Background
Rice black-streaked dwarf virus (RBSDV), a member of the genus
Fijivirus in the family
Reoviridae, is mainly transmitted by the small brown planthopper (
Laodelphax striatellus) in a persistent and propagative manner, but not transmitted via eggs [
1‐
3]. RBSDV naturally infects graminaceous plant species including rice, maize, wheat, barley, and several species of weeds, resulting in rice black-streaked dwarf disease, maize rough dwarf disease and wheat dark-green dwarf disease, respectively, [
1,
3‐
5]. The typical symptoms of diseased rice plants include stunting, darkening of leaves and white tumors or black-streaked swellings on stem and abaxial surfaces of leaves, leaf blades and sheaths [
6]. The diseased maize plants present stunted, dark green color, white tumors on stem and along the veins on abaxial surface of leaves and leaf sheaths, suppressed flowers and no ears or just nubbins [
7]. RBSDV occurs in China, Japan, and other Asian countries and causes severe yield losses in rice, maize, wheat and barley production [
3,
4,
8]. The outbreaks of RBSDV in Japan were recorded in maize during 1957–1961, and in rice and maize during 1965–1967 [
1,
3]. In China, RBSDV was reported in Zhejiang Province in 1963, and since the early 1990s, the virus caused severe damage in rice in most regions of Zhejiang Province and northern Fujian Province of China [
4,
8‐
10]. In recent years, outbreaks of the virus occurred on rice in Jiangsu Province and in maize in some maize-growing areas, causing severe losses in rice and maize production in China. The outbreak of RBSDV generally coincides with a high density of its vector, a high percentage of viruliferous planthoppers in overwintering populations during the most susceptible stage of young crops, and changes in cultivation practices [
3,
11]. Virions are localized in the phloem and gall tissues in infected plants, viroplasms, virus crystals and tubular structures in both infected plants and planthopper vector cells [
3,
12,
13].
RBSDV virions are non-enveloped, icosahedral, double-shelled particles with 75 to 80 nm in diameter and short surface spikes and contain 10 segments ranging from 1.8 to 4.5 kb of linear double-stranded genomic RNA (designated S1–S10) [
9,
14]. Most genomic segments only contain one open reading frame (ORF), while S5, S7 and S9 each contain two ORFs [
13,
15,
16]. The core particle of RBSDV consists of four proteins: P1 (RNA-dependent RNA polymerase) encoded by S1, P2 (major core capsid protein) encoded by S2, P3 (putative guanylyltransferase) encoded by S3, and P8 (a minor core capsid protein) encoded by S8 [
13,
17,
18]. The outer layer of the RBSDV particle consists of P4 (outer-shell B-spike protein) encoded by S4 and P10 (outer capsid or coat protein, CP) encoded by S10 [
13,
19]. The nonstructural protein P6 encoded by S6 functions as a viral RNA silencing suppressor [
20]. Both S7 and S9 each encode a nonstructural protein, i.e. P7-1 and P9-1. P7-1 and P9-1 are components of the tubular structures and viroplasms in infected plants and planthopper cells, respectively [
13,
21].
Currently, some approaches have been used for detection of RBSDV: reverse transcription (RT)-polymerase chain reaction assay (PCR) [
22], RT-loop-mediated isothermal amplification assay (RT-LAMP) [
22,
23], polyclonal antibodies (PAbs)-based indirect enzyme-linked immunosorbent assay (ID-ELISA) [
24], and PAbs-based double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) [
25]. Among those methods, serological methods are more suitable for routine detection of high throughput samples in the field survey. But, the results of serological methods are dependent on the quality and availability of antibodies. In this study, three highly sensitive and specific murine monoclonal antibodies (MAbs) against RBSDV antigens were produced using the hybridoma technology, and two MAb-based serological methods, antigen-coated-plate enzyme-linked immunosorbent assay (ACP-ELISA) and dot enzyme-linked immunosorbent assay (dot-ELISA) were developed for sensitive and specific detection of RBSDV in field samples. The detection results of field samples by the established two serological methods demonstrated that RBSDV is widespread in rice, maize and wheat crops in Jiangsu, Zhejiang and Shandong provinces of China.
Discussion
RBSDV causes severe yield losses in rice and maize production in many Asian countries [
3,
4,
8]. After the rice and maize crops are harvested in RBSDV prevalent regions, viruliferous planthopper vector moves first to weeds and then to barley and wheat, where it transmits the virus and oviposits [
3]. The following generation acquires the virus on infected plants, and moves to newly planted rice or maize next year to transmit the virus [
3]. Hence, it is an effective preventive measure against RBSDV disease to alter the planting time to avoid the viruliferous vectors, while rapid and effective detection of RBSDV in overwintering plants and planthopper populations would permit to select more appropriate planting time. Moreover, rapid detection of RBSDV in field planthopper populations can help to time the spraying of insecticides to efficiently control the viral vectors. So, it is urgent to develop rapid serological methods for RBSDV routine detection in field plant and insect vector.
Virions of fijiviruses are labile and readily break down during the purification processes. In general, most purified RBSDV particles lack the outer capsid and do not to elicit antibodies that could recognize the intact virions when injected into rabbits [
26]. Due to the instability of RBSDV virions and their phloem-restriction in host plants, it is very difficult to obtain intact virus particles in sufficient quantity for the preparation of high quality antibodies for the virus detection. The major outer capsid of RBSDV expressed in
Escherichia coli was used to prepare antiserum, and an ID-ELISA was developed for detecting RBSDV in wheat samples with the antiserum, But, no serological method was developed for RBSDV detection in insect vector [
24]. Furthermore, based on the high similarity of outer capsids of Southern rice black-streaked dwarf virus (SRBSDV) and RBSDV, it can be assumed that the antiserum do not distinguish RBSDV from SRBSDV. In Takahashi’s work, serological methods including DAS-ELISA were established and could successfully detect RBSDV in rice samples, but failed to detect RBSDV in vector samples because of nonspecific reaction of the antiserum [
25].
In the present study, we used the crude extract from white tumors containing high titre virions and viral non-structure proteins of RBSDV-infected maize plants as the immunogen, which allowed to obtain intact virions and viral non-structure proteins suitable for eliciting antibodies. Three RBSDV-specific MAbs (12E10, 18F10 and 5G5) were then developed. Three MAbs reacted with the crude extracts from the RBSDV-infected plant tissues and viruliferous planthopper, but not with RDV-, SRBSDV-, RRSV- or RSV-infected rice plants, healthy plant tissues or non-viruliferous vectors.
SRBSDV that is a new species in the genus
Fijivirus in the family
Reoviridae, is transmitted to rice and maize by the white backed planthopper (
Sogatella furcifera) in a persistent manner [
27,
28]. Recently, SRBSDV became one of the most important viruses of rice and maize in Southeast Asian countries [
22,
27‐
29]. Both RBSDV and SRBSDV share many similarities in genomic structure and sequence [
27‐
29], virion morphology, serology [
27], symptoms and host ranges [
22,
28]. Hence, in the process of this research, most of the MAbs screened by us can simultaneously react with RBSDV and SRBSDV (data not shown). With many time screenings and selections, we obtained MAbs only reacting with RBSDV but no with SRBSDV.
In order to forecast and control the disease, we have developed two reliable, rapid and efficient serological methods for specific and sensitive detection of RBSDV. Because RBSDV infection is an important problem in cereal crop production in Southeast Asian countries, the serological detection methods developed in this work will support further investigations on the epidemiology of RBSDV and detect the virus more efficiently and economically than the previously available assays.
It is well known that the specificity of MAb is better than PAbs. The dilution endpoints of the DAS-ELISA for RBSDV detection in rice reported by Takahashi et al. [
24] and the ID-ELISA for RBSDV detection in wheat reported by Wang et al. [
25] both are 1: 1,280 (w/v, g mL
-1). In this study, the ACP-ELISA could detect RBSDV in infected plant tissue extracts at the dilution of above 1: 10,240 (w/v, g mL
-1) and in individual viruliferous vector extracts at the dilution of 1:19200 (individual planthopper/μL), while dot-ELISA could detect RBSDV in infected plant tissue extracts at the dilution of 1: 320 (w/v, g mL
-1) and in individual viruliferous vector extracts at dilution of 1:1,600 (individual planthopper/μL) (Figures
3 and
4). To our knowledge, no serological methods were reported to detect RBSDV in plants and vectors with such lower detection limits.
RT-PCR and serological methods are frequently applied to detect plant RNA viruses. It is well known that the RT-PCR for RNA virus detection is more sensitive than serological methods and our results proved that again. However, the RT-PCR method is complicated, time-consuming, expensive, not suitable for large-scale samples. So serological methods are more often used in the routing detection of plant viruses.
Field plant and small brown rice planthopper samples were screened for the presence of RBSDV with the newly developed serological methods and the detection results showed that RBSDV is widespread in Jiangsu, Zhejiang and Shandong provinces of China (Table
2). We did not detect RBSDV in field samples collected in Hainan and Yunnan provinces of China, but we could not determine whether RBSDV occur in those two provinces and other non-detected provinces of China. Further tests are necessary to confirm the absence of the pathogen in these provinces.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
JW, YN and HL did most experiments and drafted the manuscript. LR did the RT-PCR for RBSDV detection. XZ and YZ conceived of the study, and participated in its design and coordination. XZ, YZ and JW proof-read and finalized the manuscript. All authors read and approved the final manuscript.