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广西三种甜瓜病毒分离物的分子检测与鉴定 总被引：1，自引：0，他引：1
瓜类褪绿黄化病毒Cucurbits chlorotic yellows virus(CCYV)、甜瓜黄化斑点病毒Melon yellow spot virus(MYSV)及甜瓜坏死斑点病毒Melon necrotic spot virus(MNSV)是近年来报道侵染瓜类作物的新病毒,在个别种植区大面积发生,对生产构成严重威胁。为了解3种病毒在广西的发生情况,先后到各西甜瓜种植区进行了调查,并采集疑似CCYV、MYSV及MNSV的甜瓜病叶样品,提取病叶总RNA,通过特异性引物分别进行一步法RTPCR扩增,电泳结果显示RT-PCR产物与预期大小一致的序列条带;将扩增产物分别连接到pMD19-T克隆载体上,挑选阳性克隆子进行序列测定及比对分析。结果表明:CCYV、MYSV和MNSV广西分离物的CP基因序列与其他已报道核苷酸序列一致性分别达95.1%~100%、96.5%~99%和83.7%~92.5%。 相似文献
2.
两种甜瓜病毒寿光分离物的分子检测与鉴定 总被引：3，自引：0，他引：3
甜瓜坏死斑点病毒(Melon necrotic spot virus,MNSV)及瓜类褪绿黄化病毒(Cucurbit chlorotic yellows virus,CCYV)近年来在瓜类种植区均大面积发生,危害较为严重,已成为制约甜瓜生产的重要因素。本研究广泛收集疑似感染MNSV及CCYV的甜瓜病叶,从中提取植物总RNA进行RT-PCR扩增,将产物分别连接到pEASYT1Simple克隆载体上,对含有目的片段的重组子进行测序及比对分析。结果显示得到了与预期结果一致的DNA序列,其扩增产物大小分别为673bp(MNSV)和877bp(CCYV)。同源性分析结果表明,MNSV和CCYV寿光分离物的核苷酸序列与中国其他地区或一些国家已报道的分离物同源性达99%~100%。 相似文献
3.
为明确分离自山东省寿光市甜瓜上的瓜类褪绿黄化病毒(cucurbit chlorotic yellows virus,CCYV)分离物的全基因组序列信息和遗传变异情况,利用毛形病毒属Crinivirus简并引物进行RTPCR检测,利用RACE技术结合RT-PCR方法克隆CCYV山东分离物2条RNA链的全基因组序列,通过与GenBank中其它地区CCYV分离物的全长序列进行比对分析其同源性,并基于CP基因序列构建系统进化树分析其遗传变异情况。结果表明,山东分离物经RT-PCR检测和测序后确定为CCYV。CCYV山东分离物与其它CCYV分离物的RNA1链和RNA2链的全基因组序列一致性的平均值分别为99.82%和99.88%,且2条链的5′末端均比较保守,没有碱基突变的情况发生;RNA1链3′末端存在2个碱基变异,RNA2链3′末端存在1个碱基变异。CCYV不同地区分离物主要分为3个簇群,其中山东分离物和中国其它地区分离物、日本分离物、苏丹分离物、黎巴嫩分离物和塞浦路斯分离物聚类在一起。研究表明CCYV基因组序列比较保守,该病毒的分化可能与地理来源存在一定的相关性。 相似文献
4.
瓜类褪绿黄化病毒新疆分离物基因组分析 总被引：2，自引：0，他引：2
2016年秋季,新疆维吾尔自治区吐鲁番市哈密瓜大面积发生叶片黄化和褪绿的病害,症状呈瓜类褪绿黄化病毒(Cucurbit chlorotic yellows virus,CCYV)引起的典型特征。通过CCYV的特异性引物对该疑似样本进行了RT-PCR检测,获得了预期大小的扩增片段。随后,对新疆的CCYV分离物(CCYV-Xinjiang)全长基因组进行克隆并测序,获得了CCYV-Xinjiang分离物的RNA1和RNA2全长序列信息。对CCYV-Xinjiang和来自Gen Bank数据库的其他CCYV分离物的基因组序列分析,不同分离物RNA1和RNA2两条链的一致性分别是99.65%~99.93%和99.68%~99.89%。单独对基因组的各个非翻译区(UTRs)和编码区的核苷酸变异分析,数据表明不同区域其变异程度不同,其中RNA2的3'UTR区域变异最大,而RNA1的2个UTRs区域变异最小。对已登录Gen Bank的所有CCYV分离物基因组系统进化树分析结果表明,所有分离物聚在同一个组。另外,对来自Gen Bank的18个分离物的外壳蛋白(CP)氨基酸序列进行系统进化树分析,表明来自伊朗的3个分离物分在组2(GroupⅡ),而来自亚洲其他地区的分离物均被分到组1(GroupⅠ)。基于CCYV基因组的信息,发现来自亚洲地区的CCYV的群体遗传变异很小。 相似文献
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瓜类褪绿黄化病毒(cucurbit chlorotic yellows virus, CCYV)给我国葫芦科作物造成了严重的经济损失。2014—2019年持续调查山东葫芦科作物产区CCYV的发生情况。调查结果表明：CCYV可以自然侵染西瓜、甜瓜、南瓜、黄瓜和西葫芦,样品检出率由2014年的1.7%增加到2019年的29.1%,西葫芦地块发病严重有进一步蔓延的趋势。利用RT-PCR结合RACE技术扩增CCYV西葫芦分离物全基因组序列。结果表明：CCYV西葫芦分离株的基因组RNA1和RNA2与GenBank分离株的相似性均为99.6%～100%,基于CCYV全基因组的系统发育进化树均聚类在一个分支上,与各个蛋白的进化树和相似性分析结果一致,CCYV在传播的过程中与寄主无关但可能跟地理位置有关。本研究扩增分析了CCYV西葫芦分离物的基因组序列,对CCYV的变异现状和进化趋势研究具有一定的参考价值。 相似文献
8.
瓜类褪绿黄化病毒(cucurbit chlorotic yellows virus, CCYV)是近些年来出现的一种烟粉虱 Bemisia tabaci传播的植物病毒,在瓜类生产中造成了严重损失。为了解该病毒外壳蛋白(coat protein, CP)在病毒侵染寄主过程中的亚细胞定位和致病作用,本研究以CCYV山东黄瓜分离物为研究对象,构建了荧光表达载体CP-YFP和异源表达载体pGR-CP。浸润荧光表达载体48 h后的本氏烟Nicotiana benthamiana叶片在激光共聚焦显微镜下可以观察到荧光信号在细胞质和细胞核内均有分布;浸润异源表达载体的本氏烟植株7 d后上部叶片开始显现花叶症状,13 d后顶部新叶出现严重皱缩和坏死斑点。以上结果表明CCYV CP蛋白参与了病毒对寄主的侵染过程,可能是症状形成的致病相关因子。本研究为进一步解析该病毒的致病机理提供了初步的理论支持。 相似文献
9.
瓜类褪绿黄化病毒外壳蛋白基因在大肠杆菌中的表达及抗血清的制备 总被引：1，自引：0，他引：1
根据已报道的瓜类褪绿黄化病毒(Cucurbit chlorotic yellows virus, CCYV)外壳蛋白(CP)基因的核苷酸序列设计引物,利用RT-PCR方法从感病甜瓜中扩增得到长度为753 bp的CP基因,将其克隆到原核表达载体pGEX-4T-3上,获得重组载体pGexCP,在大肠杆菌BL21菌株中诱导表达,SDS-PAGE分析表明,经IPTG诱导,CP基因在大肠杆菌BL21中得到了高效表达。以表达的蛋白为抗原,免疫家兔,制备了CCYV外壳蛋白的特异性抗血清。ACP-ELISA检测结果表明,当CP蛋白浓度为2.5 μg/mL时,血清效价高达5.12×105。Western blot分析表明制备的抗体检测CCYV侵染的甜瓜叶片得到特异性的条带,表明该抗体的特异性较好。ACP-ELISA检测结果表明,制备的抗血清可用于田间甜瓜病样的检测。本试验为CCYV的快速检测提供了重要的研究材料。 相似文献
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Gil Atzmon Hadassa van Oss Henryk Czosnek 《European journal of plant pathology / European Foundation for Plant Pathology》1998,104(2):189-194
DNA of tomato yellow leaf curl virus (TYLCV), a geminivirus transmitted by the whitefly Bemisia tabaci, was amplified from squashes of infected tomato plants and of viruliferous vectors using the polymerase chain reaction (PCR). Samples of infected tissues as small as 1 mm2 were squashed onto a nylon membrane. A 1 × 2 mm strip containing the squash was introduced into a 25 µl PCR reaction mix. The reaction products were subjected to gel electrophoresis, blotted and hybridized with a radiolabeled virus-specific DNA probe. TYLCV DNA was amplified from squashes of leaves, roots, and stem of infected tomato and from individual viruliferous whiteflies. The same squash could be used several times to amplify different virus DNA fragments with various sets of primers. Thus plant and insect squashes can be used as templates for the amplification of geminiviral DNA with no need to prepare tissue extracts or purify nucleic acids. The squash-PCR procedure was applied to study whitefly transmission of TYLCV. Tomato plants were inoculated by placing a single viruliferous insect in the center of a young leaflet. In some plants TYLCV DNA was detected at the site of inoculation as early as 5 min after the beginning of the access feeding and in all plants after 30 min. The squash-PCR procedure also was applied to the study of TYLCV acquisition by the insect vector. TYLCV DNA was detected in the head of whiteflies as early as 5 min after the beginning of the access feeding on infected tomato plants. Viral DNA was detected in the thorax after 10 min and in the abdomen after 25 min. 相似文献
12.
<正>0引言地黄(Rehmannia glutinosa Libosch．)为玄参科(Scrophulariaceae)多年生草本植物,根部为传统中药材,主要产区分布在山东、山西、河南、河北、北京等地。地黄栽培中通常使用块根进行无性繁殖,易导致病毒病积累发生,从而严重影响地黄的产量与质量[1]。目前侵染地黄的植物病毒主要有烟草花叶病毒(tobacco mosaic virus,TMV)、地黄花叶病毒(rehmannia mosaic virus,ReMV)、油菜花叶病毒(youcai mosaic virus,YoMV)、蚕豆萎蔫病毒2号(broad bean wilt virus 2,BBWV2)、车前草花叶病毒(plantago asiatica mosaic virus,P1AMV)等[2]。 相似文献
13.
Jun Ohnishi Toshio Kitamura Fumihiro Terami Ken-ichiro Honda 《Journal of General Plant Pathology》2011,77(1):54-59
The ability of the whitefly Bemisia tabaci to transmit two strains of Tomato yellow leaf curl virus, the Israel and Mild strains, was studied after serial transfers of individual whiteflies that were viruliferous for both
strains to tomato plants. After single whiteflies had successive acquisition feedings first on a single plant infected with
one strain and then on a plant infected with the other strain, the single whiteflies later transmitted intermittently one,
the other, or both strains to the test plants during serial transfers at 1-day intervals. Because both strains were found
in the head, abdomen, and legs dissected from whiteflies during the retention period after the two successive acquisition
feedings, both strains apparently circulate from midgut cells to salivary glands through the hemolymph. 相似文献
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B型烟粉虱近20年来入侵许多国家和地区,已成为重要的世界性作物害虫,其主要危害方式之一是传播双生病毒,造成作物病毒病大发生.应用PCR技术,研究了入侵我国的B型烟粉虱个体获取、存留及传播烟草曲茎病毒（Tobacco curly shoot virus,TbCSV）的能力.B型烟粉虱在感染TbCSV的普通烟毒株上饲毒30 min时,就可在40%的个体内检测到TbCSV DNA,个体带毒率随饲毒时间延长而增加,饲毒12 h后,带毒率达100%;TbCSV DNA在B型烟粉虱体内可存留10天左右,但不能终身存留;传毒处理植株的发病症状及PCR检测结果表明,B型烟粉虱是TbCSV的传播媒介. 相似文献
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Basic studies carried out in India showed that the incubation period of TLCV in plants varied from 8 days in August to 90 days in winter. The acquisition threshold for the whitefly,Bemisia tabaci Gen., was 31 min; it resulted in 3% transmission. An acquisition access of 24 h for a female whitefly on a TLCV source resulted in 30% transmission. A minimum feeding period of 32 min was required by a viruliferous whitefly to cause infection on tomato test plants; this gave 4% transmission. With inoculation access of 24 h on tomato test plants, the transmission rose to 24%. Starving the vector for 1 h pre-acquisition or 1 h pre-inoculation resulted in higher levels of transmission of TLCV: 36 and 40%, respectively, compared with 20% for non-starved whiteflies. Extending the fasting period beyond 1 h resulted in a reduced transmission level. The whiteflies could acquire the virus from the cotyledonary leaves of an infected tomato plant, with a resultant 28% transmission; but infection did not occur when the whiteflies had an inoculation access to such leaves. Higher transmission rates were obtained when the younger leaves on tomato plants were used for acquisition and inoculation. Transmission was 8 and 38% when five whiteflies per plant were allowed 24 h of acquisition access to 11- and 2-month-old virus sources, respectively. After an acquisition access of 24 h to a TLCV source, male and female whiteflies retained their infectivity for 5 and 53 days, respectively. Nymphs can acquire and transmit the virus. When ten whiteflies of each sex were given 24 h of acquisition and of inoculation access, the subsequent transmission rate of males and females was 56 and 86%, respectively. This virus is not transovarially transmitted. Whitefly colonies raised on brinjal were more efficient (70 and 84% transmission in two experiments) than those raised on chilli, cotton, cowpea, tobacco or tomato. 相似文献
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The tobacco whitefly,Bemisia tabaci Gennadius, was found to differentiate between pH values at the leyel of 0.25. It had a clear preference for media with pH values of 6.0 to 7.25 offeredin vitro, in both choice and no-choice situations, when “resting whiteflies” or survival was measured. The whiteflies showed a clear preference for a sucrose concentration of 15%. The addition of 10% sucrose to buffers at various pH values did not change the pattern of their pH preference. Inin vivo experiments whiteflies preferred old cotton leaves (120 days) to younger leaves (60 days). The pH of old leaves was 6.8 while that of young leaves was 5.9. These results may explain the fact that whiteflies attacked cotton plants in commercial fields only late in the season, when the pH values of the cotton leaves exceeded pH 6. 相似文献
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A. E. Aboul-Ata M. A. E. Awad S. Abdel-Aziz D. Peters H. Megahed A. Sabik 《EPPO Bulletin》2000,30(2):297-300
Tomato yellow leaf curl begomovirus (TYLCV) severely invaded tomato plantations in Egypt (Lower and Middle Egypt) in 1989. This study aimed to discover the relationship between TYLCV and other epidemic-associated factors in the Fayium area. The rate of TYLCV infection was inspected visually for three successive years (1994/1996) in the Fayium area. During the same period, whiteflies were collected for virus detection using bait-plant and DNA hybridization techniques. DAS-ELISA was used to detect mixed virus infections in tomato plants. TYLCV infection was prevalent (60–68%) and severe (2.1–3.0) in the Fayium fields. Cucumber mosaic cucumovirus (CMV) was found in some fields (5–28%) with moderate severity (1.0–20). Potato Y potyvirus (PVY) and potato leaf roll polerovirus (PLRV) were found in few fields (5–19% and 5% respectively) at very low severity. There was a negative correlation between TYLCV occurrence and distance from the source of infection, and a positive correlation (98%) between TYLCV intensity and percentage of viruliferous whiteflies in 1994 and 1995. There was no positive correlation between TYLCV and the total population of whiteflies caught during the same period. Five percent of viruliferous whiteflies, as proved by cDNA hybridization, led to 46% TYLCV infection. The same percentage of whiteflies, as shown by bioassay, led to 68% TYLCV infection. Monitoring of viruliferous whiteflies could be used for early prediction of TYLCV infection. 相似文献
18.
Pymetrozine, a novel compound belonging to the class pyridine-azomethines, is a feeding inhibitor labeled for use against
plant pests in the order Hemiptera. Pymetrozine was evaluated for its ability to interfere with whitefly transmission of the
begomovirusTomato yellow leaf curl virus (TYLCV). Pymetrozine was applied as Fulfill
TM
50 WG at two rates (0.291 and 0.582 g formulationl
−1) to tomato seedlings with four to six true leaves. Viruliferous whiteflies (three to five per plant) were added 1, 4, 7 and
11 d after a single application of pymetrozine, and transmission rates were determined 4 wk after the addition of whiteflies.
Pymetrozine provided protection against transmission of TYLCV by viruliferous whiteflies for up to 1 wk after a single apliation.
No phytotoxicity was observed on tomato transplants. These results indicate that pymetrozine could be an effective tool for
tomato transplant producers to protect susceptible transplants from infection by begomoviruses, such as TYLCV. Pymetrozine
might also work well as part of an integrated approach to begomovirus management in greenhouse tomato fruit production.
http://www.phytoparasitica.org positing Oct. 20, 2003. 相似文献