A Rapid and Simple Method for the Detection of Rice Dwarf Virus by Aqueous Extract

Rice dwarf disease caused by rice dwarf virus （RDV） is one of the major rice virus diseases in China,which widely distributes in the rice area of China.A simple and rapid method for detection of RDV in rice plants and Nephotettix cincticeps was investigated in this study,and the whole detection only lasted for 20 min.After diseased leaves of rice （10 mg） and leaves of Nephotettix cincticeps （10 mg） both infected by RDV were ground by sterile water （100 μl）,the supernatant was analyzed by 1% agarose gel electrophoresis to steadily detect five specific electrophoretic bands with 1 000 bp to 5 000 bp.However,there was no electrophoretic band in the healthy rice leaves and aqueous extract of non-viruliferous N.cincticeps.Therefore,this method was more rapid and simple to detect RDV in rice plants and Nephotettix cincticeps,which avoided expensive reagents and tedious steps of conventional serological testing and molecular detection （RT-PCR and Western-blot）.

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.

Ecological Fitness of Non-vector Planthopper Sogatella furcifera on Rice Plants Infected with Rice Black Streaked Dwarf Virus

We evaluated the effects of rice black streak dwarf virus （RBSDV）-infested rice plants on the ecological parameters and its relevant defensive and detoxification enzymes of white-backed planthopper （WBPH） in laboratory for exploring the relationship between RBSDV and the non-vector planthopper. The results showed that nymph survival rate, female adult weight and fecundity, and egg hatchability of WBPH fed on RBSDV-infested rice plants did not markedly differ from those on healthy plants, whereas the female adult longevity and egg duration significantly shortened on diseased plants. Furthermore, significantly higher activities of defensive enzymes （dismutase, catalase and peroxidase） and detoxification enzymes （acetylcholinesterase, carboxylesterase and glutathione S-transferase） were found in WBPH adults fed on infected plants. Results implied that infestation by RBSDV increased the ecological fitness of non-vector planthopper population.

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.

Sequence Analysis of Segment 8 of Five Chinese Isolates of Rice Gall Dwarf Virus and Expression of a Main Outer Capsid Protein in Escherichia coli

The rice gall dwarf disease, caused by the Rice gall dwarf virus (RGDV) is a serious disease occurring in rice in many regions of Guangdong province. As a basis to control the disease we have studied the genomic diversity of a variety of isolates from different locations. Genome segment 8(S8), encoding a main outer capsid protein (Pns8) of RGDV five isolates (BL, CH, DQ, GZ, XY) from Guangdong province was cloned and sequenced. The results revealed that all the S8 segments of the five isolates consisted of 1 578 nucleotides and had a single open reading frame (ORF) extending for 1 301 nucleotides from nucleotide 21 which encoded a polypeptide of 426 amino acids with an estimated molecular weight of 47.4 kDa. The S8 full-length sequence and the ORF sequence shared 97.3%-98.8% and 97.3%-99.1% nucleotide sequence identities within the five Chinese isolates, and shared 94.8%-95.6% and 95.0%-96.0% identities with those of the Thailand isolate respectively. The deduced amino acid sequence of Pns8 in GZ isolate was identical to that in the Thailand isolate, while the amino acid sequence variability of Pns8 within five Chinese isolates ranged from 0.5% to 2.1%. These results indicate that the S8 segment of RGDV is highly conserved in different isolates from different locations. The S8 cDNA from the XY isolate was cloned into the plasmid vector pET-28b(+) and a fused expression protein with an apparent molecular mass of 51kDa was specifically detected in an analysis of Escherichia coli Rossetta(DE3)Ⅱcells. To our knowledge, this is the first report on analysis of the RGDV segment 8 sequence and genetic comparison of different RGDV isolates and their protein expression.

Three-dimensional Structure of Rice Dwarf Virus by Electron Cryomicroscopy

The three-dimensional(3D)structure of intact rice dwarf virus(RDV)has been deter-mined to 2.6 nm resolution by 400kV electron cryomicroscopy and computer reconstructiontechniques.The intact virion of 69.8 nm in diameter was seen to be made up of an outer shellof 6.9 nm in thickness and an inner shell of 2.5 nm in thickness at a diameter of 54 nm.Theouter shell surface forms a T=13 icosahedral lattice with holes at 5-and 6-coordinated posi-tions.The 780 capsomeres at local 3-fold positions appear trimeric.The interface betweenthese two shells could be clearly delineated and the interaction was maintained by a cylindri-cal density whose upper domain connects subunits of the trimeric capsomeres by an interlock-ing mechanism.The 3D structure of inner shell was also reconstructed to 3.3 nm from100kV images of purified inner shell particles,confirming our interpretation of the multilayerstructure of intact RDV.

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.

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.

Expression of Rice Gall Dwarf Virus Outer Coat Protein Gene (S8) in Insect Cells

To obtain the P8 protein of Rice gall dwarf virus (RGDV) with biological activity,its outer coat protein gene S8 was expressed in Spodoptera frugiperda (Sf9) insect cells using the baculovirus expression system.The S8 gene was subcloned into the pFastBacTM1 vector,to produce the recombinant baculovirus transfer vector pFB-S8.After transformation,pFB-S8 was introduced into the competent cells (E.coli DH10Bac) containing a shuttle vector,Bacmid,generating the recombinant bacmid rbpFB-S8.After being infected by recombinant baculovirus rvpFB-S8 at different multiplicities of infection,Sf9 cells were collected at different times and analyzed by SDS-PAGE,Western blotting and immunofluorescence microscopy.The expression level of the P8 protein was highest between 48-72 h after transfection of Sf9 cells.Immunofluorescence microscopy showed that P8 protein of RGDV formed punctate structures in the cytoplasm of Sf9 cells.

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.

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.

水稻矮缩病毒(RDV)基因组cDNA克隆和部分DNA序列分析

采用氯仿处理、聚乙二醇沉淀和蔗糖密度梯度离心的方法,从感病的水稻叶片中分离、纯化了水稻矮缩病毒(RDV)。在琼脂糖凝胶板上电泳分离到了9个 RNA 片段的 RDV 基因组。用电泳回收的方法纯化了 RDV 基因组的各 RNA 片段。利用 ollgo dT 作为引物,合成并克隆了其中几个片段的 cDNA。通过对 cDNA 克隆的物理图谱分析,建立了亚克隆,测定了它们的 cDNA 序列。这里报告其中第8条片段的 cDNA 部分序列。

单引物法同时克隆RDV基因组片段S11、S12及其序列分析

水稻矮缩病毒(Rice dwarf wrus,RDV)6个地方分离物基因组的lO%SDS-PAGE电泳图谱显示:松溪分离物基因片段S11、S12相对迁移速率明显不同。运用单引物法同时克隆该分离物的基因组片段S11、s12,并测定它们的全序列,结果表明s11、S12全长分别是1036bp、l066bp,基因结构和报道的RDV福州分离物基本一致,但分别突变了34、24个碱基,同源性分别为97.01%、97.86%。同时单引物法也为dsRNA病毒基因组的克隆提供了一种方法。

Influence of rice black streaked dwarf virus on the ecological fitness of non-vector planthopper Nilaparvata lugens （Hemiptera： Delphacidae）

Rice black streak dwarf virus （RBSDV） is transmitted by the small brown planthopper （SBPH）, Laodelphax striatellus （Fallen）. Non-vector rice brown planthopper （BPH）, Nilaparvata lugens （Stal）, shares the same host rice plants with SBPH in paddy fields. The changes in nutritional composition of rice plants infected by RBSDV and the ecological fitness of BPH feeding on the infected plants were studied under both artificial climate chamber and field conditions. Contents of 16 detected amino acids and soluble sugar in RBSDV infected rice plants were higher than those in the healthy ones. On the diseased plants BPH had significantly higher nymphal survival rates, nymphal duration of the males, weight of the female adults, as well as egg hatchability compared to BPH being fed on healthy plants. However, there was no obvious difference in female nymph duration, longevity and fecundity. Defense enzymes （superoxidase dismutase, SOD and catalase, CAT） and detoxifying enzymes （carboxylesterase, CAE and glutathione S-transferase, GST） in BPH adults fed on diseased plants had markedly higher activities. The results indicate rice plants infected by RBSDV improved the ecological fitness of the brown planthopper, a serious pest but not a transmitter of the RBSDV virus.

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.

Identification of Pns12 as the second silencing suppressor of Rice gall dwarf virus

RNA silencing is a conserved mechanism found ubiquitously in eukaryotic organisms.It has been used to regulate gene expression and development.In addition,RNA silencing serves as an important mechanism in plants' defense against invasive nucleic acids,such as viruses,transposons,and transgenes.As a counter-defense,most plants,and some animal viruses,encode RNA silencing suppressors to interfere at one or several points of the silencing pathway.In this study,we showed that Pns12 of RGDV (Rice gall dwarf virus) exhibits silencing suppressor activity on the reporter green fluorescent protein in transgenic Nicotiana benthamiana line 16c.Pns12 of RGDV suppressed local silencing induced by sense RNA but had no effect on that induced by dsRNA.Expression of Pns12 also enhanced Potato virus X pathogenicity in N.benthamiana.Collectively,these results suggested that RGDV Pns12 functions as a virus suppressor of RNA silencing,which might target an upstream step of dsRNA formation in the RNA silencing pathway.Furthermore,we showed that Pns12 is localized mainly in the nucleus of N.benthamiana leaf cells.

An assembly model of Rice dwarf virus particle An assembly model of Rice dwarf virus particle

The Phytoreovirus rice dwarf virus (RDV) has a complex nucleocapsid architecture composed of multiple proteins and RNAs. However, specific RNA-protein and protein-protein interactions involved in virion packaging have not been entirely elucidated. In order to define mechanisms governing RDV particle assembly, interactions between individual components were analyzed both in vivo and in vitro. The P7 core protein binds specifically and with high affinity to all 12 genomic RDV dsRNAs. P1, a putative RNA polymerase, P5, a putative guanyltransferase and P7 are encapsidated within the virion and also bind viral transcripts based upon in vitro binding assays. P1, P5, P7 and genomic dsRNAs were lacking in empty particles purified from infected tissues that also yielded fractions containing intact, infectious particles. In addition, P7 forms complexes with P1 and P3, a core capsid protein, in viral particles. These results indicate the possibility that core proteins and dsRNAs interact as one unit suggesting a mechanism for assortment of viral RNAs and subsequent packaging into core particles.

Suppressor of RNA silencing encoded by Rice gall dwarf virus genome segment 11

Rice gall dwarf virus(RGDV)is an important rice pathogen in China and Southeast Asia.However,little is known about the molecular mechanisms of RGDV interactions with plant cells.Here,we have identi-fied an RGDV protein,Pns11,which acts as a suppressor of RNA silencing in coinfiltration assays with the reporter,green fluorescent protein(GFP)in transgenic Nicotiana benthamiana line 16c carrying GFP.Pns11 suppressed local and systemic silencing induced by sense RNA.The spread of mobile RNA si-lencing signals was blocked or inactivated by Pns11.Expression of Pns11 also enhanced Potato virus X pathogenicity in N.benthamiana.This suppressor could reduce,but not eliminate,siRNA in the local and systemic RNA silencing suppression assays,suggesting that Pns11 functions by interfering with initial stages of RNA silencing.

Two virus-encoded RNA silencing suppressors,P14 of Beet necrotic yellow vein virus and S6 of Rice black streak dwarf virus

Functional analysis for gene silencing suppres- sor of P14 gene of Beet necrotic yellow vein virus and S6 gene of Rice black streak dwarf virus was carried out by agro- in- filtration with recombinant vectors of Potato virus X. The phenotype observation of green fluorescent protein (GFP) expression and Northern blot showed that the gene silencing of gfp transgenic Nicotiana benthamiana induced by ho- mologous sequence was strongly suppressed by the immix- ture infiltration of either the P14 or the S6. In the suppressed plants, the gfp mRNA accumulation was higher than that in the non-suppressed controls and the symptoms caused by PVX infection became more severe, especially the gfp DNA methylation of plant genome was significantly inhabited when co-infiltrated with RBSDV S6 gene. These results sug- gested that these two virus genes were potentially to encode for proteins as RNA silencing suppressors.