Southern Rice Black-streaked Dwarf Virus Research Articles

Interaction research on an antiviral molecule that targets the coat protein of southern rice black-streaked dwarf virus

Southern rice black-streaked dwarf virus (SRBSDV) coat protein (P10) is the key protein required for viral transmission and host plant infection and is thus a promising target for anti-SRBSDV agent screening. In this study, P10 was obtained from Escherichia coli through cloning, expression, and purification. The antiviral agent Ningnanmycin was selected as control, and a series of antiviral compounds based on the structural scaffold of ferulic acid were analyzed. Size-exclusion chromatography analysis results showed that compound F27 can alter the aggregation of P10 proteins. Furthermore, fluorescence titration and microscale thermophoresis assay results indicated that F27 binds to P10 with KA of 5.75×105M−1 and KD of 7.81μM. The ligand- and receptor-based three-dimensional quantitative structure–activity analyses were performed to determine the requirements for the interaction between the carboxyl structures and P10s. On the basis of the obtained models and information, we provided insights regarding the design and optimization of novel molecules as anti-SRBSDV agents.

The Influence of Sogatella furcifera (Hemiptera: Delphacidae) Migratory Events on the Southern Rice Black-Streaked Dwarf Virus Epidemics.

Southern rice black-streaked dwarf virus (SRBSDV) caused serious rice losses. After the first outbreak in 2009 in northern Vietnam and southern China, the virus ravaged crops again on enormous scales in 2010, but infections have decreased sharply since 2011. We presumed that the sudden epidemics and fadeout of SRBSDV would be closely related to the migratory events of the insect vector, Sogatella furcifera. This study sought the source area of SRBSDV using the trajectory analysis method, and revealed the relationship between SRBSDV dynamics and migration of S. furcifera populations via an in-depth analysis of meteorological background of S. furcifera migration fields. The results showed that Northern Vietnam was the direct virus source area of the SRBSDV infection in China, and South Central Coast of Vietnam was the original source area of SRBSDV. Southwesterly winds were prevalent in spring of 2010 and carried large numbers of viruliferous S. furcifera to China from northern Vietnam. This infestation of S. furcifera was the direct cause of the SRBSDV outbreak in China in 2010. In 2011, the winter-spring temperatures were abnormally low and southeasterly and easterly winds dominated; therefore, the number of viruliferous S. furcifera that entered China was small, and consequently, the occurrence area of SRBSDV was rapidly reduced. The return of viruliferous S. furcifera to South Central Coast of Vietnam was an important factor that affected the occurrence scale of SRBSDV in the following year.

Evaluation of Rice Resistance to Southern Rice Black-Streaked Dwarf Virus and Rice Ragged Stunt Virus through Combined Field Tests, Quantitative Real-Time PCR, and Proteome Analysis.

Diseases caused by southern rice black-streaked dwarf virus (SRBSDV) and rice ragged stunt virus (RRSV) considerably decrease grain yield. Therefore, determining rice cultivars with high resistance to SRBSDV and RRSV is necessary. In this study, rice cultivars with high resistance to SRBSDV and RRSV were evaluated through field trials in Shidian and Mangshi county, Yunnan province, China. SYBR Green I-based quantitative real-time polymerase chain reaction (qRT-PCR) analysis was used to quantitatively detect virus gene expression levels in different rice varieties. The following parameters were applied to evaluate rice resistance: acre yield (A.Y.), incidence of infected plants (I.I.P.), virus load (V.L.), disease index (D.I.), and insect quantity (I.Q.) per 100 clusters. Zhongzheyou1 (Z1) and Liangyou2186 (L2186) were considered the most suitable varieties with integrated higher A.Y., lower I.I.P., V.L., D.I. and I.Q. features. In order to investigate the mechanism of rice resistance, comparative label-free shotgun liquid chromatography tandem-mass spectrometry (LC-MS/MS) proteomic approaches were applied to comprehensively describe the proteomics of rice varieties’ SRBSDV tolerance. Systemic acquired resistance (SAR)-related proteins in Z1 and L2186 may result in the superior resistance of these varieties compared with Fengyouxiangzhan (FYXZ).

Characteristics of siRNAs derived from Southern rice black-streaked dwarf virus in infected rice and their potential role in host gene regulation

BackgroundVirus-derived siRNAs (vsiRNAs)-mediated RNA silencing plays important roles in interaction between plant viruses and their hosts. Southern rice black-streaked dwarf virus (SRBSDV) is a newly emerged devastating rice reovirus with ten dsRNA genomic segments. The characteristics of SRBSDV-derived siRNAs and their biological implications in SRBSDV-rice interaction remain unexplored.MethodsVsiRNAs profiling from SRBSDV-infected rice samples was done via small RNA deep sequencing. The putative rice targets of abundantly expressed vsiRNAs were bioinformatically predicted and subjected to functional annotation. Differential expression analysis of rice targets and RNA silencing components between infected and healthy samples was done using RT-qPCR.ResultsThe vsiRNA was barely detectable at 14 days post infection (dpi) but abundantly present along with elevated expression level of the viral genome at 28 dpi. From the 28-dpi sample, 70,878 reads of 18 ~ 30-nt vsiRNAs were recognized (which mostly were 21-nt and 22-nt), covering 75 ~ 91% of the length of the ten genomic segments respectively. 86% of the vsiRNAs had a <50% GC content and 79% of them were 5’-uridylated or adenylated. The production of vsiRNAs had no strand polarity but varied among segment origins. Each segment had a few hotspot regions where vsiRNAs of high abundance were produced. 151 most abundant vsiRNAs were predicted to target 844 rice genes, including several types of host resistance or pathogenesis related genes encoding F-box/LRR proteins, receptor-like protein kinases, universal stress proteins, tobamovirus multiplication proteins, and RNA silencing components OsDCL2a and OsAGO17 respectively, some of which showed down regulation in infected plants in RT-qPCR. GO and KEGG classification showed that a majority of the predicted targets were related to cell parts and cellular processes and involved in carbohydrate metabolism, translation, and signal transduction. The silencing component genes OsDCL2a, OsDCL2b, OsDCL4, and OsAGO18 were down regulated, while OsAGO1d, OsAGO2, OsRDR1 and OsRDR6 were up regulated, significantly, upon SRBSDV infection.ConclusionsSRBSDV can regulate the expression of rice RNA silencing pathway components and the virus might compromise host defense and influence host pathogenesis via siRNA pathways.

Interaction between southern rice black-streaked dwarf virus minor core protein P8 and a rice zinc finger transcription factor.

The fijivirus southern rice black-streaked dwarf virus (SRBSDV) causes one of the most serious viral diseases of rice in China and Vietnam. To better understand the molecular basis of SRBSDV infection, a yeast two-hybrid screen of a rice cDNA library was carried out using P8, a minor core protein of SRBSDV, as the bait. A rice Cys2His2-type zinc finger protein (OsZFP) was found to interact with SRBSDV P8. A strong interaction between SRBSDV P8 and OsZFP was then confirmed by pull-down assays, and bimolecular fluorescence complementation assays showed that the in vivo interaction was specifically localized in the nucleus of plant cells. Using a series of deletion mutants, it was shown that both the NTP-binding region of P8 and the first two zinc fingers of OsZFP were crucial for their interaction in plant cells. The localization in the nucleus and activation of transcription in yeast supports the notion that OsZFP is a transcription factor. SRBSDV P8 may play an important role in fijiviral infection and symptom development by interfering with the host transcription activity of OsZFP.

Genomic and phylogenetic evidence that Maize rough dwarf and Rice black-streaked dwarf fijiviruses should be classified as different geographic strains of a single species.

Maize rough dwarf disease (MRDD) has long been known as one of the most devastating viral diseases of maize worldwide and is caused by single or complex infection by four fijiviruses: Maize rough dwarf virus (MRDV) in Europe and the Middle East, Mal de Rio Cuarto virus (MRCV) in South America, rice black-streaked dwarf virus (RBSDV), and Southern rice black-streaked dwarf virus (SRBSDV or Rice black-streaked dwarf virus 2, RBSDV-2) in East Asia. These are currently classified as four distinct species in the genus Fijivirus, family Reoviridae, but their taxonomic status has been questioned. To help resolve this, the nucleotide sequences of the ten genomic segments of an Italian isolate of MRDV have been determined, providing the first complete genomic sequence of this virus. Its genome has 29144 nucleotides and is similar in organization to those of RBSDV, SRBSDV, and MRCV. The 13 ORFs always share highest identities (81.3-97.2%) with the corresponding ORFs of RBSDV and phylogenetic analyses of the different genome segments and ORFs all confirm that MRDV clusters most closely with RBSDV and that MRCV and SRBSDV are slightly more distantly related. The results suggest that MRDV and RBSDV should be classified as different geographic strains of the same virus species and we suggest the name cereal black-streaked dwarf fijivirus (CBSDV) for consideration.

Virus altered rice attractiveness to planthoppers is mediated by volatiles and related to virus titre and expression of defence and volatile-biosynthesis genes.

Viruses may induce changes in plant hosts and vectors to enhance their transmission. The white-backed planthopper (WBPH) and brown planthopper (BPH) are vectors of Southern rice black-streaked dwarf virus (SRBSDV) and Rice ragged stunt virus (RRSV), respectively, which cause serious rice diseases. We herein describe the effects of SRBSDV and RRSV infections on host-selection behaviour of vector and non-vector planthoppers at different disease stages. The Y-tube olfactometer choice and free-choice tests indicated that SRBSDV and RRSV infections altered the attractiveness of rice plants to vector and non-vector planthoppers. The attractiveness was mainly mediated by rice volatiles, and varied with disease progression. The attractiveness of the SRBSDV- or RRSV-infected rice plants to the virus-free WBPHs or BPHs initially decreased, then increased, and finally decreased again. For the viruliferous WBPHs and BPHs, SRBSDV or RRSV infection increased the attractiveness of plants more for the non-vector than for the vector planthoppers. Furthermore, we observed that the attractiveness of infected plants to planthoppers was positively correlated with the virus titres. The titre effects were greater for virus-free than for viruliferous planthoppers. Down-regulated defence genes OsAOS1, OsICS, and OsACS2 and up-regulated volatile-biosynthesis genes OsLIS, OsCAS, and OsHPL3 expression in infected plants may influence their attractiveness.

An epidemic model of a rice virus transmitted by a migratory planthopper

Recent outbreaks of viral diseases of rice in east Asia are caused by migratory planthoppers. Epidemic models of these viral diseases are required to predict the annual emergence of the viruses based on established forecasting systems of outbreaks of the planthoppers. Here, we develop an epidemic model for southern rice black-streaked dwarf virus (SRBSDV), which is transmitted by the white-backed planthopper, Sogatella furcifera, as the first epidemic model of viruses borne by migratory planthoppers. The epidemic model, consisting of a Leslie model for the population dynamics of S. furcifera and a transmission model for SRBSDV, predicted a high contribution of the density of viruliferous immigrant S. furcifera to SRBSDV emergence. Early immigration of S. furcifera into paddy fields after transplanting of rice also promoted the virus epidemic primarily because of the emergence of viruliferous old nymphs during the period during which rice plants are susceptible to the virus. The epidemic model developed could be expanded to other planthopper-borne viruses and is effective for understanding the ecological aspects of virus epidemics in the field and for assessing the risk of viral diseases transmitted by migratory vectors.

Biology of Southern rice black‐streaked dwarf virus: a novel fijivirus emerging in East Asia

Southern rice black‐streaked dwarf virus (SRBSDV) was first reported in southern China in 2001 and causes a striking disease on rice and maize that leads to serious yield losses in several East Asian countries, such as China, Vietnam and Japan. A large research effort has been directed to understanding the virus and controlling the disease. Its geographic distribution, disease cycle via its insect vector, genome organization, relationship with host plants, and epidemiology are summarized in this review and the important role played by the vector, the white‐backed planthopper (Sogatella furcifera), is emphasized. Countermeasures to control the disease that have been developed and applied include molecular detection for precise forecasting, chemical, physical, and ecological pest management. There is widespread insecticide resistance in the vector population but it is hoped that current efforts to develop rice cultivars resistant to the virus will eventually provide effective and cost‐effective control.

Understanding the immune system architecture and transcriptome responses to southern rice black-streaked dwarf virus in Sogatella furcifera.

Sogatella furcifera, the white-backed planthopper (WBPH), has become one of the most destructive pests in rice production owing to its plant sap-sucking behavior and efficient transmission of Southern rice black-streaked dwarf virus (SRBSDV) in a circulative, propagative and persistent manner. The dynamic and complex SRBSDV-WBPH-rice plant interaction is still poorly understood. In this study, based on a homology-based genome-wide analysis, 348 immune-related genes belonging to 28 families were identified in WBPH. A transcriptome analysis of non-viruliferous (NVF) and viruliferous groups with high viral titers (HVT) and median viral titers (MVT) revealed that feeding on SRBSDV-infected rice plants has a significant impact on gene expression, regardless of viral titers in insects. We identified 278 up-regulated and 406 down-regulated genes shared among the NVF, MVT, and HVT groups and detected significant down-regulation of primary metabolism-related genes and oxidoreductase. In viruliferous WBPH with viral titer-specific transcriptome changes, 1,906 and 1,467 genes exhibited strict monotonically increasing and decreasing expression, respectively. The RNAi pathway was the major antiviral response to increasing viral titers among diverse immune responses. These results clarify the responses of immune genes and the transcriptome of WBPH to SRBSDV and improve our knowledge of the functional relationship between pathogen, vector, and host.

The speed of tubule formation of two fijiviruses corresponds with their dissemination efficiency in their insect vectors.

Background Rice black-streaked dwarf virus (RBSDV) and Southern rice black-streaked dwarf virus (SRBSDV) are two closely related fijiviruses transmitted by the small brown planthopper (SBPH) and white-backed planthopper (WBPH), respectively. SRBSDV has a latent period 4 days shorter than that of RBSDV, implying a more efficient spread in insect vector. Currently, the mechanisms underlying this higher efficiency are poorly understood. However, our recent studies have implicated a role of virus induced tubular structures in the dissemination of fijiiruses within their insect vectors.MethodsImmunofluorescence labeling was performed to visualize and compare the dynamics of P7-1 tubule formation of the RBSDV and SRBSDV in their own vector insects and nonhost Spodoptera frugiperda (Sf9) cells.ResultsTubule formation of SRBSDV P7-1 was faster than that of RBSDV P7-1. For RBSDV, P7-1 formed tubules were observed at 3-days post-first access to diseased plants (padp) in SBPH. For SRBSDV, these structures were detected as early as 1 day padp in WBPH. Importantly, similar phenomena were observed when P7-1 proteins from the two viruses were expressed alone in Sf9 cells.ConclusionsOur research revealed a relationship between the speed of P7-1 tubule formation and virus dissemination efficiency and also supports a role of such tubular structures in the spread of reoviruses within insect vectors.

Asymmetric Spread of SRBSDV between Rice and Corn Plants by the Vector Sogatella furcifera (Hemiptera: Delphacidae).

Plant viruses are mostly transmitted by sucking insects via their piercing behaviors, which may differ due to host plant species and their developmental stages. We characterized the transmission of a fijivirus, southern rice black-streaked dwarf virus (SRBSDV), by the planthopper vector Sogatella furcifera Horváth (Hemiptera: Delphacidae), between rice and corn plants of varying developmental stages. SRBSDV was transmitted from infected rice to uninfected corn plants as efficiently as its transmission between rice plants, while was acquired by S. furcifera nymphs at a much lower rate from infected corn plants than from infected rice plants. We also recorded a high mortality of S. furcifera nymphs on corn plants. It is evident that young stages of both the virus donor and recipient plants added to the transmission efficiency of SRBSDV from rice to corn plants. Feeding behaviors of the vector recorded by electrical penetration graph showed that phloem sap ingestion, the behavioral event that is linked with plant virus acquisition, was impaired on corn plants, which accounts for the high mortality of and low virus acquisition by S. furcifera nymphs on corn plants. Our results reveal an asymmetric spread of SRBSDV between its two host plants and the underlying behavioral mechanism, which is of significance for assessing SRBSDV transmission risks and field epidemiology, and for developing integrated management approaches for SRBSDV disease.

Investigation of viruses infecting rice in southern China using a multiplex RT-PCR assay

Rice black streaked dwarf virus (RBSDV), southern rice black streaked dwarf virus (SRBSDV), rice ragged stunt virus (RRSV) and rice gall dwarf virus (RGDV) are common pathogens that affect rice in eastern and southeastern Asia. They are transmitted by the white-backed planthopper, brown planthopper, leafhopper and small brown planthopper, respectively, and require different control strategies. Although rice plants develop characteristic symptoms when infected by each virus, infected plants may also exhibit similar symptoms, which can hinder accurate symptom-based diagnoses. Thus, a one-step multiplex reverse transcription-PCR assay was developed for the simultaneous detection of the four viruses. The amplification products in singly or co-infected plants were easily distinguished during agarose gel electrophoresis. The optimized multiplex reverse transcription-PCR method was used to analyze 582 field samples collected from southern China between 2013 and 2015. RGDV was the most common virus, detected in ∼36.1% of the samples, while RRSV, SRBSDV, and RBSDV were detected in ∼34.0%, ∼30.1%, and ∼0.5% of the samples, respectively. There were also differences in the geographical distributions of the four viruses. RRSV mostly occurred in southern Hainan, RGDV was mainly distributed in southwestern and northeastern Guangdong, SRBSDV was detected across southern China, and RBSDV was detected only in the northern parts of southern China.

Analysis of Sogatella furcifera proteome that interact with P10 protein of Southern rice black-streaked dwarf virus.

Southern rice black-streaked dwarf virus (SRBSDV) is transmitted efficiently only by white-backed planthopper (WBPH, Sogatella furcifera) in a persistent propagative manner. Here we used a yeast two-hybrid system to investigate the interactions between the SRBSDV- P10 and the cDNA library of WBPH. Of 130 proteins identified as putative interactors, 28 were further tested in a retransformation analysis and β-galactosidase assay to confirm the interaction. The full-length gene sequences of 5 candidate proteins: vesicle-associated membrane protein 7 (VAMP7), vesicle transport V-SNARE protein (Vti1A), growth hormone-inducible transmembrane protein (Ghitm), nascent polypeptide-associated complex subunit alpha, and ATP synthase lipid-binding protein) were amplified by 5′ rapid amplification of cDNA ends (RACE) and used in a GST fusion protein pull-down assay. Three of these proteins interacted with SRBSDV-P10 in vitro experiment GST pull-down assay. In a gene expression analysis of 3 different growth stages and 6 different tissue organs of S. furcifera, the mRNA level of VAMP7 was high in adult males and gut. Vti1A was abundant in adult female, and malpighian tubule, gut and ovary. Ghitm was predominantly found in adult male and the malpighian tubule. These research findings are greatly helpful to understand the interaction between SRBSDV and insect vector.

Tolerance and responsive gene expression of Sogatella furcifera under extreme temperature stresses are altered by its vectored plant virus.

Southern rice black-streaked dwarf virus (SRBSDV), a newly emerged fijivirus causing great loss to rice production in eastern and southeastern Asian countries in recent years, is efficiently transmitted by a rice pest, white-backed planthopper (WBPH, Sogatella furcifera) in a persistent, circulative propagative manner and can be considered as an insect virus. In this study, SRBSDV infection in WBPH was found to increase the vector’s death rate under extreme cold stress but improve its survival rate under extreme heat stress. Digital gene expression profiling based on RNA-Seq revealed different gene regulation patterns in WBPH under viral and/or temperature stress. Under cold stress, the virus infection upregulated 1540 genes and downregulated 131 genes in the insect, most of which were related to membrane properties and biological processes of actin and cytoskeleton; whereas under heat stress, it upregulated 363 genes and downregulated 548 genes, most of which were associated to metabolism and intracellular organelles. Several types of stress-responsive genes involving intestinal mucin, cuticle protein, ubiquitin protease, immune response, RNA interference and heat shock response, were largely upregulated under cold stress, but largely downregulated under heat stress, by SRBSDV infection. Our results suggest two distinct mechanisms of virus-altered vector insect tolerance to temperature stress.

EPG Recordings Reveal Differential Feeding Behaviors in Sogatella furcifera in Response to Plant Virus Infection and Transmission Success.

Plant viruses are primarily transmitted by insect vectors and virus infection may influence on the vectors’ feeding behaviors. Using an electrical penetration graph, we detected that infection with the Southern rice black-streaked dwarf virus (SRBSDV) in the white-backed planthopper (WBPH) and in rice plants both altered the vector’s feeding behavior. When viruliferous WBPH (carrying SRBSDV) were fed on uninfected plants, they spent more time in salivation and phloem sap ingestion than non-viruliferous insects. In comparison with uninfected plants, infected plants showed an arrestant effect on non-viruliferous WBPH for phloem sap ingestion. Differential feeding behaviors were also detected between the WBPH that inoculated or acquired SRBSDV and those that failed to. The WBPH that inoculated SRBSDV exhibited more probing bouts, salivation events and phloem sap ingestion events and longer salivation than those that failed to. The WBPH that acquired SRBSDV were quicker to reach phloem and spent more time in phloem sap ingestion than those that failed to. These behavior alterations in the vector may have adaptive advantages for SRBSDV transmission and spread success because greater salivation by viruliferous vectors on uninfected hosts will promote virus inoculation, whereas more sap ingestion by non-viruliferous vectors on infected hosts will promote virus acquisition.

Effects of SRBSDV-infected rice plants on the fitness of vector and non-vector rice planthoppers

Southern rice black-streaked dwarf virus (SRBSDV) is a new member of the genus Fijivirus and transmitted by whitebacked planthopper (WBPH), Sogatella furcifera (Horváth). The rice area affected by SRBSDV and the subsequent damages are increasing rapidly. In current study, we evaluated the effects of SRBSDV-infected plants on the ecological fitness and the tolerance to adverse environmental factors of vector WBPH and non-vector brown planthopper (BPH), Nilaparvata lugens (Stål). Our study revealed that the fitness of WBPH and BPH were both influenced by feeding on SRBSDV-infected rice. Fecundity of WBPH feeding on the SRBSDV-infected rice plants were higher than those on healthy plants, nymphal duration of male WBPH prolonged markedly by 0.77d (P<0.05), while the weight of brachypterous female adults significantly decreased (P<0.05), and the average survival time shortened both at 26°C and 31°C, respectively. Median lethal time of WBPH on SRBSDV-infected and healthy rice plants were 80h and 112h at 26°C, 64h and 88h at 31°C, respectively. As for non-vector BPH, nymph survival rate decreased significantly by 11.22% (P<0.05) and the longevity of female adults also shortened (P<0.05), while hatchability of BPH eggs increased (P<0.05). Survival time of BPH feeding on healthy rice at 26°C was significant longer than those feeding on healthy and infected rice at 31°C. The above results imply that the vector WBPH suffers much stronger effects than non-vector BPH from feeding on SRBSDV-infected rice plants.

Identification and characterization of microRNAs in the white-backed planthopper,Sogatella furcifera

MicroRNAs (miRNAs) are a novel class of small, non-coding endogenous RNAs that play critical regulatory roles in many metabolic activities in eukaryotes. Reports of the identification of miRNAs in Sogatella furcifera (white-backed planthopper), the insect that acts as the only confirmed vector of the southern rice black-streaked dwarf virus (SRBSDV), are limited. In this study, a total of 382 miRNAs were identified in S. furcifera, including 106 conserved and 276 novel miRNAs, using high-throughput sequencing based on two small RNA libraries from viruliferous and non-viruliferous S. furcifera, and these miRNAs belonged to 52 conserved miRNA families and 58 S. furcifera-specific families, respectively. Comparison with miRNAs from 26 insect species and five other species in miRBase showed that more than half of the conserved miRNA families are highly conserved in Hexapoda, while other miRNAs are only conserved in non-dipterans. Furthermore, 4117 target genes predicted for the 382 identified miRNAs could be categorized into 45 functional groups annotated by Gene Ontology. Compared with non-viruliferous cells, eight up-regulated miRNAs and four down-regulated miRNAs were identified in cells inoculated with SRBSDV, among which miR-14 and miR-n98a may be involved in the immune response to SRBSDV infection. Analyses of the identified miRNAs will provide insights into the roles of these miRNAs in the regulation and expression of genes involved in the metabolism, development and viral infection of S. furcifera.

Reverse transcription loop-mediated isothermal amplification to rapidly detect Rice ragged stunt virus.

Rice ragged stunt virus (RRSV) is a very important virus that infects rice and causes serious yield losses in Asian countries and other major rice planting areas. Thus, it is urgent to establish an efficient and practical approach for identification and diagnosis in the field. Our results indicated that reverse transcription loop-mediated isothermal amplification (RT-LAMP) reactions are more efficient and sensitive than RT-PCR for RRSV detection. The optimal LAMP conditions were as follows: 0.4–1.2 μM internal primers, 0.2–0.25 μM external primers, 0.8 μM loop primers, and incubation at 62 °C or 63 °C for 30 min. Furthermore, the RT-LAMP primers specifically targeted RRSV virus and resulted in typical waterfall-like bands by gel electrophoresis and sigmoidal amplification curves. The primers could not be used to amplify other common plant viruses including Papaya ringspot virus (PRSV), Rice yellow stunt virus (RYSV), Sorghum mosaic virus (SrMV), Cactus virus X (CVX), Melon yellow spot virus (MYSV) and Southern rice black-streaked dwarf virus (SRBSDV). Ten-fold serial dilutions of RRSV cDNA indicated that RT-LAMP is much faster and at least ten times more sensitive than RT-PCR in detecting the virus. The waterfall-like product bands could be observed within one hour. In the field study, about 77% samples were identified as RRSV. RT-LAMP has many benefits over RT-PCR such as low cost and high accuracy, sensitivity, and specificity. This technology meets the requirements for rapid diagnosis of plant virus diseases in the field to best guide management practices for growers.

Reference Gene Selection and Evaluation for Gene Expression Studies Using qRT-PCR in the White-Backed Planthopper, Sogatella furcifera (Hemiptera: Delphacidae).

The white-backed planthopper, Sogatella furcifera (Hemiptera, Delphacidae), is one of the most devastating rice pests. For a better control strategy, various genetic studies have been conducted using reverse-transcription quantitative real-time polymerase chain reaction (qRT-PCR). The appropriate application of qRT-PCR requires reliable endogenous controls; however, studies on this aspect of the white-backed planthopper are lacking. In the present study, nine commonly used reference genes, elongation factor 1-α (EF1-α), polyubiquitin (UB), ribosomal protein S18 (RPS18), actin 1 (ACT), α-1 tubulin (TUB), glyceraldehyde-3-phosphate (GAPDH), ribosomal protein L9 (RPL9), ribosomal protein L10 (RPL10), and 18S ribosomal RNA (18S), were evaluated by qRT-PCR for their expression stability under four different experimental conditions (different developmental stages, acquisition of Southern rice black-streaked dwarf virus (SRBSDV), different tissues, and different temperature stress). These results were analyzed using four software programs (geNorm, NormFinder, BestKeeper, and the delta Ct method) and a Web-based comprehensive tool RefFinder to compare and rank candidate reference genes. According to the results of RefFinder analysis, which integrates the abovementioned four software programs, TUB was ranked as the most suitable reference gene at different developmental stages and under different temperature stress, and GAPDH and RPL9 showed the highest stability for acquisition of SRBSDV and different tissues, respectively. These results will provide a solid foundation for future gene expression study on the white-backed planthopper, and also will give aids in establishing a standardized qRT-PCR procedure for other related insects.