子产孢量的影响。结果表明,稻曲病菌薄壁分生孢子在培养第7天基本达到最大孢子量;该菌最适宜产孢的培养基为马铃薯煮汁,在煮汁中添加蔗糖可大幅提高产孢量;适宜的产孢温度为26~28℃;静止培养不利于产孢,振荡培养有利于产孢,并表现为转速越高产孢量越多;光照条件对产孢量没有影响。     

甘薯脉花叶病毒外壳蛋白基因在大肠杆菌中的表达及其抗血清的制备

 根据已报道的甘薯脉花叶病毒(Sweet potato vein mosaic virus,SPVMV)外壳蛋白（CP）基因的核苷酸序列合成引物,利用RT-PCR方法克隆了SPVMV河南分离物（SPVMV-HN）基因组3′端1.8 kb的基因片段,包括部分NIb 基因序列和完整的CP基因及3′端非编码区序列(3′UTR)。序列分析表明,SPVMV-HN的CP基因由996个核苷酸组成（GenBank登录号为FJ687211）,编码332个氨基酸残基。与已发表的SPVMV其他分离物相比,其推导的氨基酸序列一致性为95.2%～98.5%,与 SPVMV广东分离物的氨基酸序列一致性为97.9%。将CP基因克隆到原核表达载体pET-28a（+）上,SDS-PAGE分析表明,经IPTG诱导,CP基因在大肠杆菌BL21(DE3) pLysS中得到了高效表达。以表达的蛋白为抗原,免疫家兔,制备了SPVMV外壳蛋白的特异性抗血清。ACP-ELISA检测结果表明,制备的抗血清可用于田间甘薯样品的检测。利用SPVMV的抗血清,对采自全国14个省（市）的田间甘薯样品以及嫁接的巴西牵牛样品进行了检测,结果表明,SPVMV在我国甘薯上普遍存在。     

马铃薯A病毒CP基因的克隆与序列分析

利用根据马铃薯A病毒 (PVA)外壳蛋白 (CP)基因序列设计合成的一对引物 ,以带毒植物总RNA为模板 ,RT-PCR扩增得到长 0.8kb的目的片段。将目的片段转入大肠杆菌并进行了序列测定。测序结果与PVA其他分离物CP基因序列比较 ,发现其核苷酸同源性最高可达 99%。依据CP序列建立了PVA病毒的系统进化树并对PVA不同分离物CP氨基酸序列差异性做了分析     

甘薯潜隐病毒外壳蛋白基因的克隆、表达及其抗血清的制备

 根据已报道的甘薯潜隐病毒(Sweet potato latent virus,SPLV)外壳蛋白(CP)基因的核苷酸序列合成引物,利用RT-PCR方法克隆了SPLV河南分离物(SPLV-HN)的CP基因及部分3'端非编码区序列,序列分析表明,SPLV-HN CP基因由879个核苷酸组成(GenBank登录号为DQ399862),编码293个氨基酸残基。与GenBank中SPLV-CH(X84011)和SPLV-T(X84012)分离物的核苷酸序列相似性分别为96.8%和93.0%;与日本分离物(E15420)的核苷酸序列相似性为83.6%。将CP基因克隆到原核表达载体pET-30a(+)上,SDS-PAGE分析表明,经IPTG诱导,CP基因在大肠杆菌BL21(DE3)pLysS中得到了高效表达。以表达的蛋白为抗原,免疫家兔,制备了SPLV外壳蛋白的特异性抗血清。ACP-ELISA检测结果表明,制备的抗血清可用于田间甘薯样品的检测。     

甘薯羽状斑驳病毒外壳蛋白基因的分子变异

应用单链构象多态性(single-strand conformation polymorphism,SSCP)技术结合核苷酸序列测定的方法,对我国甘薯主产区11个省份的甘薯羽状斑驳病毒(Sweet potato feathery mottle virus,SPFMV)外壳蛋白(CP)基因的分子变异情况进行了研究.结果表明,SPFMV CP基因的RT-PCR产物表现了较丰富的图谱类型,50个分离物共产生9种主要的SSCP带型;对显示不同带型的20个样品的CP基因进行了序列测定和进化树分析,CP基因核苷酸序列一致性为77.2％～99.9％.说明这些样品的SPFMV的CP基因存在较大的分子变异,可划分为EA、RC、O和C4个株系.     

Pectobacterium spp. associated with bacterial stem rot syndrome of potato in Canada

Pectobacterium atrosepticum, P. carotovorum subsp. brasiliensis, P. carotovorum subsp. carotovorum, and P. wasabiae were detected in potato stems with blackleg symptoms using species- and subspecies-specific polymerase chain reaction (PCR). The tests included a new assay for P. wasabiae based on the phytase gene sequence. Identification of isolates from diseased stems by biochemical or physiological characterization, PCR, and multi-locus sequence typing (MLST) largely confirmed the PCR detection of Pectobacterium spp. in stem samples. P. atrosepticum was most commonly present but was the sole Pectobacterium sp. detected in only 52% of the diseased stems. P. wasabiae was most frequently present in combination with P. atrosepticum and was the sole Pectobacterium sp. detected in 13% of diseased stems. Pathogenicity of P. wasabiae on potato and its capacity to cause blackleg disease were demonstrated by stem inoculation and its isolation as the sole Pectobacterium sp. from field-grown diseased plants produced from inoculated seed tubers. Incidence of P. carotovorum subsp. brasiliensis was low in diseased stems, and the ability of Canadian strains to cause blackleg in plants grown from inoculated tubers was not confirmed. Canadian isolates of P. carotovorum subsp. brasiliensis differed from Brazilian isolates in diagnostic biochemical tests but conformed to the subspecies in PCR specificity and typing by MLST.     

一个马铃薯X病毒分离物的外壳蛋白基因序列分析与株系鉴定

对山东省侵染马铃薯的一个马铃薯X病毒(PVX)分离物PVX—SD1的外壳蛋白(CP)基因进行了克隆和序列分析。以提纯的病毒RNA为模板，应用RT-PCR扩增目的基因，通过常规的基因克隆法将扩增的CP基因导入pUC19载体，测序。结果表明，PVX—SD1的CP基因长719bp，可编码248个氨基酸；与Gen—Bank中报道的15个有代表性的株系或分离物相比较，核苷酸同源性在80．1％-99．7％，氨基酸同源性在89．8％-100％；与欧洲株系UK3仅1个核苷酸不同，同源性为99．7％，氨基酸同源性达100％，表明它们可能为同一株系，属于X^3组．     

Biological, serological, and molecular variabilities of clover yellow vein virus

ABSTRACT A comparative study was made on the host reactions, serological properties, and nucleotide sequences of the coat protein (CP) gene of 10 clover yellow vein virus (C1YVV) isolates and one bean yellow mosaic virus (BYMV) isolate collected from different host plant species and locations in Japan. Two strains of C1YVV isolates, grouped on the basis of host reactions on Chenopodium amaranticolor, C. quinoa, Nicotianaclevelandii, N. benthamiana, Vicia faba, and Trifolium repens, corresponded to two serotypes determined by double-antibody sandwich- and triple-antibody sandwich-enzyme-linked immunosorbent assay using three polyclonal and nine monoclonal antibodies. These results were also confirmed by nucleotide sequence analysis of the CP gene. The CP gene of C1YVV isolates of strain 1, including the Australian isolate C1YVV-B, had 93 to 98% nucleotide identities and 97 to 99.6% amino acid identities. The CP of C1YVV isolates of strain 2, including the New Zealand isolate C1YVV-NZ, had 92 to 98% nucleotide identities and 95 to 98% amino acid identities. The nucleotide identities and the amino acid identities between the two C1YVV strains were 82 to 84%, and 90 to 94%, respectively. When compared with the CP sequences of 12 C1YVV isolates, the CP sequence of the BYMV isolate had 71 to 73% nucleotide identity and 73 to 77% amino acid identity. Amino acid sequence differences among C1YVV isolates from strains 1 and 2 were located mostly at the N-terminal regions of the CP. Our results indicated that the C1YVV isolates studied could be separated into two strains on the basis of host reactions, serology, and the nucleotide sequence of the CP gene.     

Biological, serological, and molecular differences among isolates of potato a potyvirus

ABSTRACT Sequences of the coat protein (CP) and 3'-end nontranslated region (3'NTR) of 13 isolates and the helper component proteinase (HC) of nine isolates of potato A potyvirus (PVA) were determined and compared with the eight previously determined PVA CP and 3'NTR sequences and one HC sequence. CP amino acid (aa), 3'NTR nucleotide, and HC aa sequence identities were 92.9, 93.4, and 94.8%, respectively. Sequence data, serological tests, and the necrotic local lesions induced in the leaves of the potato hybrid 'A6' confirmed that tamarillo mosaic virus is a strain of PVA. The aa substitutions A6T and G7S in the CP N-terminus were correlated with loss of aphid transmissibility. Development of necrotic lesions or nonnecrotic symptoms in the systemically infected leaves or lack of systemic spread in potato cv. King Edward were used to place the PVA isolates into four strain groups, but this grouping was not correlated with any differences in CP, HC, or 3'NTR. Recognition of CP by three monoclonal antibodies was used to place the PVA isolates into three groups different from the four groups above. The epitopes of two mono-clonal antibodies were mapped by site-directed mutagenesis to the same lysine residue at the CP aa 34.     

甘薯褪绿斑病毒外壳蛋白的分子变异及其特异抗血清制备

 甘薯褪绿斑病毒(Sweet potato chlorotic fleck virus,SPCFV)是侵染甘薯的主要病毒之一。本研究利用RT-PCR方法克隆了SPCFV中国4个分离物的外壳蛋白(CP)基因。序列分析表明,cp基因全长900 bp,编码299个氨基酸残基。4个分离物cp基因的核苷酸序列一致性为78.3%～89.9%,推导的氨基酸序列一致性为91.3%～95.7%,存在较大的分子变异。不同分离物CP氨基酸序列N末端的第3-32位氨基酸为多变区。将四川分离物的cp基因克隆到原核表达载体pET-28a(+)上,SDS-PAGE分析表明,经IPTG诱导,cp基因在大肠杆菌BL21(DE3)中得到了高效表达。以表达的蛋白为抗原免疫家兔,制备了SPCFV CP的特异性抗血清。ACP-ELISA检测结果表明,制备的抗血清效价达1∶128 000,可用于田间甘薯样品的检测。     

大麦黄矮病毒PAV中国分离物外壳蛋白基因的克隆及同源性分析

 大麦黄矮病毒PAV株系由麦长管蚜和禾谷缢管蚜传毒。本研究通过RT-PCR、克隆和序列测定后,确认所得到的我国小麦PAV分离物的外壳蛋白基因片段由600个核苷酸组成,编码199个氨基酸。序列同源性比较结果显示,与BYDV的其它株系典型分离物的外壳蛋白基因同源性最高为74.5%,而与国外发表的PAV 8个分离物的CP基因核苷酸同源性为81%左右,且同源性比较的分值也较其它株系高。氨基酸序列的比较中,仅在46到60位氨基酸差别较大。     

葡萄卷叶伴随病毒1号和3号宁夏分离物部分基因序列分析

为明确葡萄卷叶伴随病毒(GLRaVs)在宁夏贺兰山东麓酿酒葡萄上的侵染状况,采用RT-PCR技术对40份酿酒葡萄样品中的GLRaV-1~GLRaV-5进行了外壳蛋白(CP)、复制酶(RdRp)和热激蛋白(HSP70)基因序列的克隆和分析。检测结果表明,在所检测的5种病毒中,除GLRaV-2和GLRaV-4未检测到外,GLRaV-1和GLRaV-3的检出率最高,分别为20.0%和32.5%,GLRaV-5的检出率仅为5.0%;有6个样品存在GLRaV-1和GLRaV-3两种病毒复合侵染。序列分析表明,GLRaV-1宁夏分离物的部分CP基因序列长度为232nt,其两种分离物间的核苷酸序列同源率为90%,与已报道的国内外其他分离物CP基因序列相比,其同源率为90%~99%;GLRaV-3宁夏分离物的CP基因序列长度为942nt,其两种分离物间的核苷酸序列同源率为40%,与已报道的国内外其他分离物CP基因序列相比,其同源率为40%~99%;GLRaV-3宁夏分离物的RdRp基因序列长度为683nt,其各分离物间的核苷酸序列同源率为90%以上,与已报道的国内外其他分离物RdRp基因序列相比,其同源率为90%~99%;GLRaV-3宁夏分离物的HSP70基因序列长度为546nt,其两种分离物间的核苷酸序列同源率为96%,与已报道的国内外其他分离物HSP70基因序列相比,其同源率为96%~99%。     

进口大豆上菜豆荚斑驳病毒的免疫捕获巢式RT-PCR检测

利用DAS-ELISA对进口大豆上BPMV进行检测发现,从美国进口的部分大豆样品对BPMV的多抗血清呈阳性反应;利用免疫吸附电镜观察发现,ELISA检测阳性的大豆种皮病汁液中存在直径约30nm的球状病毒粒子.根据BPMV外壳蛋白（CP）基因的保守序列设计了2对嵌合引物,建立了BPMV的高灵敏的免疫捕获巢式RT-PCR（IC-nested RT-PCR）检测方法.该方法经免疫捕获、反转录和2轮PCR扩增,能从带毒大豆种子中扩增到预期大小的DNA条带.序列测定与分析表明此条带的序列为BPMV部分CP基因,在系统关系树上与BPMV的其它分离物形成一簇亲缘关系很近.实验表明从进境大豆上检测到了BPMV.     

甘肃省18种药用植物病毒病调查及2种病毒病的鉴定

2012年6月至2013年9月对甘肃省宕昌县、漳县、岷县、渭源县、陇西县、临洮县等地区种植的具有疑似病毒病症状的半夏等18种药用植物进行调查及采样。采用黄瓜花叶病毒(CMV)、苜蓿花叶病毒(AMV)、烟草花叶病毒(TMV)、番茄花叶病毒(ToMV)、马铃薯Y病毒(PVY)、马铃薯X病毒(PVX)、芜菁花叶病毒(TuMV)等7种双抗体夹心免疫酶联检测(DAS-ELISA)试剂盒初检, 并对CMV和ToMV采用反转录PCR(RT-PCR)方法复检, 结果表明, 半夏、掌叶大黄和红花3种药用植物受到黄瓜花叶病毒(CMV)侵染; 马兜铃、土贝母及当归3种植物受到番茄花叶病毒(ToMV)侵染; 样品中未检测到上述其他病毒种类, 其余12种病毒样品的病原尚未确定。本研究为国内首次报道CMV病毒侵染掌叶大黄、红花, ToMV病毒侵染马兜铃、当归和土贝母。此研究为防治上述病毒病提供了依据。     

白草花叶病毒承德玉米分离物3′-cDNA 片段序列分析

 采自河北承德11 个表现矮花叶症状的玉米样品,用甘蔗花叶病毒(Sugarcane mosaic virus, SCMV)和白草花叶病毒
(Pennisetum mosaic virus, PenMV)简并引物扩增了基因组3′ 端约2. 1 kb 的片段并进行测序。Blast 结果表明其中8 个样
品含有PenMV。扩增到的PenMV 序列均为2 135 nt,包括部分NIb 基因(985 nt)、完整的CP 基因(909 nt)和3′-UTR(241
nt)。这8 个分离物CP 基因和3′-UTR 与GenBank 上其他PenMV 分离物相应序列的核苷酸一致率分别为89. 8% ~ 93． 4%
和95. 9% ~ 97. 9% 。根据扩增的2 135 nt 序列和CP 基因序列构建系统发育树,8 个分离物与GenBank 上其他PenMV 分离
物都分为2 个组:山西组和承德组。重组分析表明CD9 的CP 基因存在重组。     

Diversity of Plum pox virus isolates in Bosnia and Herzegovina

Sixteen Plum pox virus (PPV) isolates from several stone fruit cultivars, host species, orchards and geographical areas of Bosnia and Herzegovina were selected for typing, using serotype-specific monoclonal antibodies (MAbs) and PCR–RFLP, targeting the 3' terminal region of the coat protein (CP) and P3-6K₁ with restriction enzymes Rsa I and Dde I. Four PPV isolates were identified as PPV-M by serology and PCR; eight isolates were identified as PPV-D based on PCR–RFLP on both genomic regions, but were not recognized by the D-specific MAb4DG5. Four isolates from plum were identified as natural D/M recombinants (PPV-Rec), based on conflicting results of CP and P3-6K₁ typing. To investigate the genetic diversity of Bosnian PPV isolates in more detail, five isolates (three PPV-Rec, one PPV-M and one PPV-D) were partially sequenced in the region spanning the 3' terminal part of the NIb gene and the 5'-terminal part of the CP gene, corresponding to nucleotides 8056–8884. Nucleotide sequence alignment of recombinant isolates showed that they were closely related at the molecular level to previously characterized recombinants from other European countries, and shared the same recombination break point in the 3' terminal part of the NIb gene. This is the first report of naturally infected Prunus trees with PPV-M, PPV-D and PPV-Rec in Bosnia and Herzegovina. The high variability of the Bosnian PPV isolates fits with the presence of this virus in the country over a long period.     

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?     

苜蓿花叶病毒ELISA检测方法的建立及其在大豆病毒调查和抗性资源鉴定中的应用

苜蓿花叶病毒(alfalfa mosaic virus, AMV)是一种世界性分布、宿主范围广、具有严重危害性的植物病毒,能引起大豆的严重病害。本研究利用原核表达的AMV CP蛋白制备的抗血清,建立了高效、准确的AMV间接ELISA检测方法,并应用于病害调查和抗性鉴定,结果表明制备的3份抗血清对重组蛋白和AMV感染的大豆植物粗提液的效价均达到256 000倍,血清特异性分析结果显示3份抗血清仅识别感染AMV的大豆叶片,不识别感染大豆花叶病毒(soybean mosaic virus, SMV)的大豆叶片。通过建立的AMV间接ELISA与常规RT-PCR同时对采集的50份疑似感染AMV的大豆样品进行检测,有46份样品检测结果一致,符合率达92%。利用建立的AMV ELISA方法和课题组已建立的SMV ELISA方法对吉林省大豆主产区的大豆样品进行病毒检测的结果表明,病毒检出率为38.30%,SMV的检出率达30.85%,AMV的检出率达17.06%,复合侵染率为9.61%。对接种AMV的40个大豆品种进行抗性鉴定,结果显示40份大豆全部感染AMV,但是病毒载量存在差异,部分品种表现出AM...