| Capsicum chinense Jacq., also known as Yellow Lantern chilli pepper, or Yellow pepper, is a special type of Hainan chilli pepper, among the most spicy peppers in the world. Its sauce has become a product as popular as coconut and coconut products for tourists visiting Hainan. With the Capsicum chinense acreage expanding rapidly, many problems, especially viral disease, have become a limiting factor in its production. Capsicum chinense is more easily infected virus, producing more severe symptoms than other pepper varieties, often leading to heavy losses or no harvest. The viral disease has become the most devastating factor that affect the Capsicum chinense farming. In comparison with the rapid increase in the Capsicum chinense acreage, viral disease control and related research such as identification, diagnosis, and resistance etc have lagged behind, greatly limiting the expansion of Capsicum chinense industry in Hainan. This study focuses on the viral diseases of Capsicum chinense in Hainan, and reports for the first time a comprehensive description of the basic characteristics of the viral diseases.Four types of viral symptoms were observed in field surveys of Capsicum chinense plants:mottle/malformation, tip necrosis, yellowing, and dwarfing, with the first symptom type most common, accounting for62.5%. The mottle/malformation symptoms include mild mottle, veinal banding mottle, and leaf distortion while dwarfing includes plant dwarf and etch symptoms. All the symptoms were more apparent in the hot season than in the cool season. The Disease Index of shaded and mulching film-covered plants was significantly higher than that of plants with no shading and no film cover. There was no significant difference between plants planted in barrels and those planted directly in fields. Barrel cultivation did not provide controls for the viral diseases.Three different viruses were identified from plants with three different symptoms: veinal banding mottle, mosaic-malformation and dwarf-etch. The identification was accomplished by direct observation of virus particles, Mass Spectrum analysis of viral capsid proteins, and sequencing analysis of viral genomes. Chilli ring spot virus (ChiRSV) was identified in China for the first time. Its particle was a flexuous rod of12～13nm×750nm, and the Pinwheel-Like inclusions were found in the infected mesophyll cells by electron microscopy. ChiRSV capsid protein was purified by the SDS-PAGE and subsequently identified by mass spectrometry. Its spectra matched those of polypeptide of ChiRSV, and the closest viral protein ID was ABL09414.1, an isolate of ChiRSV-VN/C9from Vietnam. The identification score of572was71points that the significant threshold, and the coverage is35.38%. Viral RNA was extracted from the purified virions, and a611bp fragment was amplified by RT-PCR. Blast results showed that the sequence of this fragment shared92%and90%homology to the Vietnam isolates of ChiRSV-VN/C9, and VN/C8(DQ925439and DQ925438), respectively. The complete genome of virus was then sequenced. When the genomic sequence (GenBank Accession No. JN008909) was aligned with Potyvirus sequences, it is closely related to Tobacco vein banding virus (TVbMV). However, the two viruses shared only64.8%homology at the nucleotide level and79.7%homology at the protein level, below the species threshold value of85%and80%. Based on these data, we identified the virus as ChiRSV in the Potyvirus genus. This is the first report of ChiRSV in China. Using the same methods, we also isolated Chilli veinal mottle virus (ChiVMV, Potyvirus) and Cucumber mosaic virus (CMV, Cucumovirus) from the mottle/malformed and dwarf-etched Capsicum chinense plants, respectively.Methods for ChiRSV detection were developed. First, we immunized rabbits with purified virus and obtained ChiRSV polyclonal antiserum with a titer of1:25000in indirect ELISA (ID-ELISA). Then, RT-PCR for molecular detection of ChiRSV was established, which was capable of detecting12.5pg of viral RNA, equivalent to250ng of diseased tissues.Results of random testing by virus detection methods were established, showed that we found the following viruses on Capsicum chinense:ChiRSV, CMV, ChiVMV, and TMV. ChiRSV, CMV and ChiVMV were detected in62%,59%,27%samples, respectively.48.6%of samples were co-infection by these virues. CMV should played an important role in the co-infection by inference. CMV and TMV co-infection led to the tip necrosis and death. CMV and Potyvirus (ChiRSV or ChiVMV) co-infection led to serious mottle/malformation. CMV alone caused dwarfing and etching; Infection of ChiRSV and ChiVMV alone or together resulted in mottle-mosaic. Seeds of Capsicum chinense carried CMV and TMV, but not ChiVMV and ChiRSV.An exploratory study of bacterium-mediated dsRNA to control viral diseases was performed on Capsicum chinense. We constructed dsRNA vectors to express in bacteria dsRNA corresponding to ChiRSV HC-Pro, CP, PIPO fragments. Preliminary results showed promises of this new biological control strategy based on RNAi.On disease prevention and control, Mulching has become one of the commonly used Capsicum cultivation techniques among Hainan farmers as a result of our studies suggestions. The growers have abandoned the barrels planted mode to reduce production costs. |
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