The Efficiency of Southern rice black-streaked dwarf virus Transmission by the Vector Sogatella furcifera to Different Host Plant Species

Southern rice black-streaked dwarf disease is a new rice disease that severely affects rice production in South China.To understand transmission capacity of the vector Sogatella furcifera to Southern rice black-streaked dwarf virus（SRBSDV） among different host plant species,potential host plants of SRBSDV collected from the diseased rice field and/or adjacent to the field in Hunan Province,China,were determined by RT-PCR,and the transmission rates of SRBSDV by S.furcifera among different host plant species were investigated.The results showed that host plants of SRBSDV in the rice fields were five of family Gramineae（Oryza sativa,Echinochloa crusgalli,Zea mays,Paspalum distichum,Alopecurus aequali） and two of family Cyperaceae（Juncellus serotinus and Cyperus difformis）.S.furcifera could not transmit SRBSDV between gramineous plants and cyperaceous plants,and could not transmit SRBSDV between the gramineous plants,J.serotinus and C.difformis as well.However,SRBSDV could be transmitted by S.furcifera within gramineous plants.S.furcifera could transmit SRBSDV between interspecies among three species plants（O.sativa,E.crusgalli and Z.mays）,and between P.distichum and A.aequali.At 15,20,25,30,and 35°C,both macropterous and brachypterous adult of S.furcifera could transmit SRBSDV from the plants（e.g.,E.crusgalli,Z.mays and O.sativa） infected with SRBSDV to rice seedlings.The transmission rates were first increased and then decreased with the increase of temperature.Macropterous adults transmitted SRBSDV from the viruliferous E.crusgalli,Z.may and rice plants to the healthy rice seedlings,and the infected rates of rice seedlings were 26.2,18.8 and 23.7% at 15°C,56.6,64.6 and 53.6% at 25°C,and was 11.2,10.2 and 7.3% at 35°C,respectively.Transmission capacity of brachypterous adults was significantly higher than that of macropterous adults at 15,20 and 25°C（P0.05）,while transmission capacity of brachypterous adults was relatively lower compared with that of macropterous ones at 35°C.These results offer evidence on the transmission of SRBSDV via the vector S.furcifer among different host plants,which can be helpful to control Southern rice black-streaked dwarf disease by the appropriate cultural measures in South China.

Molecular Characterization of Segments S7 to S10 of a Southern Rice Black-streaked Dwarf Virus Isolate from Maize in Northern China

Southern rice black-streaked dwarf virus (SRBSDV) is a novel Fijivirus prevalent in rice in southern and central China,and northern Vietnam. Its genome has 10 segments of double-stranded RNA named S1 to S10 according to their size. An isolate of SRBSDV,JNi4,was obtained from naturally infected maize plants from Ji'ning,Shandong province,in the 2008 maize season. Segments S7 to S10 of JNi4 share nucleotide identities of 72.6%-73.1%,72.3%-73%,73.9%-74.5% and 77.3%-79%,respectively,with corresponding segments of Rice black-streaked dwarf virus isolates,and identities of 99.7%,99.1%-99.7%,98.9%-99.5%,and 98.6%-99.2% with those of SRBSDV isolates HN and GD. JNi4 forms a separate branch with GD and HN in the phylogenetic trees constructed with genomic sequences of S7 to S10. These results confirm the proposed taxonomic status of SRBSDV as a distinct species of the genus Fijivirus and indicate that JNi4 is an isolate of SRBSDV. Shandong is so far the northernmost region where SRBSDV is found in China.

Transmission of Rice Black-Streaked Dwarf Virus from Frozen Infected Leaves to Healthy Rice Plants by Small Brown Planthopper (Laodelphax striatellus)

In order to preserve virus for identifying the resistance of rice varieties against rice black-streaked dwarf disease, a simple and reliable method was developed, through which virus-free small brown planthopper （SBPH） acquired rice black-streaked dwarf virus （RBSDV） from frozen infected leaves and the virus was transmitted to healthy rice plants. The experimental results showed that SBPH could obtain RBSDV from frozen infected rice leaves and the virus could be transmitted to a susceptible rice variety. For the ability to acquire RBSDV and transmit the virus to healthy plants by SBPH, there was no significant difference between frozen infected leaves and in vitro infected leaves. The novel method could be applied to identification of rice variety resistance to rice black-streaked dwarf disease, facilitating the breeding process for rice black-streaked dwarf disease resistance.

Resistance of Major Rice Cultivars in Hangzhou Area of China against Southern Rice Black-streaked Dwarf Virus

Resistance of 17 major rice cultivars in Hangzhou region of China against southern rice black-streaked dwarf virus was tested in the paper. The results showed that the incidence rate of southern rice black-streWed dwarf virus had certain positive correlation with quantity of white-backed planthopper （Sogatellafurci-fera） and resistance among various cultivars was significant. Two varieties, Yueyou 9113 and Tianyouhuazhan, were most susceptible ; Yongyou 8, Jiayou 2 and Xi-ushui 134 had best resistance and could be used as disease-resistant varieties to prevent the disease.

Prevalence Characteristics of Rice Black-streaked Dwarf Virus Disease and Continuous Control Strategies

Through summarizing the prevalence characteristics of rice black-streaked dwarf virus disease(RBSDVD)in Linyi City of Shandong Province,this paper analyzed its prevalence is related to changes in farming and cultivation systems,the increase in the population of venomous Laodelphax striatellus Fallén and its own migration and spread,the poor disease resistance of cultivated varieties,and inadequate time of prevention and control.Besides,based on the practice of local control,it came up with some comprehensive control measures including strengthening monitoring,early warning and forecasting,planting resistant(tolerant)rice varieties according to local conditions,appropriately delaying the sowing(planting)period,supplemented by insect nets to cover seedlings,and making scientific use of chemical control.It is expected to provide a reference for the prevention and control of RBSDVD.

Screening of Rice Genes Interacting with p5b of Rice BlackStreaked Dwarf Virus

Rice black-streaked dwarf virus （RBSDV） is a recognized member of the genus Fijivirus, family Reoviridae. Its genome has ten double-stranded RNA （dsRNA） segments （$1-$10）, in which the fifth genome segment （$5） contains two open reading frames （ORFs） with a partially overlapping region. The second ORF of RBSDV S5 encodes a viral nonstructural protein named p5b with unknown function. To reveal the function of p5b, its gene was ligated into the bait plasmid pGBKT7 and an expression library containing rice cDNAs was constructed using plasmid pGADT7 for yeast two-hybrid assay. The bait protein p5b was detected in yeast by western blot, and the result of an auto-activation test showed that p5b could not autonomously activate the expression of reporter genes in yeast. Then the bait protein p5b was used for screening the cDNA expression libraries of rice. Gene fragments of some pivotal enzymes involved in photosynthesis, respiration and other important metabolic processes, were identified to interact with p5b in yeast, suggesting that these interactions may play roles in symptom development in infected plants.

Disaster Prevention and Mitigation Technologies of Rice Virus Diseases Spread by Small Brown Planthopper (SBPH)

Rice stripe virus disease （RSVD） and rice black-streaked dwarf virus disease （RBSDVD） are two epidemic diseases in Yancheng City, Jiangsu Province in the last decade. The authors investigated the disaster regularity, prevention and control technology of RSVD and RBSDVD systematically. The occurrence and virus transmission of SBPH and disaster regularity of virus diseases were studied; the resistance of some rice varieties was cleared; the effects of physical and agricultural measures such as insect net blocking, appropriate late sowing and plowing on controlling occurrence and virus transmission of SBPH were figured out; a hatch of chemical agents were screened, providing efficient and harmless pesticides for effective control against SBPH and prevention against virus diseases. A set of disaster control and mitigation technologies was proposed in this paper, which was practical, sustainable, and easy to operate for the local planting patterns.

Epidemiological Characteristics of Maize Rough Dwarf Disease in Huang-Huai-Hai Plain

Maize rough dwarf disease is a common epidemic disease in large areas.Its epidemic and occurrence mechanism is a complex process.In this paper,the epidemiological characteristics and influencing factors of maize rough dwarf disease in Huang-Huai-Hai plain were elaborated based on the research results of maize rough dwarf disease at home and abroad for many years.The epidemic of maize rough dwarf disease is affected by many factors,such as the occurrence and virus carrying rate of the first generation small brown planthopper,accumulation of virus sources on gramineous crops and weed hosts,maize variety resistance,maize sowing date,maize growth period,crop layout,tillage system,climate and ecological environment.The key factors causing the outbreak and epidemic of maize rough dwarf disease are the planting of maize susceptible varieties,the meeting of maize seedling stage and the peak period of adult spread of the first generation of small brown planthopper.

南方水稻黑条矮缩病抗病遗传资源与分子机理研究进展

南方水稻黑条矮缩病(Souther rice black-streaked dwarf virus disease,SRBSDVD)是依靠白背飞虱(Sogatella furcifera)为传播介体,由南方水稻黑条矮缩病毒(Southern rice black-streaked dwarf virus,SRBSDV)侵染引起的一种水稻病毒病。该病害于2001年在我国华南地区首次被发现,随后发生面积不断扩大,危害逐年加重,已成为近年来危害中国南方稻区水稻生产的重要病害之一。白背飞虱迁飞性强、种群数量大、传毒持久等特点,导致化学农药防虫治病效果并不理想,至今还未有可持续和绿色控制SRBSDVD的手段。根据病原病毒的爆发性及其传播介体白背飞虱的生物学特性,预计该病害在未来较长的一段时期内将是我国最严重的水稻病害之一,很有可能再次暴发流行。因此,当前生产上急需培育出能够抵御SRBSDVD的抗性水稻品种。本文综述了SRBSDVD的发病规律、抗病种质资源的收集与评估、抗病基因或数量性状位点(QTL)的精确定位,以及这些抗性基因的分子作用机制,进一步探讨了利用现代分子生物学技术,包括分子标记辅助选择、基因沉默(RNAi)和基因编辑等技术加快SRBSDVD抗病品种的选育,可为今后有效控制SRBSDVD提供基础。

南方水稻黑条矮缩病毒快速检测

南方水稻黑条矮缩病毒(southern rice black-streaked dwarf virus,SRBSDV)引起的新水稻矮缩病近年在越南北部和我国南方各省爆发,准确快速地检测病毒是病害预测的关键。本文针对该病毒的S10序列设计了一对新的引物S10F/S10R,利用一步法RT-PCR,对2010年5~8月间湖南省下属各县送来的疑似感染该病毒引起的水稻矮缩病样本进行了检测,结果显示,新引物能有效地区分阳性样品和非阳性样品。同时对PCR产物进行了序列测定,结果表明序列均与已发表的南方水稻黑条矮缩病毒序列(登录号:EU523360和EU784840)达约99%的同源性。还在此基础上构建了系统发育树,发现SRBSDV湖南、广东和海南分离物病毒位于一个相对独立的分支。研究发现相对以往的巢式RT-PCR,本文采用的新引物及一步法RT-PCR能快速检测南方水稻黑条矮缩病毒,简化了操作步骤,缩短了检测时间,适合在SRBSDV检测和病害预测中应用。

南方水稻黑条矮缩病毒外壳蛋白p10的原核表达和抗血清制备及应用

南方水稻黑条矮缩病毒(SRBSDV)p10蛋白由其基因组片段S10编码,根据SRBSD海南分离物S10序列(EU523360)设计特异性引物扩增编码p10蛋白的片段,并亚克隆至原核表达载体pET-28a+,以大肠杆菌BL21plysS为宿主菌进行高水平表达,利用纯化的p10蛋白免疫兔子,制备了p10蛋白的特异性抗血清。ELISA检测显示,p10蛋白的抗血清可与来自湖南不同地区的白背飞虱病汁液发生强烈的血清学反应,表明该抗血清适用于田间SRBSDV的快速诊断。

毒氟磷防治南方水稻黑条矮缩病药效研究

为明确自主创制新抗病毒药剂——30%毒氟磷WP对南方水稻黑条矮缩病的防控效果以及探讨防治该病的理想药剂组合,采用喷雾施药法对30%毒氟磷WP、25%吡蚜·噻虫嗪SC、10%醚菊脂MG、8%宁南霉素AS的单用或混用处理进行研究。结果表明,在大田分蘖初期,以25%吡蚜·噻虫嗪SC375mL/hm2和30%毒氟磷WP990g/hm2的药剂组合的防治效果最好,防效为66.67%,25%吡蚜·噻虫嗪SC、10%醚菊脂MG、30%毒氟磷WP和8%宁南霉素AS的防效分别为50.51%、33.33%、33.33%、17.12%;在收割期,以25%吡蚜·噻虫嗪SC375mL/hm2和30%毒氟磷WP990g/hm2的药剂组合的防治效果最好,防效为73.24%,25%吡蚜·噻虫嗪SC375mL/hm2、10%醚菊脂MG450g/hm2、30%毒氟磷WP990g/hm2和8%宁南霉素AS750mL/hm2处理组的防效依次是46.48%、45.07%、43.66%、42.25%。同时,测产试验结果表明:与CK相比,增产效果最明显的是25%吡蚜·噻虫嗪SC375mL/hm2和30%毒氟磷WP990g/hm2组合,增产值为292.35%,而25%吡蚜·噻虫嗪SC、10%醚菊脂MG、30%毒氟磷WP和8%宁南霉素AS的增产值为249.75%、230.7%、208.65%、104.7%。因此,为有效防治南方水稻黑条矮缩病,可采用本研究中的杀虫剂和抗病毒剂的联合处理,特别是在秧田期用药。

浙江武义2009年南方水稻黑条矮缩病的毒源地分析

2009年,浙江省境内首次出现南方水稻黑条矮缩病毒(Southern rice black-streaked dwarf virus,SRBSDV),且仅武义县有发病现象。因为该病毒病是一种虫媒病毒,且白背飞虱Sogatella furcifera(Horváth)是主要传毒介体,所以本文通过白背飞虱灯下诱虫情况调查、迁飞轨迹模拟、天气学背景分析以及毒源地分析,阐释了2009年浙江省武义县发现的南方水稻黑条矮缩病的供毒源地分布情况,以及白背飞虱携毒的传递路径,并讨论了轨迹模拟中各生物学参数的设定方法,从而为剖析该病毒的宏观流行规律奠定科学基础。结果显示:(1)通过对白背飞虱迁入武义的主要虫源地与经鉴定的南方水稻黑条矮缩病发病区域的叠加分析,明确了浙江武义的可能毒源地分布于两广、闽南、赣南四省区境内;(2)西南低空急流及偏南气流是白背飞虱将我国南方的病毒远距离传送到武义县境内的动力源;(3)白背飞虱随下沉气流和降雨在武义境内的集中降落是南方水稻黑条矮缩病在当地暴发的触发条件。

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。

南方水稻黑条矮缩病毒江西分离物CP基因克隆及序列分析

根据GenBank已登陆的SRBSDV S10核苷酸序列设计引物,克隆SRBSDV江西分离物外壳蛋白(CP)基因,结合已公布的序列,通过生物信息学方法进行序列分析。结果表明,SRBSDV江西分离物CP基因全长1 674个核苷酸,推测编码557个氨基酸,与已报道的SRBSDV CP基因之间核苷酸同源性为97.5%～100%,氨基酸同源性为97.7%～100%。SRBSDV CP基因核苷酸序列高度保守,保守位点占全部位点的83.5%。没有发现重组事件。本研究首次报道根据系统发育进化树,SRBSDV可分为2大组群,不存在明显的地域和寄主分化。

南方水稻黑条矮缩病毒诱导的水稻挥发物及白背飞虱成虫对其组分的行为反应

【目的】南方水稻黑条矮缩病毒(Southern rice black-streaked dwarf virus,SRBSDV)是白背飞虱Sogatella furcifera在稻苗间传播的一种持久性病毒。本研究旨在调查SRBSDV是否诱导水稻挥发物的含量及成分发生变化,并探究变化的组分是否对白背飞虱具引诱或驱避作用。【方法】采用动态顶空法收集3个水稻品种病/健植株的挥发物,利用气相色谱-质谱联用仪(GC-MS)分析各品种不同生育期病/健株挥发物的组分,使用嗅觉测定仪测定了白背飞虱对病株特有挥发物的嗅觉行为反应。【结果】从健株检测到11大类化合物共36种;从病株检测到12大类化合物共37种。病株特有挥发物12种,即正十六烷、癸醚、butyl octyl phthalate、正十八烷、己二酸二异丁酯、正十五碳醛、十六烷酸甲酯、2-methylhexacosane、二苯甲酮、雪松醇、正二十烷和叶绿醇;病/健株中共有但含量差异显著的挥发物6种,即1-tricosene、octadecanal、萘、植酮、正十二烷醇和2,6-二叔丁基对甲酚。嗅觉选择实验表明,在50μL/L浓度下,仅叶绿醇对白背飞虱成虫(健康与饲毒)表现出显著的驱避活性。【结论】SRBSDV的侵染改变了水稻挥发物的组分,且不同品种、不同生育期挥发物组分均具差异。叶绿醇为孕穗期荣优华占病株特有挥发物,对健康与饲毒白背飞虱均有驱避活性,是否有助于促进该病毒传播,尚需进一步验证。

感染和未感染南方水稻黑条矮缩病毒的白背飞虱成虫唾液腺转录组比较分析

【目的】白背飞虱Sogatella furcifera是南方水稻黑条矮缩病毒(Southern rice black-streaked dwarf virus,SRBSDV)传播的主要介体,其唾液腺在取食和传毒的过程中都发挥着重要作用,本研究旨在通过对带毒与不带毒白背飞虱成虫的唾液腺进行转录组测序,进而比较分析找出差异表达基因,推测出唾液腺中与传毒相关的基因。【方法】利用Ion ProtonⅡ,PGM平台进行带毒与不带毒白背飞虱成虫唾液腺转录组测序,用SOAPdenovo软件从头组装,通过blast X比对进行基因注释。采用Blast2go和Blastall软件对所测基因的GO功能富集和KEGG代谢途径进行分析;釆用RPKM分析差异表达基因。【结果】带毒和不带毒白背飞虱的唾液腺转录组样本的原始数据经过软件处理后分别获得52 062和51 407条unigenes序列(GenBank登录号分别为SRS851833和SRS843978),平均长度分别为639和647 bp。通过NR数据库比对,共18 431个unigenes具有同源序列,与赤拟谷盗Tribolium castaneum的同源序列最多(16.23%)。所有unigenes富集到52个GO类群,240个KEGG信号通路。白背飞虱在感染南方水稻黑条矮缩病毒后,唾液腺有89个unigenes序列的表达发生变化。【结论】本研究通过比较分析带毒与不带毒白背飞虱唾液腺转录组样本后,发现有部分基因序列的表达存在差异,这些基因序列可能和病毒与载体的互作有关。转录组数据的获得为研究与取食和传毒相关的基因提供了有用的信息,从而在分子水平上为后续研究白背飞虱与SRBSDV互作机制奠定基础。

南方水稻黑条矮缩病毒P6抗体的制备及应用

为了建立快速准确检测田间介体白背飞虱带毒率的方法,利用Gateway重组技术将与病毒早期复制有关的P6基因的N端993 bp构建原核表达载体pDEST17-P6,并将诱导表达的目的蛋白免疫注射新西兰大白兔制备多克隆抗体。结果表明:Western blot检测制备的P6抗体可特异性检测SRBSDV侵染水稻的P6蛋白,免疫荧光标记技术可检测南方水稻黑条矮缩病毒(Southern rice black streaked dwarf virus,SRBSDV)侵染白背飞虱体内的P6蛋白,且P6抗体可直观高效准确地用于免疫荧光标记检测昆虫带毒率。以上研究表明,所制备的P6抗体可用于SRBSDV的田间介体昆虫带毒率快速检测,有助于病毒病害的监测和预报。

白背飞虱酵母双杂交cDNA文库的构建

白背飞虱(Sogatella furcifera Horváth)作为一种迁飞性害虫,不仅自身危害水稻,而且以持久性方式传播南方水稻黑条矮缩病毒(Southern rice black-streaked dwarf virus,SRBSDV)。为了研究白背飞虱与SRBSDV之间的相互作用,需要构建一个高质量的酵母筛选文库,试验利用Gateway技术构建了白背飞虱cDNA酵母表达文库,检测分析显示,白背飞虱初级文库库容量约为1.72×10~7CFU,cDNA插入片段主要分布在500~2 000 bp,重组率约为95.8%;次级文库库容量约为1.73×10~7CFU,cDNA插入片段主要分布在400~2 000bp,重组率为100%;Y187酵母菌株的白背飞虱酵母双杂交cDNA文库库容量约5.09×10~7CFU,插入片段大小在750~2 000 bp,重组率100%,再随机挑选36个单克隆进行测序分析,所得结果与Gen Bank数据库比对显示36个克隆中含有22个不同的基因,长度在750~2 000 bp,这些分析表明,该文库可以用于酵母双杂交的后续筛选,为研究白背飞虱与SRBSDV之间的互作奠定了基础。

粤西地区南方水稻黑条矮缩病的流行规律及防治

南方水稻黑条矮缩病为白背飞虱传播的一种毁灭性的水稻病毒病。笔者根据2007—2016年化州地区南方水稻黑条矮缩病发生和危害情况,通过对南方水稻黑条矮缩病病害症状和介体昆虫的认识,分析南方水稻黑条矮缩病发生的轻重与介体数量、毒源量、水稻品种、种植方式、水稻播期、秧田位置及气候条件等因素有关。提出全面实施"防控技术前移,切断毒源,治虫防病"的防治策略;形成适合粤西稻区特点的南方水稻黑条矮缩病综合防控的技术体系,并在生产上推广应用,有效预防控制南方水稻黑条矮缩病发生流行危害,取得了显著的社会经济效益。