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.

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.

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.

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.

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.

The Control Effect of Prevention and Control Technology Combination against Southern Rice Black-Streaked Dwarf Disease

Objective] This study aimed to explore a better prevention and control technology combination against southern rice black-streaked dwarf disease. [Method] The control effects of seed dressing, antiviral agents and fly net along with 25%pymetrozine against southern rice black streaked dwarf disease were determined. [Result] When the seeds were dressed with 60% imidacloprid （FS, 2 g a.i./kg）, and the rice seedlings were sprayed once with 25% pymetrozine （WP, 360 g/hm2） 10 d before the transplanting and sprayed twice with 30% Dufulin （WP, 800×） at three-leaf stage and 7 d after the transplanting respectively, the control efficiency reached 88.05%. When fly net and pesticides were applied simultaneously, the fly net was lifted and 25% pymetrozine （WP） was spayed once on the rice seedlings at six-leaf stage and the seedlings were hardened for 3 d and sprayed once with 25%pymetrozine （WP） 10 d after the transplanting, the control efficiency reached 80.50%. [Conclusion] Seed dressing or applying antiviral agents alone can not better control diseases. The southern rice black-streaked dwarf disease can be better con-trol ed by seed dressing, along with spraying of planthopper-kil ing agents at two-leaf stage, 3 d before the transplanting and 7 d after the transplanting, respectively. If condition al ow, fly net can be instal ed to achieve better control effect.

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.

Southern rice black-streaked dwarf virus:A new proposed Fijivirus species in the family Reoviridae

For the past several years, a novel dwarf disease has been observed on rice (Oryza sativa) in some regions of Guangdong Province and Hainan Province, southern China. Infected plants showed stunting, dark leaf and small enations on stem and leaf back. Typical Fijivirus viroplasma containing crystalline arrayed spherical virons approximately 70―75 nm in diameter and tubular structures were detected in ultrathin sections by an electron microscope in parenchyma phloem cells of the infected plants. The virus was transmitted to rice seedlings by white-backed planthoppers, Sogatella furcifera (Hemiptera: Delphacidae), collected in the diseased fields. Analysis of dsRNA extracts from infected plants revealed ten linear segments, which were similar to the electrophoretic profile of Rice black-streaked dwarf virus (RBSDV). RT-PCR with a single primer which matched to a linker sequence ligated to both 3′ ends of the viral genomic dsRNAs resulted in amplification of genome segments 9 (S9) and 10 (S10) cDNA products. The complete nucleotide sequences of S9 and S10 were obtained from clones of the RT-PCR amplicon exhibited characteristic properties of Fijivirus including low GC content (34.5% and 35.6%), genus conserved 5′ and 3′ termini sequences and similar genome organization. Blast searches indicated that the sequences of S9 and S10 shared 68.8%―74.9% and 67.1%―77.4% nucleotide identities with those of viruses in the Fijivirus group 2, respectively. These values were similar to those among other viruses in the Fijivirus group 2 and considerably lower than those among RBSDV isolates. Phylogenetic trees based on S9 and S10 nucleotide sequences and their putative amino acid sequences showed that this virus represented a separate branch among other Fijiviruses. The virus was also detected by a nested RT-PCR assay in corn (Zea mays), barnyard grass (Echinochloa crusgalli), Juncellus serotinus and flaccidgrass (Pennisetum flaccidum) in and/or adjacent to the infected rice fields. It is proposed that this virus be considered as a new species, Southern rice black-streaked dwarf virus, in the group 2 of the genus Fijivirus in the family Reoviridae.

Detection,Occurrence,and Survey of Rice Stripe and BlackStreaked Dwarf Diseases in Zhejiang Province,China

The major viral diseases that occur on rice plants in Zhejiang Province, eastern China, are stripe and rice black-streaked dwarf diseases. Rice stripe disease is only caused by rice stripe tenuivirus （RSV）, while rice black-streaked dwarf disease can be caused by rice black-streaked dwarf fijivirus （RBSDV） and/or southem rice black-streaked dwarf fijivirus （SRBSDV）. Here we review the characterization of these viruses, methods for their detection, and extensive surveys showing their occurrence and spread in the province.

气象因子对河池市南方水稻黑条矮缩病发展的影响

利用河池市2005—2023年南方水稻黑条矮缩病(SRBSDV)的发病面积比例数据和期间气象数据,通过卡方分布的概率密度函数拟合流行周期内的发病趋势概率,分离出发病浮动概率,探究关键气象因子与发病浮动概率的相关性,并基于显著相关气象因子构建发病气象评价模型。结果表明:(1)2010年为河池市SRBSDV的暴发期,2010—2023年为SRBSDV的一个流行周期,发病概率符合自由度为3的卡方分布的概率密度函数;(2)发病浮动概率与12月—次年2月的平均气温、6—7月的降水量和相对湿度呈显著正相关,与安全播种期日序数负相关;(3)发病气象评价模型在预测暴发期的发病程度时预测偏轻,其他年份均预测正确,模型在一个流行周期内的定性评价效果较好。通过气象发病评价模型可定性评价流行周期内的发病程度,研究结论可为河池市的水稻病害防治工作提供参考依据。

饲食感染SRBSDV的水稻病株对传毒介体白背飞虱相关能源物质含量的影响

南方水稻黑条矮缩病毒(Southern rice black-streaked dwarf virus,SRBSDV)近年对我国南方稻区水稻生产造成了极大危害。为探讨SRBSDV发生流行的原因,研究了饲食感染SRBSDV水稻病株对其介体昆虫白背飞虱体内能源物质含量的影响。结果表明,饲食感染SRBSDV水稻病株对白背飞虱若虫体内能源物质含量没有明显的影响,但是对雌成虫有明显的影响;饲食感染SRBSDV水稻病株使白背飞虱3日龄和5日龄雌成虫体内可溶性糖及游离氨基酸含量显著升高;此外,饲食感染SRBSDV水稻病株使5日龄雌成虫体内甘油酯含量和可溶性蛋白含量含量明显升高。上述结果证明饲食感染SRBSDV水稻病株会促进白背飞虱雌成虫体内糖类、脂类、氨基酸等物质的积累,有可能会导致其迁飞能力的提升,增加SRBSDV暴发流行的可能性。

南方水稻黑条矮缩病毒在广西兴安的迁入与侵染动态

广西兴安地处白背飞虱Sogatella furcifera(Horváth)迁入廊道要冲,南方水稻黑条矮缩病毒(southern rice black-streaked dwarf virus,SRBSDV)依赖白背飞虱携传。本研究采用探照灯诱集高空白背飞虱,同时采集田间白背飞虱和稻叶,采用dot-ELISA法检测白背飞虱及水稻的带毒状况,以明确SRBSDV的迁入与侵染动态。综合来看,2021年-2022年间兴安的带毒白背飞虱迁入始期为5月下旬,迁入白背飞虱的带毒率高峰为6月中下旬;田间白背飞虱7月上旬-7月下旬带毒率较高(10%~15%);水稻7月中旬-8月上旬带毒率较高(10%~14%)。水稻带毒率与田间白背飞虱带毒率之间存在显著相关关系。本文结果有助于桂北和长江中下游地区开展白背飞虱携传SRBSDV的监测预警。

不同提取方法对检测单头白背飞虱携带SRBSDV灵敏度的影响

为找到较好的白背飞虱RNA提取方法用于大规模带毒率检测、SRBSDV的早期监测及科学防控提供参考,采用Trizol试剂盒法、改良Trizol法、改良CTAB-LiCl法、改良异硫氰酸胍法和NaOH粗提法5种方法提取单头白背飞虱的总RNA,进行检测比较其提取速率和稳定性。结果表明:采用Trizol试剂盒法、改良Trizol法、改良CTAB-LiCl法、改良异硫氰酸胍法和NaOH粗提法5种方法提取单头白背飞虱的总RNA浓度分别为113.08ng/μL、222.43ng/μL、57.68ng/μL、102.16ng/μL和31.06ng/μL,改良Trizol法和改良CTAB-LiCl法提取的RNA纯度较高。5种方法提取的RNA均能用于SRBSDV的检测,其中改良Trizol法RT-PCR扩增的灵敏度最高,稀释梯度为10-4,NaOH粗提法灵敏度最差,稀释梯度仅为10-1,其余3种均能达到10-3。改良Trizol法提取的虫体RNA较完整且扩增效果稳定,但试剂价格较昂贵,不适合做大量样品提取;NaOH粗提法的灵敏度虽相对较低,但由于操作简便,经济快速,更适合用于大量样品的检测。

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。

水稻OsAGO家族成员特征及对RGDV和SRBSDV的响应

为初步探究OsAGO家族在水稻抗病毒通路中的功能,对水稻OsAGO蛋白的基因组结构、系统发育关系、氨基酸序列及水稻瘤矮病毒(rice gall dwarf virus,RGDV)和南方水稻黑条矮缩病毒(southern rice black streaked dwarf virus,SRBSDV)侵染后的转录组数据进行分析,同时采用实时荧光定量PCR(quantitative real-time PCR,qPCR)技术对这2种病毒侵染后OsAGO基因的相对表达量变化进行验证。结果表明,19个水稻OsAGO蛋白的外显子数量、内含子数量及编码区长度存在较大差异,且这19个OsAGO蛋白均匀分布在3个分支中;OsAGO蛋白PAZ结构域中与小RNA结合相关的YF(酪氨酸-苯丙氨酸)基序在OsAGO2、OsAGO3和OsAGO5中变成了YY(酪氨酸-酪氨酸),OsAGO蛋白PIWI结构域中与OsAGO蛋白切割活性相关的DDX(X代表H或D,即天冬氨酸-天冬氨酸-组氨酸/天冬氨酸)基序在OsAGO13中替换为LDH(亮氨酸-天冬氨酸-组氨酸)基序,而在OsAGO17中不包含YF基序且DDX基序替换为HDR(组氨酸-天冬氨酸-精氨酸)基序。RGDV侵染后OsAGO5和OsAGO12基因的转录组分析结果与qPCR结果一致,其中OsAGO5上调表达,OsAGO12下调表达;SRBSDV侵染后OsAGO1a、OsAGO1b、OsAGO1c、OsAGO1d和OsAGO4b基因的转录组分析结果与qPCR结果一致,均上调表达。表明大多数OsAGO均能响应RGDV和SRBSDV的侵染。

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。

水稻新病害南方水稻黑条矮缩病发生特点及危害趋势分析

由斐济病毒属(Fijivirus)暂定新种南方水稻黑条矮缩病毒(Southern rice black-streaked dwarf virus,SRBSDV)引起的水稻矮缩病,自2001年在广东省阳西县被发现以来,已迅速扩散至我国南方广大稻区。2009年,该病害在我国南部及越南晚季稻上暴发成灾,造成严重的产量损失。本文简介了该病害的症状、危害特点、病原病毒及其传毒介体特征,对其发生趋势进行了讨论,提出了以秧苗期治虫为重点的病害防控应急措施。

一种快速同步检测水稻黑条矮缩病毒和南方水稻黑条矮缩病毒的方法

水稻黑条矮缩病毒和南方水稻黑条矮缩病毒是近年来在水稻上危害日益严重的两种病毒,由于两者同属于呼肠孤病毒科斐济病毒属,在症状、粒体形状、传播介体及寄主等方面非常相似,由此也给诊断及鉴定造成了困难。针对这一问题,通过设计简并引物,并优化体系,建立了一种快速、简便、灵敏、特异的方法,为这两种病毒的检测及鉴别提供了方便。