Southern Rice Black-streaked Dwarf Virus Research Articles

Changes in Transcriptome and Gene Expression in Sogatella furcifera (Hemiptera: Delphacidae) in Response to Cycloxaprid.

The white-backed planthopper, Sogatella furcifera (Horváth), causes substantial damage to crops by direct feeding or virus transmission, especially southern rice black-streaked dwarf virus, which poses a serious threat to rice production. Cycloxaprid, a novel cis-nitromethylene neonicotinoid insecticide, has high efficacy against rice planthoppers, including imidacloprid-resistant populations. However, information about the influence of cycloxaprid on S. furcifera (Hemiptera: Delphacidae) at the molecular level is limited. Here, by de novo transcriptome sequencing and assembly, we constructed two transcriptomes of S. furcifera and profiled the changes in gene expression in response to cycloxaprid at the transcription level. We identified 157,906,456 nucleotides and 131,601 unigenes using the Illumina technology from cycloxaprid-treated and untreated S. furcifera. In total, 38,534 unigenes matched known proteins in at least one database, accounting for 29.28% of the total unigenes. The number of coding DNA sequences was 28,546 and that of amino acid sequences in the coding region was 22,299. In total, 15,868 simple sequence repeats (SSRs) were identified. The trinucleotide repeats accounted for 45.1% (7,157) of the total SSRs and (AAG/CTT)n were the most frequent motif. There were 359 differentially expressed genes that might have been induced by cycloxaprid. There were 131 upregulated and 228 downregulated genes. Twenty-two unigenes might be involved in resistance against cycloxaprid, such as cytochrome P450, glutathione S-transferase (GST), acid phosphatase (ACP), and cadherin. Our study provides vital information on cycloxaprid-induced resistance mechanisms, which will be useful to analyze the molecular mechanisms of cycloxaprid resistance and may lead to the development of novel strategies to manage S. furcifera.

Development of marker-free rice with stable and high resistance to rice black-streaked dwarf virus disease through RNA interference.

The rice black‐streaked dwarf virus (RBSDV) disease causes severe rice yield losses in Asia. RNA interference (RNAi) has been widely applied to develop antiviral varieties in plants. So far, only a few studies reported the application of RNAi in rice against RBSDV and most of them are lack of enough data to support its breeding potential, which limited the progress on developing RBSDV‐resistant variety. In this study, we generated three RNAi constructs to specifically target three RBSDV genes (S1, S2 and S6), respectively. We confirmed that RNAi targeting RBSDV S6 conferred rice with almost full immunity to RBSDV through phenotyping test in eight consecutive years in both artificial inoculation and field trials, while RNAi of S1 or S2 only leads to partially increased resistance. The S6RNAi was also found conferring strong resistance to southern rice black‐streaked dwarf virus (SRBSDV), a novel species closely related to RBSDV that outbroke recently in Southern China. In particular, no adverse effects on agronomical and developmental traits were found in S6RNAi transgenic lines. The marker‐free transgenic lines with S6RNAi, driven by either maize ubiquitin‐1 promoter or rice rbcS green tissue expression promoter, in elite rice background should have great potential in breeding of resistant varieties to both RBSDV and SRBSDV and provide a basis for further safety evaluation and commercial application.

Emergence of Southern Rice Black-Streaked Dwarf Virus in the Centuries-Old Chinese Yuanyang Agrosystem of Rice Landraces.

Southern rice black-streaked dwarf virus (SRBSDV), which causes severe disease symptoms in rice (Oriza sativa L.) has been emerging in the last decade throughout northern Vietnam, southern Japan and southern, central and eastern China. Here we attempt to quantify the prevalence of SRBSDV in the Honghe Hani rice terraces system (HHRTS)—a Chinese 1300-year-old traditional rice production system. We first confirm that genetically diverse rice varieties are still being cultivated in the HHRTS and categorize these varieties into three main genetic clusters, including the modern hybrid varieties group (MH), the Hongyang improved modern variety group (HY) and the traditional indica landraces group (TIL). We also show over a 2-year period that SRBSDV remains prevalent in the HHRTS (20.1% prevalence) and that both the TIL (17.9% prevalence) and the MH varieties (5.1% prevalence) were less affected by SRBSDV than were the HY varieties (30.2% prevalence). Collectively we suggest that SRBSDV isolates are freely moving within the HHRTS and that TIL, HY and MH rice genetic clusters are not being preferentially infected by particular SRBSDV lineages. Given that SRBSDV can cause 30–50% rice yield losses, our study emphasizes both the need to better monitor the disease in the HHRTS, and the need to start considering ways to reduce its burden on rice production.

Dufulin Intervenes the Viroplasmic Proteins as the Mechanism of Action against Southern Rice Black-Streaked Dwarf Virus.

Southern rice black-streaked dwarf virus (SRBSDV) causes disease in crops, which reduces the quality and yield. Several commercial antiviral agents are available to control the SRBSDV induced disease. However, the mechanism of antiviral agents controlling SRBSDV is largely unknown. Identifying targets in SRBSDV is a key step of antiviral agent discovery. Here, we investigated the potential protein target of the antiviral agent dufulin. We cloned and expressed a soluble viroplasmic P6 protein in the prokaryote Escherichia coli and the eukaryote Spodoptera frugiperda 9. The dissociation constants of dufulin with the purified P6 protein from E. coli and S. frugiperda 9 expression systems were 4.49 and 4.95 μM, respectively, indicating a strong binding affinity between dufulin and P6 protein. In vivo, dufulin significantly inhibited the expression of both P6 protein and P6 gene in the SRBSDV-infected rice leaves. This inhibition on P6 protein expression was also observed in transformed Nicotiana benthamiana where the P6 was overexpressed. Our data also showed that dufulin inhibited the duplication of SRBSDV in a dose-dependent manner in infected rice leaves with a half maximum effective concentration of 3.32 mM. It is therefore concluded that dufulin targets the viroplasmic protein P6 to inhibit the virulence of SRBSDV.

Odorant-Binding Protein 2 is Involved in the Preference of Sogatella furcifera (Hemiptera: Delphacidae) for Rice Plants Infected with the Southern Rice Black-Streaked Dwarf Virus

Southern rice black-streaked dwarf virus, transmitted by the white-backed planthopper (Sogatella furcifera [Horváth]) (Hemiptera: Delphacidae) was first found in Guangdong Province, China. A previous study has demonstrated that the host plant preferences of S. furcifera are altered by infection with the virus, with virus-free S. furcifera preferring virus-infected rice plants over healthy rice plants, whereas viruliferous S. furcifera prefer uninfected plants. To test the hypothesis that odorant-binding proteins (OBPs) are involved in the preference of S. furcifera for virus-infected rice plants, we first compared the expression levels of SfurOBP2 and SfurOBP11 in virus-free and viruliferous S. furcifera. The results show that mRNA transcript of these 2 genes were significantly reduced in viruliferous S. furcifera. We then used RNAi-mediated gene silencing to confirm the function of these 2 odorant-binding proteins in host selection of S. furcifera. The results showed that silencing of the SfurOBP2 gene caused virus-free S. furcifera to no longer prefer virus-infected rice plants over uninfected rice plants, but the ability to locate host plants was maintained. These results indicate that SfurOBP2 appears to play a crucial role in the preference of S. furcifera for virus-infected rice plants.

Evaluation of reference genes and expression of key genes involved in the isoprenoid metabolic pathway of rice leaves after infection by the Southern rice black-streaked dwarf virus

The Southern rice black-streaked dwarf virus (SRBSDV), a novel Fijivirus, poses a major threat to rice production in East Asia. Analysis of the gene expression of SRBSDV-infected rice plants may reveal the molecular basis of interactions between the virus, its vector and rice plants. Reliable reference genes are required for accurate qRT-PCR analysis. However, no reliable, valid reference genes for examining gene expression in SRBSDV-infected rice plants have so far been identified. We examined the expression of eight candidate reference genes in the leaves of SRBSDV-infected, and healthy, rice plants at different points in time after virus inoculation. We used four dedicated algorithms, geNorm, BestKeeper, NormFinder and RefFinder, to evaluate the performance of these candidate genes. The RefFinder results indicate that 18S, EF1α and UBQ10 are suitable reference genes. In addition, we used these three reference genes to analyze the expression of key genes involved in the isoprenoid metabolic pathway in rice leaves after infection by SRBSDV. The results of this analysis reveal that SRBSDV may suppress the production of the rice plant volatiles that attract natural enemies of its vector Sogatella furcifera, thereby increasing the likelihood of pathogen transmission.

Close evolutionary relationship between rice black-streaked dwarf virus and southern rice black-streaked dwarf virus based on analysis of their bicistronic RNAs

BackgroundRice black-streaked dwarf virus (RBSDV) and Southern rice black-streaked dwarf virus (SRBSDV) seriously interfered in the production of rice and maize in China. These two viruses are members of the genus Fijivirus in the family Reoviridae and can cause similar dwarf symptoms in rice. Although some studies have reported the phylogenetic analysis on RBSDV or SRBSDV, the evolutionary relationship between these viruses is scarce.MethodsIn this study, we analyzed the evolutionary relationships between RBSDV and SRBSDV based on the data from the analysis of codon usage, RNA recombination and phylogenetic relationship, selection pressure and genetic characteristics of the bicistronic RNAs (S5, S7 and S9).ResultsRBSDV and SRBSDV showed similar patterns of codon preference: open reading frames (ORFs) in S7 and S5 had with higher and lower codon usage bias, respectively. Some isolates from RBSDV and SRBSDV formed a clade in the phylogenetic tree of S7 and S9. In addition, some recombination events in S9 occurred between RBSDV and SRBSDV.ConclusionsOur results suggest close evolutionary relationships between RBSDV and SRBSDV. Selection pressure, gene flow, and neutrality tests also supported the evolutionary relationships.

A Microinjection Method for Infecting the Planthopper Sogatella furcifera (Hemiptera: Delphacidae) with the Southern Rice Black-Streaked Dwarf Virus.

The southern rice black-streaked dwarf virus (SRBSDV) causes significant economic damage to rice crops. This virus is transmitted to rice plants by the planthopper Sogatella furcifera (Horváth) in a persistent, circular, and propagative manner. Researchers currently lack suitable methods for assaying the activity of SRBSDV in vitro and preserving the virus all year. We used a microinjection method to directly inject SRBSDV extracts into the hemocoel of S. furcifera nymphs. SRBSDV was subsequently detected by Reverse Transcription-Polymerase Chain Reaction in more than 56.7% of the insects after 5 d and 60% of healthy rice plants fed by these insects also became SRBSDV infected. Moreover, injecting planthopper with an extract of SRBSDV-infected rice plant that had been frozen at -80°C for 220 d caused 63.3% to become viruliferous. These results indicate that SRBSDV can be successfully transmitted to S. furcifera by microinjection, and that extracts of SRBSDV-infected rice plants frozen at -80°C for 220 d still contain sufficient active SRBSDV to infect S. furcifera. We provide a novel way to preserve SRBSDV all year by injecting S. furcifera with the SRBSDV extract.

Rice black-streaked dwarf virus P10 acts as either a synergistic or antagonistic determinant during superinfection with related or unrelated virus.

Summary Rice black‐streaked dwarf virus (RBSDV), a member of the genus Fijivirus, is a devastating pathogen of crop plants. RBSDV S10 encodes a capsid protein (P10) that is an important component of the double‐layered particle. However, little information is available on the roles of RBSDV P10 in viral infection or in interactions with other viruses. Here, we demonstrate that the expression of P10 in plants alleviates the symptoms of both RBSDV and the closely related Southern rice black‐streaked dwarf virus (SRBSDV), and reduces the disease incidence, but renders the plants more susceptible to the unrelated Rice stripe virus (RSV). Further experiments suggest that P10‐mediated resistance to RBSDV and SRBSDV operates at the protein level, rather than the RNA level, and is not a result of post‐transcriptional gene silencing. Transcriptomic data reveal that the expression of P10 in plants significantly suppresses the expression of rice defence‐related genes, which may play important roles in resistance to RSV infection. After infection with RBSDV, plants are more resistant to subsequent challenge by SRBSDV, but more susceptible to RSV. Overall, these results indicate that P10 acts as an important effector in virus interactions.

Impact of Two Reoviruses and Their Coinfection on the Rice RNAi System and vsiRNA Production.

Both Southern rice black-streaked dwarf virus (SRBSDV) and Rice ragged stunt virus (RRSV) belong to the family Reoviridae, and synergistic infection of these two viruses commonly occurs in the field. This study revealed that both SRBSDV and RRSV affect the RNA interference (RNAi) pathway and form different virus-derived interfering RNA (vsiRNA) profiles in rice. Co-infection of rice by SRBSDV and RRSV up-regulated the expression of rice DICER-like (DCL) proteins but down-regulated the expression of rice RNA-dependent RNA polymerases (RDRs), and the accumulation of vsiRNAs of either RBSDV or RRSV was decreased compared with that in singly infected plants. The majority of SRBSDV vsiRNAs were 21 nt or 22 nt in length, whether plants were singly infected with SRBSDV or co-infected with RRSV. On the other hand, the majority of RRSV vsiRNAs were 20 nt, 21 nt, or 22 nt in length, among which those 20 nt in length accounted for the largest proportion; co-infection with SRBSDV further increased the proportion of 20 nt vsiRNAs and decreased the proportion of 21 nt vsiRNAs. Co-infection had no effects on the strand favoritism and hot spots of the vsiRNAs, but changed the bias of the 5′ terminal nucleotide significantly. This study provides a reference for further study on the pathogenesis and synergistic mechanism of SRBSDV and RRSV.

Metabolomic Changes in Sogatella furcifera under Southern rice black-streaked dwarf virus Infection and Temperature Stress.

Southern rice black-streaked dwarf virus (SRBSDV) is a devastating newly emerged rice reovirus in Eastern and Southeastern Asia transmitted by a long-distance migratory pest, the white-backed planthopper (WBPH). We previously showed that SRBSDV infection decreased the cold tolerance but improved the heat tolerance of its vector, WBPH. Comparative metabolomic analysis was used to explore the potential mechanisms underlying these changes in temperature stress response. Fourth-generation WBPH nymphs were treated with SRBSDV and/or extreme temperature stress and were analyzed using gas chromatography-time of flight-mass spectrometry. A total of 605 distinguishable peaks were identified and 165, 207, and 202 differentially accumulated metabolites were identified in WBPH after virus infection, cold, or heat stress, respectively. The nucleic acids and fatty acids were the major categories of metabolites regulated by SRBSDV infection, whereas temperature stress regulated tricarboxylic acid cycle compounds, sugars, and polyols. For the WBPH samples infected with SRBSDV and subjected to temperature stress, amino acids, sugars, and polyols were the most significant regulated metabolites. The metabolomics study suggests that SRBSDV may influence the extreme temperature tolerance of WBPH by regulating the accumulation of amino acids, sugars, and polyols in the insect body.

A virus plays a role in partially suppressing plant defenses induced by the viruliferous vectors

Herbivorous attack induces plant defenses. There is evidence that some pests suppress these defenses by interfering with signaling pathways. We here report that infestation by the white-backed planthopper, Sogatella furcifera, induces defense responses in rice and infection of the southern rice black-streaked dwarf virus in the planthoppers partially suppresses the planthopper-induced plant defenses. Salicylic acid (SA) levels generally showed a temporal increase pattern while jasmonic acid (JA) levels generally exhibited a decrease pattern in the planthopper-infested plants, irrespective of virus infection status in the insects. The increase in SA was less while the decrease in JA was more in the viruliferous insect-infested plants than in the nonviruliferous insect-infested plants at both 48 and 72 h post infestation. The phytohormone levels corresponded to the patterns of relative expression levels of SA-marker genes (ICS1 and NPR1) and JA-marker gene (AOS2) in the plant treatments. Planthoppers performed better on the uninfested plants than on the previously infested plants and were of not significant increase in performance on the plants previously attacked by viruliferous planthoppers in comparison with the plants previously attacked by nonviruliferous insects. Our results indicate that the virus plays a role in partially suppressing the plant defenses induced by the planthopper. These findings provide a new perspective on plant–virus-vector interactions.

Analysis and application of the SRBSDV P9-1 octamer crystal structure for the target of α-amino phosphonate derivatives

// Yan Ding 1 , Xiangyang Li 1 , Kai Chen 1 , Guoping Zhang 1 , Liangzhi Luo 1 , Deyu Hu 1 and Baoan Song 1 1 State Key Laboratory Breeding Base of Green Pesticide and Agricultural Bioengineering, Key Laboratory of Green Pesticide and Agricultural Bioengineering, Ministry of Education, Guizhou University, Huaxi District, Guiyang 550025, PR China Correspondence to: Baoan Song, email: basong@gzu.edu.cn Xiangyang Li, email: xyli1@gzu.edu.cn Keywords: targets; SRBSDV P9-1; octamers; bind to; α-amino phosphonate derivatives Received: October 17, 2017      Accepted: December 05, 2017      Published: January 13, 2018 ABSTRACT Southern rice black-streaked dwarf virus (SRBSDV) P9-1 octameric protein accumulates viroplasms in SRBSDV-infected plant and insect cells, our previous studies found α-amino phosphonate drug—dufulin had a micromole affinity with SRBSDV P9-1. Now we focus our studies on the SRBSDV P9-1 crystal structure and use it as the target for α-amino phosphonate derivatives. The structure of the SRBSDV P9-1 cylindrical octamer was determined to a 2.2 Å resolution using X-ray crystallography, this structure was composed of nine α-helices, nine β -sheets and a series of interconnecting loops. The structures of all eight subunits were nearly identical, except for some differences in the β -sheet core. There were six different sites (R20K, V109D, F164T, V123L, C124L and V128T) present between the SRBSDV P9-1 and the previously reported RBSDV P9-1 crystal structure. Fluorescence titration, isothermal calorimetry and microscale thermophoresis experiments showed that α-amino phosphonate derivatives GUFCC-013 and GUFCC-023 bound to SRBSDV P9-1 with micromole binding affinities. These results will provide important help for the further improvement of the lead compound and develop the potential anti-SRBSDV drugs.

Differences in Molecular Characteristics of Segment 8 in Rice black-streaked dwarf virus and Southern rice black-streaked dwarf virus.

Rice black-streaked dwarf virus (RBSDV) and Southern rice black-streaked dwarf virus (SRBSDV) cause maize rough dwarf disease (MRDD) and rice black-streaked dwarf disease (RBSDD) in China. RBSDV segment 8 (S8) contains the only deletion mutation in the genomes of these viruses, which are both members of the genus Fijivirus. To illuminate the molecular differences between the RBSDV and SRBSDV genomes and better understand the evolution of these viruses, and to determine which virus is specifically associated with MRDD and RBSDD in each region, S8 was analyzed in 66 virus isolates collected from 10 geographic locations in China and 14 S8 sequences obtained from the National Center for Biotechnology Information GenBank. Phylogenetic analysis showed that the pathogen associated with MRDD and RBSDD in the Yellow and Huai River valleys was RBSDV, whereas the pathogen associated with these diseases in Sanya was SRBSDV. Codon usage bias in S8 differed significantly between RBSDV and SRBSDV, as indicated by effective number of codons used by a gene (Nc) and GC values, Nc plots, and variation explained by the first axis in correspondence analysis. The nucleotide identities among these 66 RBSDV and SRBSDV isolates ranged from 66.2 to 68.2%, and were considerably lower than the nucleotide identities within RBSDV (from 94.1 to 99.9%) or SRBSDV (from 93.9 to 100%) isolates. Most S8 polymorphisms were identified in the region from 1,000 to 1,200 bp in RBSDV and in the region from 500 to 700 bp in SRBSDV. The difference in the lengths of RBSDV (1,936 bp) and SRBSDV (1,928 bp) was due to an 8-bp deletion in the 3'-untranslated region of SRBSDV. Six recombination events were detected in S8 in RBSDV and two recombination events were detected in S8 in SRBSDV. Recombination breakpoints were found within the region containing the deletion mutation in nine isolates. However, no recombination events were detected between RBSDV and SRBSDV. Both of these viruses were under negative and purifying selection, although the ratio of nonsynonymous mutations to synonymous mutations (Ka/Ks) for RBSDV S8 (0.0530) was not significantly lower than that of SRBSDV S8 (0.0823, P = 0.1550). We found that SRBSDV was more highly genetically differentiated (product of effective population size and the migration rate among populations < 1; values for the among-populations component of genetic variation or normalized variation > 0.33; and P values of the sequence statistic, the rank statistic, and the nearest-neighbor statistic < 0.01) than RBSDV. However, gene flow between RBSDV and SRBSDV was not frequent.

Investigation of alimentary canal ultrastructure following knockdown of the Dicer-2 gene in planthoppers reveals the potential pathogenicity of southern rice black streaked dwarf virus to its insect vector

An increasing number of studies are suggesting that plant viruses, including southern rice black-streaked dwarf virus (SRBSDV), can adversely affect biological characteristics of insect vectors by unknown mechanisms. To study the adverse effect of SRBSDV at cellular level on the insect vector, we promoted viral infection by the disruption of the small interfering RNA (siRNA) pathway. The transmission electron microscopy was utilized to describe the ultrastructural changes that occurred in insects when the core component of the siRNA pathway, Dicer-2, was knocked down. The increasing accumulation of SRBSDV in virus-infected vector, the white-backed planthoppers, caused severe cytopathology in the alimentary canal. Similar cytopathology changes in the midgut ultrastructure were characterized in the virus-infected incompetent vector, the small brown planthopper. These results not only add support to the existing evidence suggesting that the siRNA pathway has an antiviral effect, but also reveal the universal and potential ability of SRBSDV to cause damage to the insect tissues of both the vector and non-vector.

A novel method for transmitting southern rice black-streaked dwarf virus to rice without insect vector

BackgroundSouthern rice black-streaked dwarf virus (SRBSDV) has spread from the south of China to the north of Vietnam in the past few years, and has severely influenced rice production. However, previous study of traditional SRBSDV transmission method by the natural virus vector, the white-backed planthopper (WBPH, Sogatella furcifera), in the laboratory, researchers are frequently confronted with lack of enough viral samples due to the limited life span of infected vectors and rice plants and low virus acquisition and inoculation efficiency by the vector. Meanwhile, traditional mechanical inoculation of virus only apply to dicotyledon because of the higher content of lignin in the leaves of the monocot. Therefore, establishing an efficient and persistent-transmitting model, with a shorter virus transmission time and a higher virus transmission efficiency, for screening novel anti-SRBSDV drugs is an urgent need.MethodsIn this study, we firstly reported a novel method for transmitting SRBSDV in rice using the bud-cutting method. The transmission efficiency of SRBSDV in rice was investigated via the polymerase chain reaction (PCR) method and the replication of SRBSDV in rice was also investigated via the proteomics analysis.ResultsRice infected with SRBSDV using the bud-cutting method exhibited similar symptoms to those infected by the WBPH, and the transmission efficiency (>80.00%), which was determined using the PCR method, and the virus transmission time (30 min) were superior to those achieved that transmitted by the WBPH. Proteomics analysis confirmed that SRBSDV P1, P2, P3, P4, P5–1, P5–2, P6, P8, P9–1, P9–2, and P10 proteins were present in infected rice seedlings infected via the bud-cutting method.ConclusionThe results showed that SRBSDV could be successfully transmitted via the bud-cutting method and plants infected SRBSDV exhibited the symptoms were similar to those transmitted by the WBPH. Therefore, the use of the bud-cutting method to generate a cheap, efficient, reliable supply of SRBSDV-infected rice seedlings should aid the development of disease control strategies. Meanwhile, this method also could provide a new idea for the other virus transmission in monocot.

Varietal differences in ovicidal response to the white-backed planthopper Sogatella furcifera (Hemiptera: Delphacidae) and susceptibility to Southern rice black-streaked dwarf virus in rice

The recent spread of Oryza sativa L. (rice) ssp. indica-derived cultivars in East and Southeast Asia increases the risk of white-backed planthopper, Sogatella furcifera, and Southern rice black-streaked dwarf virus (SRBSDV) outbreaks. We compared the degree of ovicidal response to S. furcifera and susceptibility to SRBSDV among two Oryza sativa ssp. japonica, two Oryza sativa ssp. indica and one Oryza sativa ssp. indica-derived rice cultivars. The ovicidal response of the O. sativa ssp. indica-derived cultivar was significantly higher than that of the tested O. sativa ssp. indica cultivars and was equivalent to that of the O. sativa ssp. japonica cultivars that we used, supporting previous findings that the ovicidal response is higher in O. sativa ssp. japonica cultivars than in O. sativa ssp. indica cultivars and is a quantitative trait controlled by a few relevant genes. Contrary to the varied ovicidal response, the SRBSDV infection rate among the cultivars and the height of SRBSDV-infected plants between most pairs of cultivars did not differ significantly, although twisting of the leaf tips, a typical symptom of SRBSDV, was not observed in the O. sativa ssp. japonica cultivars. These results indicate that damage by S. furcifera and SRBSDV can occur in O. sativa ssp. japonica as well as in O. sativa ssp. indica and O. sativa ssp. indica-derived cultivars; therefore, in the cultivation of O. sativa ssp. japonica, measures should also be taken against white-backed planthopper during high levels of emergence.

Co-infection of two reoviruses increases both viruses accumulation in rice by up-regulating of viroplasm components and movement proteins bilaterally and RNA silencing suppressor unilaterally

BackgroundSynergism between southern rice black-streaked dwarf virus (SRBSDV) and rice ragged stunt virus (RRSV) not only aggravates disease symptoms but also enhances their vector acquisition efficiencies by increasing both viruses’ titers in co-infected rice plants, which may exacerbate the epidemic of both viruses and cause significant damage to rice production. The molecular mechanism of viral synergism of these two viruses remains unexplored.MethodsSingle and double infection of SRBSDV and RRSV were obtained with the viruliferous white-backed planthopper and brown planthopper inoculation on four-leaf stage rice seedlings, respectively, under experimental condition. The second upper leaf from each inoculated rice plants were collected at 9, 15, and 20 days post inoculation (dpi) and used for relative quantification of 13 SRBSDV genes and 11 RRSV genes by the reverse-transcription quantitative PCR. Viral gene expression levels were compared between singly and doubly infected samples at the same stage.ResultsThe movement protein and viroplasm matrix-related genes as well as the structural (capsid) protein genes of both viruses were remarkably up-regulated at different time points in the co-infected rice plants compared with the samples singly infected with SRBSDV or RRSV, however, the RNA silencing suppressor (P6) of only RRSV, but not of both the viruses, was up-regulated.ConclusionsThe SRBSDV-RRSV synergism promoted replication and movement of both viruses and inhibited the host immunity by enhancing the gene suppressing effect exerted by one of them (RRSV).

Sublethal effects of imidacloprid on the development, reproduction, and susceptibility of the white-backed planthopper, Sogatella furcifera (Hemiptera: Delphacidae)

Sogatella furcifera (Horváth) (white-backed planthopper, WBPH) is one of the most important pests on rice in Asia. It seriously impacts rice production by sucking phloem from the rice plant and transmitting southern rice black-streaked dwarf virus (SRBSDV). In the current study, we examined the sublethal effects of imidacloprid on the development, reproduction, and susceptibility of S. furcifera in the laboratory using the rice stem dipping method. The results showed that exposure of third-instar S. furcifera nymphs to sublethal concentrations (LC10 and LC25) of imidacloprid inhibited the rate of nymphal development in the F0 and F1 generations, and there was a significant difference between the LC25 treated group and the control group. The fecundity (eggs per female) of the F1 generation showed significant declines of 22.05% and 38.56% following exposure to LC10 and LC25, respectively compared with the control group. Emergence rate, copulation rate, and hatchability were significantly lower in the LC25-treated group. The relative fitness of S. furcifera treated with LC10 and LC25 imidacloprid was 0.46 and 0.22, respectively. These results showed that exposure to imidacloprid impacted the next generation of S. furcifera, thus affecting the population growth of this species. After third-instar S. furcifera nymphs were exposed to sublethal concentrations of imidacloprid LC10 and LC25, S. furcifera showed reduced susceptibility to treatment with thiamethoxam and buprofezin. Although the susceptibility of S. furcifera to chlorpyrifos was decreased after the exposure of third-instar nymphs to imidacloprid LC25, S. furcifera populations that had been exposed to imidacloprid LC10 showed enhanced susceptibility to chlorpyrifos. These results provided a reference point for the application of insecticides to control S. furcifera.

First report of Maize yellow mosaic virus infecting maize in Ecuador

Maize yellow mosaic virus (MaYMV) was first described in maize (Zea mays) from China and proposed as a new virus in the genus Polerovirus (Chen et al., 1). Recently, MaYMV was also detected in symptomatic maize plants in Burkina Faso (Palanga et al., 3) indicating a global distribution for the virus. In May 2016 maize plants showing leaf chlorosis and downward curling of leaf tips were collected from a large production area (c. 125,000 hectares in maize cultivation) in the Los Rios Province in Ecuador. It has been estimated that the disease causes yield losses of 10-30%. Total RNA from symptomatic leaf samples was extracted using the RNeasy Plant Mini Kit (Qiagen, USA) and subjected to deep sequencing analysis using the Illumina platform. Quality filtered paired end reads (8,777,028) were aligned with reference genomes of known plant viruses using standard bioinformatic tools. The transcriptome was highly enriched with viral RNA reads (>5%) showing significant similarity with Sugarcane mosaic virus (SCMV) and other MaYMV sequences. These short reads were assembled into contigs and full-length and nearly full-length genome sequences of SCMV (9632 nt; Genbank Accession No. K006657) and MaYMV (5488 nt; KY052793) were identified. Alignments and phylogenetic trees were performed using SeaView software (Gouy et al., 2). The Ecuadorian MaYMV isolate had the highest nucleotide identity (96%) with a Chinese isolate (KU291103). MaYMV has a typical Polerovirus organisation with six open reading frames (ORFs; 0-5) including ORF3a. ORF3a is translated from a non-standard start codon (in this case AUA) and encodes a protein responsible for long distance movement of the virus in plants (Smirnova et al., 5). ORF3a (nt 3389-3523) shows a single amino acid substitution compared with the Chinese isolate (KU291103). ORF0 (nt 42-836) shows 97% amino acid identity with MaYMV isolate KU291100 (Table 1) and harbours an essential F-box like element (Pazhouhandeh et al., 4). The phylogenetic tree (Fig. 1) constructed from ORF0 amino acid sequences indicates significant similarity to the Chinese MaYMV isolate and high variability between different species of the genus Polerovirus. Phylogenetic analysis of ORF3 from selected species of the genera Enamovirus, Luteovirus and Polerovirus places the Ecuadorian isolate within a group of poleroviruses that infect a wide range of host plants (Fig. 2). MaMYV has been reported to be generally present in mixed infections with other viruses, including Maize chlorotic mottle virus, Southern rice black-streaked dwarf virus or SCMV, but has also been found in single infections in China (Chen et al., 1) displaying characteristic disease symptoms. The significance of mixed infections with SCMV in disease pathology in Ecuador is unclear and requires further investigation. The authors are thankful to SENESCYT Ecuador (Prometeo Project) and Agrocalidad, Ecuador for their financial support.