BBTV两个株系DNA组分1的克隆及序列分析

 对在生物学特性特别是寄主范围上存在明显差异的NSP株系和NS株系的代表分离物(广州天河分离物和高州分离物)的DNA组分1进行了克隆和序列分析,结果表明:2个代表分离物的DNA组分1全序列、ORF及其编码的氨基酸序列的变异率分别为3.2%、3.1%和2.8%,从而进一步确证了可以用寄主范围来作上述2个株系的鉴定。此外,这2个株系的DNA组分1、ORF及其编码的氨基酸序列分别与肖火根等报道的中国(广东)分离物、以及亚洲组和南太平洋组各分离物进行比较,结果表明:NS株系与肖火根等报道的中国(广东)分离物的亲缘关系很密切;这2个株系与亚洲组各分离物的差异均较小,而与南太平洋组各分离物的差异均较大,它们应属亚洲组。     

芋花叶病毒的RT-PCR检测及外壳蛋白基因序列分析

 采用RT-PCR技术对采自中国湖北、浙江和山东的91份芋[Colocasia esculenta（L.）Schott]样品的芋花叶病毒（Dasheen mosaic virus,DsMV）进行了检测,检出率为26.4%。对其中14个DsMV分离物的317 bp扩增产物（为外壳蛋白基因的一部分）序列分析的结果显示,各分离物内的核苷酸变异相对较低,而分离物间存在较大的分子变异,相似性为68.3% ~ 97.8%。对来自湖北和浙江的2个DsMV分离物DsMV-SCS和DsMV-JH的外壳蛋白基因（coat protein gene,cp）进行了测序,全长分别为951 bp和987 bp,二者cp核苷酸和氨基酸序列相似性分别为79.0%和82.3%,与已报道DsMV的cp核苷酸和氨基酸序列相似性分别为73.0% ~ 92.1%和74.8% ~ 98.2%,在构建的系统发育树上聚在两个不同的簇,各DsMV分离物的系统进化关系与其寄主和地理来源无显著相关性。     

成团泛菌 Ljb-2 对番茄黄化曲叶病毒病的田间防效初步研究

 成团泛菌（Pantoe agglomerans）Ljb-2 是从番茄生境中分离得到的对番茄黄化曲叶病毒病 （tomato yellow leaf curl virus disease,TYLCD）具有较好防效的拮抗细菌,其对温室中番茄感病品种‘格 瑞斯’TYLCD 的防控效果为49.77%。2012 年4—8 月在连云港进行的田间试验结果表明,该菌株在田间 条件下对感病品种‘格瑞斯’TYLCD 的防效达到57.12%。2012 年7—12 月,将该菌株进行田间防治试 验,结果表明,番茄移栽60 d 时,Ljb-2 对番茄中抗品种‘迪芬妮’TYLCD 的防效达到45.26%。同时, 用Ljb-2 处理番茄植株后,能显著增加叶片叶绿素a、叶绿素b 及总叶绿素含量,提高果实中维生素C、 可溶性糖、可滴定酸含量及糖酸比,对果实品质具有明显的改善作用。此外,对番茄的增产效果达到 20.05%。     

A蛋白斑点免疫金染色和双抗体夹心酶联检测齿兰环斑病毒的比较

A蛋白斑点免疫金染色检测齿兰环斑病毒提纯病毒的灵敏度为1.56 ng/μl,检测病汁液的稀释倍数为640倍.同时采用碱性磷酸酯酶标记抗体IgG.IgG包被酶联板的浓度为1 μg/ml,酶标抗体以1 /1000稀释使用.DAS-ELISA技术检测ORSV提纯病毒的灵敏度为0.048 ng/μl,检测病汁液的稀释倍数为20480倍.     

番茄褪绿病毒病在山东设施西葫芦上的发生及潜在危害

番茄褪绿病毒tomato chlorosis virus (ToCV)是我国蔬菜生产的重要新发病毒,其寄主范围逐年扩大。2019年10月项目组在山东寿光西葫芦温室调查时发现部分叶片呈现黄化、脉间褪绿,类似于ToCV侵染症状,并伴有烟粉虱发生。利用特异性引物检测发现,扩增的目的片段与GenBank中登录的侵染番茄的ToCV基因序列(登录号：KC887998.1)同源性高达99.58%,充分说明西葫芦植株已被ToCV侵染。通过对病毒病发生规律和烟粉虱虫口数量调查发现,西葫芦定植后1个月表现侵染症状的植株为2.0%,定植后2个月达到4.2%,定植后4个月后高达68.2%,病毒发生呈指数增长,而烟粉虱虫口数量却维持较低密度。从济南和德州采集的西葫芦疑似病叶和烟粉虱中也检出ToCV病毒,说明该病毒可能已经在山东省设施西葫芦主要种植区普遍发生,并经烟粉虱广泛传播,需引起高度重视。     

Identification of divergent variants of Grapevine virus A

Eight isolates of Grapevine virus A (GVA), which induced different symptoms in leaves of Nicotiana benthamiana, were recovered from various grapevines. The dsRNA patterns of two isolates, which consistently induced mild vein clearing (referred here as mild isolates of GVA) were similar, but different from those of other isolates of GVA. Analysis based on overall nucleotide (nt) sequence identity in the 3 terminal part of the GVA genome, comprising part of ORF3 (putative movement protein, MP), entire ORF4 (capsid protein, CP), entire ORF5 and part of 3 UTR, revealed that GVA isolates separate into three groups (I, II, III), sharing 91.0–99.8% nt sequence identity within groups and 78.0–89.3% nt sequence identity between groups. Mild isolates of the virus were group III and shared only 78.0–79.6% nt sequence identity with the other isolates. The comparison of predicted amino acid sequences for MP and CP revealed many amino acid alterations, revealing distinct local net charges of these proteins for mild isolates of the virus. Based on both conserved and divergent nt regions in the CP and ORF5, oligonucleotide primers were designed for the simultaneous RT-PCR detection of all GVA isolates and for the specific detection of the most divergent virus variants represented here by mild isolates of the virus.     

Intra-specific variation and inheritance of BYDV-PAV transmission in the aphid <Emphasis Type="Italic">Sitobion avenae</Emphasis>

Vector efficiency of 44 clonal lines (clones) of Sitobion avenae belonging to 31 different genotypes (distinct patterns for five microsatellite loci) originating from Western France was evaluated by transmitting the isolate PAV4 of BYDV-PAV to barley seedlings. Variation in transmission rates from 3.7% to 92.5% was observed, with significant effects of the aphid clone, of the plant species on which clones were collected, and of the reproductive mode of the clones. When genotypes are considered instead of clones, a continuum in transmission rates was observed. A subset of S. avenae clones was tested for transmission of one (10 clones) and 13 (4 clones) other BYDV-PAV isolates, and a clear clone effect modulated by an isolate effect was observed. Crosses were made between clones with different vectoring phenotypes and their F1 progeny were tested for PAV4 transmission. The narrow sense heritability of the PAV transmission character was rather high in the F1 families (h²=0.5) and the segregation analyses suggested an oligo/polygenic determinism of this character. The possibility of generating new transmission variants by sexual reproduction and its consequences on transmission mechanism studies and on BYD epidemics are discussed.     

基于全基因序列的中国北方3省(区)马铃薯Y病毒遗传多样性分析

本研究以马铃薯Y病毒(PVY)全基因组为基础,分析吉林、黑龙江和内蒙古3省(区)PVY群体遗传多样性和群体分化,并评估突变、重组、选择等遗传力所起的作用。根据已报道的PVY全基因序列保守区设计4对引物,采用片段重叠法对来自内蒙古和吉林的24个PVY分离物全基因序列进行测定,并联合NCBI中已登录的9个黑龙江分离物全基因组序列进行遗传多样性参数评估、群体分化检验和分子变异等分析。结果显示,我国北方3省(区)PVY群体遗传多样性高,其中内蒙古和黑龙江PVY群体遗传多样性高于吉林群体,并且3个群体之间呈现一定程度的遗传分化。分子变异分析发现在PVY基因组中存在1 786个变异位点,表明我国北方3省(区)PVY群体变异程度较高,并且这种高变异度有85.54%来自各个马铃薯种植区内PVY个体的遗传变异。重组分析和系统发育分析发现,我国北方3省(区)PVY群体中重组株系占比高达90.3%,并具有明显的株系多样性,表明PVY重组株系已成为我国北方3省(区)马铃薯种植区的流行株系。选择压力分析显示,使用FEL和IFEL法分别检测出501个和315个净化压力选择位点,这表明3省(区)PVY群体受净化选择压力为主。以上结果表明,中国北方3省(区)PVY群体遗传多样性高,突变、重组和自然选择都对遗传多样性和群体分化存在一定影响。     

灰飞虱体内水稻条纹病毒的检测

为评价灰飞虱体内水稻条纹病毒检测方法的适用范围,利用已经制备的水稻条纹病毒(Rice stripe virus,RSV)的多克隆抗体SV21和单克隆抗体3B9,采用多孔板间接ELISA、DIBA和Western blotting等三种方法进行单头灰飞虱Laodelphax striatellus Fallén(SBPH)体内RSV检测。结果表明,检测灵敏度以多孔板间接ELISA最高,其次为DIBA,Western blotting最低;用单克隆抗体3B9检测灵敏度高于多克隆抗体SV21。RT-PCR、IC-RT-PCR和DB-RT-PCR三种方法中,RT-PCR对单头灰飞虱稀释到400倍可检测到病毒;IC-RT-PCR在多克隆抗体SV21稀释浓度大于500倍时检测不出RSV,单克隆抗体3B9稀释浓度大于800倍时检测不到RSV;DB-RT-PCR检测结果显示,单头灰飞虱在稀释400倍后均无阳性反应。