Genetic variability of rice stripe virus after its pandemic in Jiangsu.

BACKGROUND: Rice stripe virus (RSV) caused a serious disease pandemic in rice in East China between 2001 and 2010. The continuous integrated managements reduced virus epidemic year by year until it was non-epidemic. As an RNA virus, its genetic variability after undergoing a long-term non-epidemic period was meaningful to study. While in 2019, the sudden occurrence of RSV in Jiangsu provided an opportunity for the study. METHODS AND RESULTS: The complete genome of JY2019, an RSV isolate from Jiangyan, was determined. A genotype profile of 22 isolates from China, Japan and Korea indicated that the isolates from Yunnan formed the subtype II, and other isolates clustered the subtype I. RNA 1-3 of JY2019 isolate well-clustered in the subtype I clade, and RNA 4 was also in subtype I, but it had a slight separation from other intra-group isolates. After phylogenetic analyses, it was considered NSvc4 gene contributed to the tendency, because it exhibited an obvious trend towards the subtype II (Yunnan) group. High sequence identity (100%) of NSvc4 between JY2019 and barnyardgrass isolate from different regions demonstrated genetic variation of NSvc4 was consistent in RSV natural populations in Jiangsu in the non-epidemic period. In the phylogenetic tree of all 74 NSvc4 genes, JY2019 belonged to a minor subtype Ib, suggesting the subtype Ib isolates might have existed in natural populations before the non-epidemic period, but not a dominant population. CONCLUSIONS: Our results suggested that NSvc4 gene was susceptible to selection pressure, and the subtype Ib might be more adaptable for the interaction between RSV and hosts in the non-epidemic ecological conditions.

A simplified RT-PCR assay for the simultaneous detection of tomato chlorosis virus and tomato yellow leaf curl virus in tomato.

Tomato chlorosis virus (ToCV), a species of single-stranded RNA virus belonging to the Crinivirus genus, and Tomato yellow leaf curl virus (TYLCV), a species of single-stranded circular DNA virus belonging to the Begomovirus genus, are two major emerging viruses transmitted by whiteflies and are causing huge losses to tomato production worldwide. To facilitate the simultaneous detection of both viruses in co-infected plants for disease control, a duplex reverse-transcription PCR assay was developed. The assay used three primers, a degenerate reverse primer targeting a conserved region of TYLCV and the RNA2 of ToCV, and two virus-specific forward primers targeting the minor coat protein gene of ToCV and the C3 gene of TYLCV, respectively, to amplify a 762-bp and a 338-bp fragment from ToCV and TYLCV, respectively, in a single reaction. The concentration of the primers, annealing temperature and amplification cycles used in the assay were optimized, and the sensitivity of the assay was assessed. Using this assay, 150 tomato leaf samples collected from the field during 2018 were tested. The results showed that both viruses could be detected simultaneously in co-infected field samples. The assay should benefit the rapid detection of these two viruses in tomato crops and would facilitate early warning of infections for the control of the two virus diseases.

Development of a reverse-transcription loop-mediated isothermal amplification assay for the detection of Tobacco mild green mosaic virus (TMGMV).

Tobacco mild green mosaic virus (TMGMV), a member species of the genus Tobamovirus, infects pepper (Capsicum annuum) and a number of other economically important species in the Solanaceae family. TMGMV infections had seriously impacted pepper production worldwide, including China. A reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed to detect TMGMV in pepper field samples and seed. This assay was based on four primers that matched to six sequences in the C-terminal region of the TMGMV genome. RT-LAMP assay could detect the presence of the virus in 3.0 × 10-7 µg of total RNA extract from pepper leaves, which was ten times more sensitive than the corresponding reverse-transcription polymerase chain reaction (RT-PCR) assay. This method specifically detected TMGMV but not the closely related species of the same genus Pepper mild mottle virus, Cucumber green mottle mosaic virus and Tomato mosaic virus. In addition, the use of SYBR Green I facilitated the detection of the TMGMV RT-LAMP products by the naked eye. These results indicated that the RT-LAMP assay was a simple, sensitive, specific and affordable diagnostic tool that has the potential to detect and monitor TMGMV infection in field samples.

qMrdd2, a novel quantitative resistance locus for maize rough dwarf disease.

BACKGROUND: Maize rough dwarf disease (MRDD), a widespread disease caused by four pathogenic viruses, severely reduces maize yield and grain quality. Resistance against MRDD is a complex trait that controlled by many quantitative trait loci (QTL) and easily influenced by environmental conditions. So far, many studies have reported numbers of resistant QTL, however, only one QTL have been cloned, so it is especially important to map and clone more genes that confer resistance to MRDD. RESULTS: In the study, a major quantitative trait locus (QTL) qMrdd2, which confers resistance to MRDD, was identified and fine mapped. qMrdd2, located on chromosome 2, was consistently identified in a 15-Mb interval between the simple sequence repeat (SSR) markers D184 and D1600 by using a recombinant inbred line (RIL) population derived from a cross between resistant ("80007") and susceptible ("80044") inbred lines. Using a recombinant-derived progeny test strategy, qMrdd2 was delineated to an interval of 577 kb flanked by markers N31 and N42. We further demonstrated that qMrdd2 is an incompletely dominant resistance locus for MRDD that reduced the disease severity index by 20.4%. CONCLUSIONS: A major resistance QTL (qMrdd2) have been identified and successfully refined into 577 kb region. This locus will be valuable for improving maize variety resistance to MRDD via marker-assisted selection (MAS).

Single amino acid in V2 encoded by TYLCV is responsible for its self-interaction, aggregates and pathogenicity.

The V2 protein encoded by Begomovirus is essential for virus infection and is involved in multiple functions, such as virus movement and suppression of the host defence response. In this study, we reported that V2 encoded by the Tomato yellow leaf curl virus (TYLCV), which is one of the most devastating tomato-infecting begomoviruses, could interact with itself and a S71A mutation of V2 (V2S71A) abolished its self-interaction. Fluorescence results showed that V2 localized primarily in the cytoplasm and around the nucleus. Site-directed mutagenesis V2S71A had the similar subcellular localization, but V2S71A formed fewer large aggregates in the cytoplasm compared to wild-type V2, whereas the level of aggregates came to a similar after treatment with MG132, which indicates that the S71A mutation might affect 26S proteasome-mediated degradation of V2 aggregates. Meanwhile, heterologous expression of V2S71A from a Potato virus X vector induced mild symptoms compared to wild-type V2, delay of virus infection associated with mild symptoms was observed in plants inoculated with TYLCV-S71A, which indicates that the amino acid on position 71 is also involved in the pathogenicity of V2. To the best of our knowledge, this report is the first to state that the S71A mutation of V2 encoded by TYLCV affects the self-interaction, aggregate formation and pathogenicity of V2.

Viruliferous rate of small brown planthopper is a good indicator of rice stripe disease epidemics.

Rice stripe virus (RSV), its vector insect (small brown planthopper, SBPH) and climatic conditions in Jiangsu, China were monitored between 2002 and 2012 to determine key biotic and abiotic factors driving epidemics of the disease. Average disease severity, disease incidence and viruliferous rate of SBPH peaked in 2004 and then gradually decreased. Disease severity of RSV was positively correlated with viruliferous rate of the vector but not with the population density of the insect, suggesting that the proportion of vectors infected by the virus rather than the absolute number of vectors plays an important role in RSV epidemics and could be used for disease forecasting. The finding of a positive correlation of disease severity and viruliferous rate among years suggests that local infection is likely the main source of primary inoculum of RSV. Of the two main climatic factors, temperature plays a more important role than rainfall in RSV epidemics.

Complete genome analysis of a novel recombinant isolate of pepper veinal mottle virus from mainland China.

BACKGROUND: Pepper veinal mottle virus (PVMV) was well established in Africa, and also reported infecting pepper (Capsicum annuum L) in Taiwan and India. However, there is not available of PVMV in mainland China. Here, the first complete genome sequence of PVMV isolated from pepper in mainland China was reported. FINDING: The complete genomic sequence of isolate PVMV-HN isolated from pepper in mainland China is reported in this study. The genome of PVMV-HN is 9793 nucleotides (nt) excluding the poly (A) tail, shares 98-99 % nucleotide sequence identity with those two PVMV isolates from Ghana and Taiwan. Recombinant analysis showed that PVMV-HN probably represents a novel recombinant of PVMV. The phylogenetic relationship of PVMV-HN isolate to other PVMV isolates and other potyviruses based on genome or polyprotein sequence level and CP gene level, was also analyzed in this study. CONCLUSION: The current study will help to understand phylogenetic relationship of isolate PVMV-HN.

Rice stripe virus affects the viability of its vector offspring by changing developmental gene expression in embryos.

Plant viruses may affect the viability and development process of their herbivore vectors. Small brown planthopper (SBPH) is main vector of Rice stripe virus (RSV), which causes serious rice stripe disease. Here, we reported the effects of RSV on SBPH offspring by crossing experiments between viruliferous and non-viruliferous strains. The life parameters of offspring from different cross combinations were compared. The hatchability of F1 progeny from viruliferous parents decreased significantly, and viruliferous rate was completely controlled by viruliferous maternal parent. To better elucidate the underlying biological mechanisms, the morphology of eggs, viral propagation and distribution in the eggs and expression profile of embryonic development genes were investigated. The results indicated that RSV replicated and accumulated in SBPH eggs resulting in developmental stunt or delay of partial eggs; in addition, RSV was only able to infect ovum but not sperm. According to the expression profile, expression of 13 developmental genes was regulated in the eggs from viruliferous parents, in which two important regulatory genes (Ls-Dorsal and Ls-CPO) were most significantly down-regulated. In general, RSV exerts an adverse effect on SBPH, which is unfavourable for the expansion of viruliferous populations. The viewpoint is also supported by systematic monitoring of SBPH viruliferous rate.

A novel, in vivo, indoor method to preserve rice black-streaked dwarf virus in small brown planthopper using wheat seedling as a bridge host.

Rice black-streaked dwarf virus (RBSDV) naturally infects Gramineae plants through small brown planthopper (SBPH) as a vector. However, RBSDV cannot be transmitted to the SBPH offspring through transovarian transmission. Wheat plant, an important intermediate host in winter, is essential for the completion of the annual cycle of RBSDV in farm ecosystem. We developed a novel, in vivo, indoor method to preserve RBSDV in SBPH using wheat seedling as a bridge host. The temperature range of 23-27°C was initially selected to rear the insects and plants. Before initiating the scheme cycle, viruliferous SBPH was obtained by feeding the virus-free 1st to 2nd instar nymphs with RBSDV-infected rice plants. Four to six RBSDV-infected SBPH were placed per plant to inoculate wheat seedlings at two-to-four leaf stages. After 48 h of inoculation, the viruliferous SBPH were removed. Five mated, newly emerged virus-free SBPH females were then transferred onto each inoculated plant and allowed to lay eggs for 48 h. The newly hatched SBPH were raised on wheat seedlings until the 2nd instar nymph stage, and then transferred onto healthy rice seedlings for further development until 5th instar nymphs or adults. These newly obtained viruliferous SBPH can be used for inoculating new wheat seedlings in the succeeding maintenance cycles, or for further experiments. We discovered that the incubation period of RBSDV in wheat seedlings synchronized with the gestation period of SBPH eggs at four to six inoculated viruliferous SBPH per plant and lasted for approximately seven days. In addition, this period was optimal for enhancing the SBPH infection ratio because SBPH nymphs can only acquire the virus after they hatch. The RBSDV infection ratio of the SBPHs acquired through this method consistently exceeded 50%.

Distribution and genetic diversity of Southern rice black-streaked dwarf virus in China.

BACKGROUND: Rice and maize dwarf diseases caused by the newly introduced Southern rice black-streaked dwarf virus (SRBSDV) have led to severe economic losses in South China in recent years. The distribution and diversity of SRBSDV have not been investigated in the main rice and maize growing areas in China. In this study, the distribution of SRBSDV in China was determined by using reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: Between 2009 and 2010, 2404 plant samples (2294 rice, 110 maize samples, and more than 300 cultivars) with dwarf symptoms were collected from fields in 194 counties of 17 provinces in China and SRBSDV was detected. The results indicated that 1545 (64.27%) of samples (both rice and maize) were infected with SRBSDV. SRBSDV was detected widely in Hainan, Guangdong, Guangxi, Yunnan, Guizhou, Chongqing, Fujian, Jiangxi, Hunan, Hubei, Anhui, Jiangsu, and Zhejiang provinces, which suggests SRBSDV is an important pathogen causing rice dwarfing diseases in South China. Phylogenetic analysis of 15 representative virus isolates revealed that SRBSDV isolates in China had high levels of nucleotide and amino acid sequence identities (>97.8%). CONCLUSIONS: SRBSDV spreads naturally in Yangtze River basin and south region, the location of the major rice production areas. In comparison, the virus rarely spreads north of Yangtze River in North China. Distribution of SRBSDV is consistent with the migrating and existing ranges of its vector WBPH, suggesting that SRBSDV might be introduced into South China along with the migration of viruliferous WBPH.

Viroplasm protein P9-1 of Rice black-streaked dwarf virus preferentially binds to single-stranded RNA in its octamer form, and the central interior structure formed by this octamer constitutes the major RNA binding site.

The P9-1 protein of Rice black-streaked dwarf virus (RBSDV) is an essential part of the viroplasm. However, little is known about its nature or biological function in the viroplasm. In this study, the structure and function of P9-1 were analyzed for in vitro binding to nucleic acids. We found that the P9-1 protein preferentially bound to single-stranded versus double-stranded nucleic acids; however, the protein displayed no preference for RBSDV versus non-RBSDV single-stranded ssRNA (ssRNA). A gel mobility shift assay revealed that the RNA gradually shifted as increasing amounts of P9-1 were added, suggesting that multiple subunits of P9-1 bind to ssRNA. By using discontinuous blue native gel and chromatography analysis, we found that the P9-1 protein was capable of forming dimers, tetramers, and octamers. Strikingly, we demonstrated that P9-1 preferentially bound to ssRNA in the octamer, rather than the dimer, form. Deletion of the C-terminal arm resulted in P9-1 no longer forming octamers; consequently, the deletion mutant protein bound to ssRNA with significantly lower affinity and with fewer copies bound per ssRNA. Alanine substitution analysis revealed that electropositive amino acids among residues 25 to 44 are important for RNA binding and map to the central interior structure that was formed only by P9-1 octamers. Collectively, our findings provide novel insights into the structure and function of RBSDV viroplasm protein P9-1 binding to RNA.

One-step reverse transcription loop-mediated isothermal amplification for the rapid detection of cucumber green mottle mosaic virus.

Cucumber green mottle mosaic virus (CGMMV) has caused serious damage to Cucurbitaceae crops worldwide. The virus is considered one of the most serious Cucurbitaceae quarantine causes in many countries. In this study, a highly efficient and practical one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) was developed for the detection of CGMMV. The total RNA or crude RNA extracted from watermelon plants or seeds could be detected easily by this RT-LAMP assay. The RT-LAMP assay was conducted in isothermal (63°C) conditions within 1h. The amplified products of CGMMV could be detected as ladder-like bands using agarose gel electrophoresis or visualized in-tube under UV light with the addition of a fluorescent dye. The RT-LAMP amplification was specific to CGMMV, as no cross-reaction was observed with other viruses. The RT-LAMP assay was 100-fold more sensitive than that of reverse-transcription polymerase chain reaction (RT-PCR). This is the first report of the application of the RT-LAMP assay to detect CGMMV. The sensitive, specific and rapid RT-LAMP assay developed in this study can be applied widely in laboratories, the field and quarantine surveillance of CGMMV.

Mapping and validation of quantitative trait loci associated with wheat yellow mosaic bymovirus resistance in bread wheat.

Wheat yellow mosaic (WYM) caused by wheat yellow mosaic bymovirus (WYMV) has been growing as one of the most serious diseases affecting wheat production in China. In this study, the association of quantitative trait loci (QTLs) governing WYMV resistance with molecular markers was established using 164 recombinant inbred lines (RILs) derived from 'Xifeng Wheat' (highly resistant) × 'Zhen 9523' (highly susceptible). Phenotypic data of WYMV resistance of the RILs were collected from 4-year, two-location replicated field trials. A molecular marker-based linkage map, which was comprised of 273 non-redundant loci and represented all the 21 wheat chromosomes, was constructed with the JoinMap 4.0 software. Using the Windows QTL Cartographer V2.5 software, three QTLs associated with WYMV resistance, QYm.njau-3B.1, QYm.njau-5A.1 and QYm.njau-7B.1, were detected on chromosomes 3BS, 5AL, and 7BS, respectively. The favorable allele effects were all contributed by 'Xifeng Wheat'. Among the three QTLs, QYm.njau-3B.1 and QYm.njau-5A.1 were detected in all the four trials and the overall mean, and could explain 3.3-10.2% and 25.9-53.7% of the phenotypic variation, respectively, while QYm.njau-7B.1 was detected in one trial and the overall mean and explained 4.9 and 3.3% of the phenotypic variation, respectively. A large portion of the variability for WYMV response was explained by a major QTL, QYm.njau-5A.1. The relationship of the molecular markers linked with QYm.njau-5A.1 and the WYMV resistance was further validated using a secondary F(2) population. The results showed that three markers, i.e., Xwmc415.1, CINAU152, and CINAU153, were closely linked to QYm.njau-5A.1 with the genetic distances of 0.0, 0.0, and 0.1 cM, respectively, indicating they should be useful in marker-assisted selection (MAS) wheat breeding for WYMV resistance. A panel of germplasm collection consisting of 46 wheat varieties with known WYMV response phenotypes was further used to validate the presence and effects of QYm.njau-5A.1 and the above three markers. It was found that QYm.njau-5A.1 was present in 12 of the 34 WYMV-resistant varieties.

[QTL analysis for rice stripe disease resistance gene using recombinant inbred lines (RILs) derived from crossing of Kinmaze and DV85].

Rice stripe disease transmitted by small brown planthopper (Laodelphax striatellus Fall.) is one of the most serious viral diseases in East Asia. The disease is severely epidemic in most rice growing areas where the main cultivars are susceptible or moderately susceptible to rice stripe virus. In this research, a recombinant inbred lines (RILs) population of 81 lines derived from a cross of Kinmaze (japonica)/DV85(indica) by the single seed descent method was used to detect quantitative trait loci (QTL) conferring resistance to rice stripe virus(RSV). The response of the two parents and 81 RILs to RSV were investigated by inoculating seedlings with viruliferous small brown planthopper insects, and scored by the disease rate index. The quantitative trait loci for rice stripe disease resistance were analyzed by QTL Cartographer software. Three QTL controlling RSV resistance were detected on chromosomes 1, 7 and 11, respectively. Individual QTL accounted for 19.8%-30.9% of the phenotypic variance in the RILs population. The direction of the additive gene effects at two loci qStv7 and qStv11 coincided with that predicted by phenotypes of the parents. At these two loci, the DV85 alleles increased the resistance to RSV, while at qStv1, the Kinmaze alleles increased the resistance to RSV.