医用抗病毒剂脱除葡萄病毒的初步研究

以携带葡萄(Vitis spp.)斑点病毒(GFkV)、沙地葡萄茎痘伴随病毒(GRSPaV)、葡萄病毒A(GVA)、葡萄卷叶伴随病毒-1(GLRaV-1)、葡萄卷叶伴随病毒-2(GLRaV-2)、葡萄卷叶伴随病毒-3(GLRaV-3)和葡萄卷叶伴随病毒-6(GLRaV-6)等病毒的8个葡萄品种试管苗样品为材料,采用不同质量浓度的医用抗病毒剂利巴韦林注射液进行脱毒处理。结果发现,葡萄植株的死亡率较高,设置的4个质量浓度处理中,只有加入25 mg/L利巴韦林注射液处理的试管苗样品获得了再生植株。对分离成活的茎尖进行RT-PCR检测,结果表明,获得的5个葡萄品种的9株再生植株中,只有维多利亚的2株检测带有GRSPaV,其余均不带有所检病毒。     

甘肃葡萄扇叶病毒和卷叶病毒的检测

采用 DAS-ELISA 检测方法对甘肃地区的葡萄主要品种进行扇叶病毒和卷叶病毒的调查和测定。结果表明,这两种病毒病在甘肃地区的主要葡萄产区均有发生。兰州地区和一些老的葡萄产区葡萄带病毒病较严重。通过对比植株下部枝条的幼叶、幼茎、老叶和老茎病原的检测结果,说明植株下部幼嫩组织是卷叶病毒最适的检测部位。     

A sensitive SYBR Green RT-qPCR method for grapevine virus E and its application for virus detection in different grapevine sample types

To develop a rapid and high-sensitivity method for detection of grapevine virus E(GVE), a SYBR Green based real-time fluorescence quantitative RT-PCR method(RT-qPCR) was established. This method could be used to detect GVE specifically, and the sensitivity was about 100 times greater than conventional RT-PCR. An excellent linear correlation(R2=0.997) and a high amplification efficiency(E=97.5%) were obtained from the standard curve of this method. Reproducibility tests revealed that the coefficients of variation in the intra-and inter-assay results were 0.31–1.03% and 0.82–2.62%, respectively, indicating a good reproducibility. The RT-qPCR method could be used to detect GVE in a wide range of grapevine sample types. The detection rates of RT-qPCR for nearly all sample types from different positions and seasons were higher than conventional RT-PCR. The detection rates in spring, summer, autumn and winter increased gradually. Samples in autumn and winter were best for detection, and the detection rates of most samples were 80–100%, which were 10 to 40% higher than conventional RT-PCR. In general, old petioles and branches were the best tissues for GVE detection. The detection rates of these samples in each season were all 100%, which were 20 to 40% higher than conventional RT-PCR. The second highest rates were in the old leaf, with detection rates for RT-qPCR of 80–100% in all seasons, which were 20 to 40% higher than conventional RT-PCR. GVE could be difficultly detected in young leaves by conventional RT-PCR, and the detection rates were only 0–50%, while by RT-qPCR the rates could increase to 0–80%. A total of 33 out of 363 samples(belonging to 68 cultivars) from 20 regions in China were detected to be positive by RT-qPCR(9.1%), which was more than twice the rate of the conventional RT-PCR(3.9%).     

Identification of the DEAD-box RNA helicase family members in grapevine reveals that VviDEADRH25a confers tolerance to drought stress

Grapevine growing areas are increasingly affected by drought, which has greatly limited global wine production and quality. DEAD-box is one of the largest subfamilies of the RNA helicase family, and its members play key roles in the growth and development of plants and their stress responses. Previous studies have shown the potential of DEAD-box genes in the drought stress responses of Arabidopsis and tomato, rice, and other crop species. However, information about DEAD-box genes in grapevine remains limited. In this report, a total of 40 DEAD-box genes were identified in grapevine and their protein sequence characteristics and gene structures were analyzed. By comparing the expression profiles of VviDEADRHs in response to drought stress in different grapevine varieties, nine candidate genes (VviDEADRH10c, -13, -22, -25a, -25b, -33, -34, -36, and -39) were screened based on expression profiling data. Combined with qRT-PCR results, VviDEADRH25a was selected for functional verification. Heterologous overexpression of VviDEADRH25a in Arabidopsis showed the transgenic plants were more sensitive to drought stress than the control. Both electrolyte permeability and malondialdehyde content were significantly increased in transgenic plants, whereas the chlorophyll content and superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and ascorbate peroxidase (APX) enzyme activities were significantly decreased. Furthermore, VviDEADRH25a-overexpressing plants showed down-regulated expression levels of several drought stress-related marker genes, namely AtCOR15a, AtRD29A, AtERD15, and AtP5CS1, which indicated that they participated in the drought stress response. In summary, this study provides new insights into the structure, evolution, and participation of DEAD-box RNA helicase genes in the response to drought stress in grapevines.     

Effects of Partial Rootzone Drying on the Growth of Vitis vinifera cv. Malvasia Grafted on Different Rootstocks

To lay a biological foundation for rootstocks and alternate irrigation （AI） popularization, the effects of partial rootzone drying （PRD） on the growth of the grapevine Malvasia grafted on different rootstocks were investigated. Biological effects of 1/2 divided root irrigation on three combinations, i.e., Malvasia/420A, Malvasia/3309C, and Malvasia/110R, were studied by wood-boxed plants. All the plants were separated into three groups for different irrigation strategies. Mass growth of new root in alternate-irrigated plants was remarkably promoted by about 7.8-22.2% higher than the well- watered ones. However, new shoot growth, especially the internode was reduced by alternate irrigation. The average root-shoot ratio of all the three combinations was increased from 1.1 to 1.46. New root growth and internode length were decreased by fixed partial rootzone irrigation （FI） at different amount, M/3309C at 37.9 and 36.9%, M/110R at 18.4 and 22.5%, respectively. Total biomass of all the three combinations under FI decreased at the rate of 19.2-34.3% compared with well-watered ones. Water stress adaptation of grapevine mainly depends on rootstock. 110R is more efficient than 3309C and 420A in water stress adaptation. PRD-AI benefited root growth, thus improved the drought-resistant ability of grapevine.     

崇明水仙试管球脱毒技术

【目的】探索操作简单且高效的方法脱除或减弱崇明水仙携带的病毒,为脱毒球生产奠定基础。【方法】采用DHT+热处理、DHT+病毒唑处理等化学药剂与物理手段结合的方法,对崇明水仙试管球进行离体脱毒研究,脱毒结果采用指示植物法和RT-PCR检测法验证。【结果】热处理影响崇明水仙试管球生长和存活;热处理＋DHT处理后,病毒含量降低,DHT浓度小于15 mg•L-1,对试管球生长抑制作用较小。DHT30 mg•L-1+病毒唑30 mg•L-1处理,对崇明水仙NMAV病毒,起到了很好的抑制作用,用RT-PCR技术只检测到了极其微弱的病毒残留;NDV和NCLV对两种药剂较敏感,在10 mg•L-1时,RT-PCR检测不到病毒的特异谱带。【结论】热处理对崇明水仙试管球生长和存活有不利影响;DHT+热处理能明显降低病毒含量;DHT30 mg•L-1+病毒唑30 mg•L-1处理能将NMaV含量降低到极微弱的水平,不影响试管球存活,可通过增加两种药剂的浓度或多次继代来彻底脱除;DHT10 mg•L-1+病毒唑10 mg•L-1处理可以显著降低NDV和NCLV病毒含量, RT-PCR检测,没有出现病毒的特异谱带。     

抗水稻黑条矮缩病毒和南方水稻黑条矮缩病毒的双价RNA沉默载体的构建

为利用RNAi技术获取抗水稻黑条矮缩病毒(RBSDV)和南方水稻黑条矮缩病毒(SRBSDV)植株,分别针对两种病毒的S6和S10基因构建RNA沉默载体。采用重组PCR方法将RBSDV S6基因片段(R6)和SRBSDVS6基因片段(SR6)进行融合,获得600 bp的R6-SR6融合基因;将RBSDV S10基因片段(R10)和SRBSDV S10基因片段(SR10)进行融合,获得600 bp的R10-SR10融合基因。融合基因以反向重复的方式连入pBS载体,并定向插入到pCAMBIA1301载体上,获取了含有发夹结构的植物表达载体pCAMBIA1301-hp(R6-SR6)和pCAMBIA1301-hp(R10-SR10)。抗RBSDV和SRBSDV RNA沉默载体的构建为利用RNA沉默进行植物抗病毒研究奠定了基础。     

Neopestalotiopsis eucalypti,a causal agent of grapevine shoot rot in cutting nurseries in China

Grapevine (Vitis vinifera L.) is an economically important fruit crop in the world, and China ranks first in the production of grapes with approximately 15% of the world's total yield. However, diseases that cause the death of grapevine shoots pose a severe threat to the production of grapes. In this study, the fungus Neopestalotiopsis eucalypti was identified as a causal pathogen of grapevine shoot rot based on the morphology of conidia and a phylogenetic analysis. The phylogenetic analysis was performed with three isolates based on the combined sequence of internal transcribed spacer (ITS) region of ribosomal DNA, part of the translation elongation factor 1-alpha (Tef) and the β-tubulin (Tub2) genes. The three isolates were all identified as N. eucalypti. Pathogenicity tests of the three fungal isolates were conducted on grapevines shoots in vitro and in vivo. The results showed that all three fungal isolates caused severe rot lesions on the inoculated grapevine shoots, and N. eucalypti was re-isolated from the inoculated grapevine shoots. Therefore, N. eucalypti was confirmed as a causal agent of the grapevine shoot rot. This is the first report of N. eucalypti causing grapevine shoot disease in China.     

百合脱毒及病毒检测技术研究进展

百合是重要的观赏花卉，百合的繁殖主要通过无性繁殖方式进行，由于病害的侵染，常常造成植株体内的病毒积累。导致植株发生病毒病，最终严重降低百合植株的经济价值和观赏价值。因此，防止百合感染病毒病的应对策略是采用脱毒培养技术获得脱毒苗，并在各个生产环节进行严格的病毒检测。作系统地介绍了常用的脱毒技术——茎尖培养、珠芽培养、化学处理和热处理，以及主要病毒检测方法一指示植物法、电镜技术、酶联免疫法和分子生物学技术。     

硫酸盐对三氮唑核苷酸抑制番茄病毒病效果的影响

以人工接种TMV的盆栽番茄苗为受试寄主,测定添加硫酸盐对三氮唑核苷酸抑制番茄病毒病效果的影响.结果表明,5种硫酸盐对三氮唑核苷酸抑制TMV均有增效作用,其中以同时添加硫酸铜和硫酸锌[m(三氮唑核苷酸)∶m(硫酸铜)∶m(硫酸锌)=1∶10∶15]的增效作用最为显著,其毒力指数为176.根据三氮唑核苷酸和添加硫酸盐的混合物抑制番茄病毒病的测定结果,用"时间-剂量-抑制率"模型比较分析了添加硫酸盐对三氮唑核苷酸抑制病毒病的时间效应和剂量效应,结果表明,硫酸盐对三氮唑核苷酸抑制番茄病毒病的增效作用主要表现在有效时段的中、后期.     

Viral Diseases and Rehabilitation of Honeysuckle (<Emphasis Type="Italic">Lonicera caerulea</Emphasis> L.) Plants

The prevalence of viruses in honeysuckle plants depends on the location of the plantation and the variety composition. TBRV virus had the maximal prevalence (28–60%) in most blue-berried honeysuckle stands. Prevalence of the ArMV virus ranged from 0 to 26%, that of the RpRSV virus ranged from 6 to 17%, and that of the SLRSV virus ranged from 7 to 25%. Most varieties of honeysuckle (67%) were infected with a complex of viruses, whereas no viruses were detected in certain varieties. The varieties produced at the Lisavenko Research Institute for Selection and Seedage had a higher degree of viral infection than those produced at the All-Russia Vavilov Research Institute of Plant Breeding. The viruses found in blue-berried honeysuckle plants were mostly in the latent form, but green leaf mottle and interstitial chlorosis were observed on individual bushes from a number of varieties. A tendency for virus infection accumulation with the extension of plant life was observed. TBRV and SLRSV transmission through seeds was registered, which is indicative of the need for improvement of the hybrid forms involved in the breeding process. The duration of dry air thermotherapy of honeysuckle plants should not exceed 30–45 days. Combined use of thermotherapy and meristem culture provided for an 80% yield of healthy plants.     

越冬前北京和云南葡萄溃疡病组织内相关真菌的鉴定

为了解我国不同地区在冬季修剪窗口期葡萄枝干组织中与葡萄溃疡病相关的病原微生物的种类,探索葡萄溃疡病组织中病原微生物的年周期变化,从栽培和植保角度制定防控措施,采用微生物分离、生物学特性观察,以及ITS测序的方法,对北京和云南葡萄溃疡病组织内相关真菌进行分离和鉴定。从北京的材料中分离鉴定出5种真菌:45.0%葡萄座腔菌属(Botryosphaeria),30.0%镰刀菌属(Fusarium),15.0%刺盘孢属(Colletotrichum),7.5%木霉属(Trichoderma),2.5%茎点霉属(Phoma);从云南的材料中分离鉴定出7种真菌,32.0%茎点霉(Phoma),27.0%壳囊孢属(Cytospora),14.0%拟茎点霉属(Phomopsis),14.0%葡萄座腔菌属(Botryosphaeria),4.5%附球菌属(Epicoccum),4.5%镰刀菌(Fusarium),4.5%链格孢属(Alternaria)。研究结果首次揭示出北京和云南葡萄越冬前溃疡病组织具有多种植物病原微生物共同存在的特征及主要相关真菌的种类。     

湖北省蔬菜病毒病主要毒原种类检测

采用斑点酶联免疫吸附法(Dot-ELISA)和RT-PCR法对采自湖北省7个地区的966份疑似感染病毒的蔬菜样本进行黄瓜花叶病毒(Cucumber mosaic virus,CMV)、烟草花叶病毒(Tobacco mosaic virus,TMV)、番茄斑萎病毒(Tomato spotted wilt virus,TSWV)、番茄花叶病毒(Tomato mosaic virus,ToMV)、芜菁花叶病毒(Turnip mosaic virus,TuMV)、蚕豆萎蔫病毒(Broad bean wilt virus,BBWV)、黄瓜绿斑驳花叶病毒(Cucumber green mottle mosaic virus,CGMMV)共7种病毒的检测。结果显示检出阳性样本695份,占总样本数的71.9%。其中,十字花科蔬菜上主要毒原为TuMV,检出率高达86.4%,其次为CMV,检出率为26.7%;茄科、葫芦科和豆科蔬菜上主要毒原均为CMV,检出率分别为34.3%、59.2%和56.2%。CMV几乎能侵染所有蔬菜,为湖北省蔬菜病毒病的主要毒原。在检出的6种病毒中,存在TuMV+CMV、TuMV+BBWV、TuMV+CMV+BBWV、CMV+BBWV、CMV+ToMV和CMV+BBWV+TSWV+TMV等6种复合侵染类型,其中TuMV+CMV为十字花科蔬菜上的主要复合侵染类型,复合侵染率为23.8%,其他蔬菜上主要侵染类型为CMV+BBWV,复合侵染率为28.0%。     

VvFT和VvTFL1A基因在葡萄花发育中的表达特性研究

为分析''香妃’葡萄花发育中VvFT和VvTFL1A基因的功能,利用RT-PCR、原位杂交以及酵母双杂等方法研究其在葡萄不同器官、花发育中的功能和基因间的互作方式。结果显示:VvFT与VvTFL1A基因在不同器官中的表达不同,VvFT基因不但可以在花序中生成,而且参与了花发育的整个过程;VvTFL1A基因对花器官的发育影响不大。表明这2个基因在花发育中的作用可能相反。同时酵母双杂的试验表明VvFT和VvTFL1A蛋白都与VvFD蛋白产生互作,并且VvFT与VvFD蛋白之间的互作要强于VvTFL1A与VvFD蛋白,说明VvFT与VvTFL1A基因所起的功能可能都与VvFD有关。     

病毒抑制剂对蝴蝶兰病毒植株的脱毒效果

选取感染建兰花叶病毒(CyMV)和齿兰环斑病毒(ORSV)的蝴蝶兰植株进行离体培养,在芽增殖培养基中添加不同的病毒抑制剂,研究对CyMV和ORSV病毒的脱除效果.结果表明,氨基寡糖素具有很好的脱毒效果,对CyMV的脱除率为83.33％,对ORSV的脱除率为66.67％;病毒唑次之,对CyMV的脱除率为83.33％,对ORSV的脱除率为50％;中药类的脱毒效果不明显.     

宁夏地区葡萄卷叶伴随病毒遗传变异的PCR-SSCP分析

宁夏贺兰山东麓酿酒葡萄卷叶病病株率较高,严重影响了酿酒葡萄产业的健康发展,但有关其病原葡萄卷叶伴随病毒(Grapevine leafroll-associated virus, GLRaVs)的遗传变异情况则鲜有报道.利用RT-PCR技术对宁夏酿酒葡萄不同种植区的7个主栽品种的GLRaV-1和GLRaV-3进行分子检测,利用SSCP技术对扩增片段进行遗传变异分析.试验结果共获得8个GLRaV-1和13个GLRaV-3的阳性样本. 8个GLRaV-1分离物基于CP基因分析差异明显,可分为3类; 13个GLRaV-3分离物基于RdRp基因分析差异明显,可分为3类,而基于CP和HSP70基因分析可分为2类.说明宁夏酿酒葡萄不同种植区、不同品种的GLRaV-1和GLRaV-3种群分子特性存在差异且遗传变异明显.这有助于宁夏地区酿酒葡萄GLRaVs流行学调查和分子诊断分析,为该病害的防控提供理论依据.     

Molecular and biological characterization of melon-infecting squash leaf curl China virus in China

It has been reported that squash leaf curl China virus (SLCCNV) infects some Cucurbitaceae crops except for melon (Cucumis melo L.). A new disease of melon exhibiting severe leaf curl and dwarfing was observed in Hainan Province of China. In this study, the pathogen was identified as SLCCNV through biological and molecular characterization. The isolate (SLCCNV-HN) possess a bipartite genome, DNA-A (HM566112.1) with the highest nucleotide identity (99%) to SLCCNV-Hn (MF062251.1) pumpkin and SLCCNV-Hn61 (AM260205.1) squash isolates from China, whereas DNA-B (HM566113.1) with the highest nucleotide identity (99%) to SLCCNV-Hn (MF062252.1). Phylogenetic analyses based on the full-length SLCCNV-HN DNA-A and -B sequences indicated that SLCCNV-HN melon isolate is clustered with SLCCNV-Hn pumpkin, SLCCNV-Hn61 and SLCCNV-SY squash isolates from southern China, forming an independent cluster. Infectious clone of SLCCNV-HN was constructed and the melon plants were inoculated and the infection rate is 100%, the systemic symptoms in melon showed identical to those of melon plants infected in fields. Additionally, melon plants transmission of this virus by Bemisia tabaci with a transmission rate of 95% (19/20) showed leaf curl and dwarf symptoms 15 days post transmission, thereby fulfilling Koch's postulates. Analysis of genomic organization and phylogenetic trees indicated that SLCCNV-HN melon isolate belongs to the Begomovirus genus. To the best of our knowledge, this is the first characterization of melon-infecting SLCCNV through its genome, infectious clone and transmission.     

云南地区部分猪场猪呼吸道疾病综合症（PRDC）患病猪群中PRRSV，PCV-2，CSFV，PRV混合感染调查

 针对云南省不同地区不同季节猪呼吸道疾病综合征进行流行病学调查,根据其临床特征,在不同规模养殖场共采集1164份血清样品,利用ELISA和RT-PCR方法检测其猪瘟抗原CSFV,猪繁殖与呼吸综合征抗原PRRSV,猪伪狂犬病PRV gE抗体及猪圆环病毒2型PCV-2特异抗体。结果表明：各季节和不同生长阶段猪群存在一种及两种以上的病毒混合感染,四重感染相对较少。从全年看单独感染占39.10%,PCV-2感染率最高,其次是PRV,PRRSV和CSFV;二重感染占26.13%,以PCV-2+PRV最常见,其次是PCV-2+ PRRSV、PRV+PRRSV;三重感染占8.05%,PCV-2+ PRRSV+PRV最常见;有四重感染出现,仅占1.57%。从季节来看,春季和夏季混合感染最高,分别为76.53%和60.74%,冬季和秋季的混合感染率分别为59.50%和53.55%。从年龄和性别看,育肥猪的混合感染率高于仔猪,分别为68.66%和61.11%,种公猪的感染率高于母猪,分别为70.00%和55.51%。说明4种病毒有单独感染或混合感染,推测其中PCV-2在混合感染中可能充当免疫抑制的角色,混合感染使PRDC症状更明显,死亡率增高。