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.

Transcriptome analysis reveals different response of resistant and susceptible rice varieties to rice stripe virus infection

Rice stripe disease,caused by rice stripe virus (RSV) which is transmitted by small brown planthopper (SBPH,Laodelphax striatellus Fallen),resulted in serious losses to rice production during the last 2 decades.Research on the molecular differences between resistant and susceptible rice varieties and the interaction between rice and RSV remains inadequate.In this study,RNA-Seq was used to analyze the transcriptomic differences between the resistant and susceptible rice varieties at different times post RSV infection.Through Gene Ontology (GO) annotation,the differentially expressed genes (DEGs) related to transcription factors,peroxidases,and kinases of 2 varieties at 3 time points were identified.Comparing these 2 varieties,the DEGs associated with these 3 GOs were numerically less in the resistant variety than in the susceptible variety,but the expression showed a significant up-or down-regulation trend under the conditions of|log_2(Fold change)|>0&P_(adj)<0.05 by significance analysis.Then through Kyoto Encyclopedia of Genes and Genomes (KEGG) annotation,DEGs involved in some pathways that have a contribution to disease resistance including plant hormone signal transduction and plant–pathogen interaction were found.The results showed that resistance responses regulated by abscisic acid (ABA) and brassinosteroids (BR) were the same for 2 varieties,but that mediated by salicylic acid (SA) and jasmonic acid (JA)/ethylene (ET) were different.The DEGs in resistant and susceptible varieties at the 3 time points were identified in both PAMP-triggered immunity (PTI) and Effector proteintriggered immunity (ETI),with that most of the unigenes of the susceptible variety were involved in PTI,whereas most of the unigenes of the resistant variety were involved in ETI.These results revealed the different responses of resistant and susceptible varieties in the transcription level to RSV infection.

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）.

Characterization of protein-protein interactions between rice viruses and vector insects

Planthoppers are the most notorious rice pests,because they transmit various rice viruses in a persistent-propagative manner.Protein–protein interactions(PPIs)between virus and vector are crucial for virus transmission by vector insects.However,the number of known PPIs for pairs of rice viruses and planthoppers is restricted by low throughput research methods.In this study,we applied DeNovo,a virus-host sequence-based PPI predictor,to predict potential PPIs at a genome-wide scale between three planthoppers and five rice viruses.PPIs were identified at two different confidence thresholds,referred to as low and high modes.The number of PPIs for the five planthopper-virus pairs ranged from 506 to 1985 in the low mode and from 1254 to 4286 in the high mode.After eliminating the“one-too-many”redundant interacting information,the PPIs with unique planthopper proteins were reduced to 343–724 in the low mode and 758–1671 in the high mode.Homologous analysis showed that 11 sets and 31 sets of homologous planthopper proteins were shared by all planthopper-virus interactions in the two modes,indicating that they are potential conserved vector factors essential for transmission of rice viruses.Ten PPIs between small brown planthopper and rice stripe virus(RSV)were verified using glutathione-S-transferase(GST)/His-pull down or co-immunoprecipitation assay.Five of the ten PPIs were proven positive,and three of the five SBPH proteins were confirmed to interact with RSV.The predicted PPIs provide new clues for further studies of the complicated relationship between rice viruses and their vector insects.

Screening of Rice Stripe Virus(RSV)-resistant Germplasm from Landraces of Rice(Oryza sativa L.)

[Objective] The aim of this study was to screen rice strip virus （RSV）-resistant landraces. [Method] The resistance of 119 rice landraces to rice stripe virus was identified in field spontaneously infected with smal plant-hopper. [Result] There were 55 landraces resistant to rice strip disease in 56 indica rice landraces, but on-ly two resistant to rice strip disease in 63 japonica rice landraces. [Conclusion] The results revealed that there were abundant rice landscapes resistant to RSV in Chi-na, and these varieties can be used to develop more genes resistant to RSV.

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.

Surface Display of Rice Stripe Virus NSvc2 and Analysis of Its Membrane Fusion Activity

Rice stripe virus (RSV) infects rice and is transmitted in a propagative manner by the small brown planthopper. How RSV enters an insect cell to initiate the infection cycle is poorly understood. Sequence analysis revealed that the RSV NSvc2 protein was similar to the membrane glycoproteins of several members in the family Bunyaviridae and might induce cell membrane fusion. To conveniently study the membrane fusion activity of NSvc2, we constructed cell surface display vectors for expressing Nsvc2 on the insect cell surface as the membrane glycoproteins of the enveloped viruses. Our results showed that NSvc2 was successfully expressed and displayed on the surface of insect Sf9 cells. When induced by low pH, the membrane fusion was not observed in the cells that expressed NSvc2. Additionally, the membrane fusion was also not detected when co-expressing Nsvc2 and the viral capsid protein on insect cell surface. Thus, RSV NSvc2 is probably different from the phlebovirus counterparts, which could suggest different functions. RSV might enter insect cells other than by fusion with plasma or endosome membrane.

Highly Sensitive and Specific Monoclonal Antibody-Based Serological Methods for Rice Ragged Stunt Virus Detection in Rice Plants and Rice Brown Planthopper Vectors

Rice ragged stunt virus（RRSV） is a serious rice disease in Asia, causing serious yield losses on rice. The capsid protein（CP） gene of the major outer capsid protein of RRSV was expressed in Escherichia coli BL21（DE3） using the pMAL-C2 X expression vector. The recombinant protein was used as the immunogen to immunize BALB/c mice. A hybridoma cell line 8A12 secreting monoclonal antibody（MAb） against RRSV was obtained by fusing mouse myeloma cells（Sp 2/0） with spleen cells from the immunized BALB/c mice. Western blot analysis showed that the MAb 8A12 can specifically react with RRSV CP. Using the MAb, an antigen-coated-plate enzyme-linked immunosorbent assay（ACP-ELISA）, a dot enzyme-linked immunosorbent assay（dot-ELISA）, and immunocapture-RT-PCR（IC-RT-PCR） assay were developed to detect RRSV. The established ACP-ELISA, dot-blot ELISA and IC-RT-PCR methods could detect RRSV in infected rice tissue crude extracts with dilutions of 1：40 960, 1：1 280 and 1：655 360（w/v, g mL-1）, respectively. The ACP-ELISA and dot-blot ELISA methods could detect RRSV in infected insect vector crude extracts with dilutions of 1：12 800 and 1：1 600（an individual planthopper μL-1）, respectively. The field survey revealed that Rice ragged stunt disease occurs on rice in Hainan, Yunnan, Guangxi, Sichuan, Guizhou, Fujian, Hunan, Jiangxi and Zhejiang 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.

RNAi-mediated transgenic rice resistance to Rice stripe virus

Rice stripe virus（RSV） often causes severe rice yield loss in temperate regions of East Asia. Although the correlation of small interfering RNAs（si RNAs） with transgenic virus resistance of plants using RNA interference（RNAi） is known for decades, no systematical research has been done on the profiling of si RNAs from a genomic scale. Our research is aiming to systematically study the RNAi impact in RSV-resistant transgenic rice, which was generated by introducing an inverted repeat construct that targets RSV nucleocapsid protein（NCP） gene. In this paper, three independent RSV-retsistant transgenic rice lines were generated, their stable integration of the T-DNA fragment and the expression of si RNAs were confirmed by Southern blotting and Northern blotting analyses, and the majority of si RNAs were in lengths of 21, 22, and 24 nucleotides（nt）, which have validated a connection between the presence of the RSV NCP homologous si RNAs and the RSV resistance in those transgenic rice lines. In one of these transgenic lines（T4-B1）, the T-DNA fragment was found to have been inserted at chromosome 1 of the rice genome, substituting the rice genome fragment from 32 158 773 to 32 158 787 nt. Bioinformatics analysis of small RNA-Seq data on the T4-B1 line also confirmed the large population of NCP-derived si RNAs in transgenic plants, and the RSV-infected library（T4-B1-V） possessed more si RNAs than its mock inoculated libraries（T4-B1-VF）, these results indicating the inverted repeat construct and RSV could introduce abundance of si RNAs in transgenic rice. Moreover, a varied expression level of specific si RNAs was found among different segments of the NCP gene template, about 47% of NCP-derived si RNAs reads aligned with the fragment from 594 to 832 nt（239 nt in length） in NCP gene（969 nt in length） in the T4-B1-V, indicating a potential usage of hotspot regions for RNAi silencing in future research. In conclusion, as the first study to address the si RNA profile in RSV-resistant transgenic plant using next generation sequencing（NGS） technique, we confirmed that the massive abundance of si RNA derived from the inverted repeat of NCP is the major reason for RSV-resistance.

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.

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.

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.

Detection of Rice Yellow Mottle at the Asymptomatic Stage by Hyperspectral Fluorescence and Reflectance Spectroscopies

Rice yellow mottle is considered the most destructive disease threatening rice production in Africa. Early detection of this infection in rice is essential to limit its expansion and proliferation. However, there is no research devoted to the spectral detection of rice yellow mottle virus (RYMV) infection, especially in the asymptomatic or early stages. This work proposes the use of hyperspectral fluorescence and reflectance data at leaf level for the detection of this disease in asymptomatic stages. A greenhouse experiment was therefore conducted to collect hyperspectral fluorescence and reflectance data at different stages of infection. These data allowed to calculate nine vegetation indices: one from fluorescence spectra and eight from reflectance spectra. A t-test made it possible to identify, from the second day after infection, four relevant reflectance vegetation indices to discriminate healthy leaves from those infected: these are Photochemical Reflectance Index (PRI), Transformed Chlorophyll Absorption in Reflectance Index (TCARI), Structure Intensive Pigment Index (SIPI) and Simple Ratio Pigment Index (SRPI). The fluorescence index was less sensitive in detecting infection. The four significant vegetation indices for the detection of RYMV were then used to build and evaluate models for discriminating plants according to their health status by the supervised classification of support vector machine (SVM) at different stages of infection. The maximum overall accuracy is 92.5% six days after inoculation (6 DAI). The sixth day after inoculation would be the adequate day to detect RYMV. This plants discrimination was validated by the mean reflectance spectra and by the histograms showing the differences between the average reflectance vegetation indices values of the two types of plants. Our results demonstrate the feasibility of differentiating RYMV-infected samples. They suggest that support vector machine learning models could be developed to diagnose RYMV-infected plants based on vegetation indices derived from spectral profiles at early stages of disease development.

抗性品种对预防水稻条纹叶枯病发生和流行的研究

：２８～３０℃条件下，用相同数量、带毒率为５０～５１％的灰飞虱经卵毒虫，分别接种于抗水稻条纹叶枯病品种与感病品种上，表明品种间的抗性由种质决定；抗性品种的发病潜育期为１０～１３天，起始发病率为２５～３５％，最终发病率为２４～３５％，卷叶症状０～２５％；感病品种发病潜育期为５～６天，起始发病率为１７５～３３０％，最终发病率为８７～１００％，卷叶症状７０～９０％；介体昆虫在感病品种上的繁殖率分别为抗病品种盐粳２０、中国９１的１６５和１６１。

Rice St#pe Virus Interferes with S-acylation of Remorin and Induces Its Autophagic Degradation to Facilitate Virus Infection

Remorins are plant-specific membrane-associated proteins and were proposed to play crucial roles in plant-pathogen interactions. However, little is known about how pathogens counter remorin-mediated host responses. In this study, by quantitative whole-proteome analysis we found that the remorin protein （NbREM1） is downregulated early in Rice stripe virus （RSV） infection. We further discovered that the turn- over of NbREM1 is regulated by S-acylation modification and its degradation is mediated mainly through the autophagy pathway. Interestingly, RSV can interfere with the S-acylation of NbREM1, which is required to negatively regulate RSV infection by restricting virus cell-to-cell trafficking. The disruption of NbREM1 S-acylation affects its targeting to the plasma membrane microdomain, and the resulting accumulation of non-targeted NbREM1 is subjected to autophagic degradation, causing downregulation of NbREMI. Moreover, we found that RSV-encoded movement protein, NSvc4, alone can interfere with NbREM1 S-acylation through binding with the C-terminal domain of NbREM1 the S-acylation of OsREM1.4, the homologous remorin of NbREM1, and thus remorin-mediated defense against RSV in rice, the original host of RSV, indicating that downregulation of the remorin protein level by interfering with its S-acylation is a common strategy adopted by RSV to overcome remorin-mediated inhibition of virus movement.

Interaction between Rice stripe virus Disease- Specific Protein and Host PsbP Enhances Virus Symptoms

Rice stripe virus （RSV） causes severe diseases in Oryza sativa （rice） in many Eastern Asian countries. Diseasespecific protein （SP） of RSV is a non-structural protein and its accumulation level in rice plant was shown to determine the severity of RSV symptoms. Here, we present evidence that expression of RSV SP alone in rice or Nicotiana benthamiana did not produce visible symptoms. Expression of SP in these two plants, however, enhanced RSV- or Potato virus X （PVX）- induced symptoms. Through yeast two-hybrid screening, GST pull-down, and bimolecular fluorescence complementation assays, we demonstrated that RSV SP interacted with PsbP, a 23-kDa oxygen-evolving complex protein, in both rice and N. benthamiana. Furthermore, our investigation showed that silencing of PsbP expression in both plants increased disease symptom severity and virus accumulation. Confocal microscopy using N, benthamiana protoplast showed that PsbP accu- mulated predominantly in chloroplast in wild-type N. benthamiana cells. In the presence of RSV SP, most PsbP was recruited into cytoplasm of the assayed cells. In addition, accumulation of SP during RSV infection resulted in alterations of chloroplast structure and function. Our findings shed light on the molecular mechanism underlying RSV disease symptom development.