Characterization and a RT-RPA assay for rapid detection of Chilli Veinal mottle virus (ChiVMV) in tobacco

Abstract

Background: Chilli veinal mottle virus (ChiVMV), which belongs to the genus Potyvirus of the family Potyviridae, mainly infects solanaceous plants and has caused serious economic losses in Asia and Africa. Tobacco plants infected with ChiVMV suffered from punctate necrosis of leaves, leaf deformation, systemic necrosis of leaves and stems, and eventually plant death. However, ChiVMV infection could not usually be identified given the lack of rapid and efficient detection assays in tobacco plants. Therefore, an isolate of tobacco-infecting ChiVMV (ChiVMV-LZ) was obtained, and a novel isothermal amplification and detection technique, reverse transcription-recombinase polymerase amplification (RT-RPA), was established to detect ChiVMV in tobacco plants. Methods: In this study, the full-length genome of ChiVMV-LZ was obtained using reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) assays. The genome sequence of ChiVMV-LZ was characterized by sequence alignment and phylogenetic analysis. Then, a RT-RPA assay was established for rapid and sensitive detection of ChiVMV-LZ in tobacco. Additionally, the established RT-RPA assay was compared to the RT-PCR assay in aspect of sensitivity and application in field-collected tobacco samples. Results: ChiVMV-LZ was isolated from diseased tobacco in Luzhou, Sichuan, China. The tobacco plants inoculated with ChiVMV-LZ showed typical symptoms of yellow and round spots on the leaves, and curled and folded leaf margin, similar to those observed on naturally ChiVMV-infected tobacco in the field. The full-length genomic sequence of ChiVMV-LZ was determined to be 9742 nucleotides. Sequence alignment and phylogenetic analysis showed that ChiVMV-LZ was most closely related to ChiVMV-Yp8 isolated from pepper plants in Sichuan province while distantly related to ChiVMV-YN from tobacco in Yunnan province, indicating a possibly geographical differentiation of ChiVMV isolates. Additionally, a RT-RPA assay was established for rapid detection of ChiVMV in tobacco. The RT-RPA has no cross-reaction with other related tobacco viruses and is about 10-fold more sensitive than conventional RT-PCR method. Conclusion: The characterization of ChiVMV-LZ infecting tobacco was determined, and the established RT-RPA assay provides a reliable and effective method for rapid detection of ChiVMV in tobacco.

Role of Plant Extracts and Salicylic Acid for the Management of Chili Veinal Mottle Virus Disease

“…First time in history, the ChiVMV reported from Malaysia (Ong et al 1979) and then it spread to Asia and Africa (Lee et al 2013). The yield losses of more than 50 percent have been recorded in Pakistan due to this disease virus (Hameed et al 1995).It is a ssRNA virus belongs to the family Potyviridae and the genus Potyvirus (Lee et al 2017;Fei et al 2019;Jiao et al 2020;Adediji et al 2020). This virus is transmitted from one plant to another through a vector aphid(Aphis gossypii) in a non-persistent manner, through grafting, mechanically and did not transfer through seed (Shah et al 2008).The infected chilli plant could not survive happily and have shown the symptoms of vein mottling, stunting in an early stage of infection, dark-green streaks on branches and on the stem, flowers dropping and fruit deforming is the characteristic symptoms of ChiVMV (Wang et al 2006;Nono-Womdim et al 2001;Ahmad and Ashfaq 2018;Gao et al 2016).…”

Identification of viruses infecting cigar and flue-cured tobacco using next generation sequencing and establishment of multiplex RT-PCR

“…In China, plant viruses cause extensive economic losses in tobacco production in spite of efforts on different control strategies [25]. In addition to TMV, viruses belong to the potyvirus genus have become the major viral pathogens infecting tobacco [12,15]. Sichuan province is one of the main tobacco production area in China [15], where the tobacco plantation is usually surrounded or mixed with other crops, especially pepper plants, which increases the risk of multiple virus synergistic infection.…”

“…In addition to TMV, viruses belong to the potyvirus genus have become the major viral pathogens infecting tobacco [12,15]. Sichuan province is one of the main tobacco production area in China [15], where the tobacco plantation is usually surrounded or mixed with other crops, especially pepper plants, which increases the risk of multiple virus synergistic infection. Therefore, it is of great signi cance to accurately identify the species of plant viruses and establish speci c and sensitive detection techniques for virus control.…”

“…TMV is the most well investigated plant virus that cause continuous losses to tobacco production [14]. In addition to TMV, various viruses belong to the genus Potyvirus including PVY, ChiVMV, TVBMV and tobacco etch virus (TEV) can also independently or synergistically infect tobacco plants and signi cantly decrease their quality [12,15,16]. PVY is a prevalent virus with wide host range that can induce vein necrotic or leaf mosaic symptoms in tobacco based on their strains [17][18][19].…”

“…Infection of TVBMV can cause bands of dark green tissue along the veins as well as small white to brown spots on the leaves [20]. Additionally, ChiVMV induce leaf punctate necrosis with round bright yellow spots and deformation symptoms on tobacco [15,21,22].…”