Abstract
BackgroundRice black-streaked dwarf virus (RBSDV) causes great losses in rice, maize and wheat production in Asian countries. The use of serological methods for RBSDV detection depends on the availability of antibodies. In this study, three highly sensitive and specific murine monoclonal antibodies (MAbs) against RBSDV antigens were produced using crude extracts from tumors of RBSDV-infected maize as the immunogen, and two serological assays, antigen-coated-plate enzyme-linked immunosorbent assay (ACP-ELISA) and dot enzyme-linked immunosorbent assay (dot-ELISA) were developed for RBSDV detection.ResultsAll three MAbs reacted strongly and specifically with the crude extracts from RBSDV-infected plant and planthopper tissues. The detection endpoints of three MAbs (12E10, 18F10 and 5G5) in ACP-ELISA were respectively 1:40,960, 1:40,960, 1:81,920 (w/v, g mL-1) with the crude extract of infected maize, 1:10,240, 1:20,480, 1:20,480 (w/v, g mL-1) with the crude extract of infected rice, 1:5,120, 1:10,240, 1:10,240 (w/v, g mL-1) with the crude extract of infected wheat, 1:9,600, 1:9,600, 19,200 (individual planthopper/μL) with the crude extract of infected planthopper. The newly developed ACP-ELISA could detect the virus in the infected maize, wheat, rice tissue crude extracts diluted at 1:81,920, 1:20,480, 1:10,240 (w/v, g mL-1), respectively, and in individual viruliferous planthopper extract diluted at 1:19200 (individual planthopper/μL). The dot-ELISA was proved to detect the virus in the infected maize, wheat and rice tissue crude extracts diluted at 1:320 (w/v, g mL-1), and in individual viruliferous planthopper extract diluted at 1:1,600 (individual planthopper/μL), respectively. Field plants (915) and planthopper samples (594) from five provinces of China were screened for the presence of RBSDV using the two developed serological assays. The results indicated that 338 of the 915 plant samples and 19 of the 594 planthopper samples were infected by RBSDV.ConclusionsThe newly developed ACP-ELISA and dot-ELISA were highly sensitive and specific to detect RBSDV in field plant and planthopper samples. The field survey demonstrated that RBSDV is widespread in rice, maize and wheat crops in Jiangsu, Zhejiang, Shandong provinces of China.
Highlights
Rice black-streaked dwarf virus (RBSDV) causes great losses in rice, maize and wheat production in Asian countries
Preparation and characterization of monoclonal antibodies (MAbs) against RBSDV The crude extract containing RBSDV virions and its nonstructure proteins from white tumors of RBSDV-infected maize were used as the immunogen
The results revealed that the MAbs (12E10 and 5G5) reacted with a protein of approximately 40 kDa in both crude extracts from RBSDV-infected plant and insect vector tissues, while the MAb 18F10 reacted with a protein of approximately 56 kDa
Summary
Rice black-streaked dwarf virus (RBSDV) causes great losses in rice, maize and wheat production in Asian countries. 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, [1,3,4,5]. 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]. 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]
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