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A triple antibody sandwich enzyme-linked immunosorbent assay (TAS-ELISA) and an immunocapture reverse transcription-polymerase chain reaction (IC-RT-PCR) for pepper mild mottle virus (PMMoV) detection were developed based on monoclonal and polyclonal antibodies produced in this study. Production of anti-PMMoV monoclonal antibodies (MAbs) was achieved using the hybridoma technique and a recombinant protein of the PMMoV coat protein as the antigen. Production of a polyclonal antibody (PAb) against the recombinant coat protein of PMMoV was carried out in a New Zealand White rabbit. The PAb was specific to five tobamoviruses consisting of PMMoV, tobacco mosaic virus (TMV), odontoglossum ringspot virus (ORSV), tomato mosaic virus (ToMV) and ribgrass mosaic virus (RMV). Two TAS-ELISA protocols were developed based on the characteristics of the antibodies produced. One protocol, based on MAb TOBA1, could detect five tobamoviruses (TMV, PMMoV, ORSV, ToMV, and RMV), four of which infect peppers. In the other protocol, TOBA10 was used to specifically detect PMMoV. We also successfully developed an IC-RT-PCR procedure using TOBA1. The sensitivity of the IC-RT-PCR (1:2,000,000) was much higher than TAS-ELISA (1:5120) for detection of the virus in plant sap. We anticipate these assays to facilitate the screening process of tobamovirus-resistant pepper cultivars in breeding programs.

Melon yellow spot virus (MYSV) is one of the most devastating plant viruses in the genus Tospovirus and adversely affects productivity, and quality of cucumber and melon. The locally made polyclonal antibody against nucleocapsid (N) protein of Thai isolate of MYSV has been previously used for the diagnosis of tospovirus species in serogroup IV as well as MYSV, therefore, causing problems for specific MYSV diagnosis. In this research, polyclonal antibody against MYSV- non structural (NSs) protein was produced and evaluated for use in MYSV diagnosis. The N and NSs genes were directly amplified by RT-PCR from MYSV-infecting melon and cloned. Analysis of N gene indicated MYSV infection in diseased melon (designated as isolate MYSV-SUT). The cloned NSs gene of MYSV-SUT is composed of 1,410 nucleotides which encoded for 470 amino acid residues. In vitro protein expression of NSs gene inserted in pQE80L expression vector yielded a recombinant 6xHis-NSs polypeptide. The rabbit polyclonal antibodies against the expressed 6xHis-NSs protein (PAb-MYSV-NSs) had titer up to 1:12,800 when tested with 3 μg/ml of the 6xHis-NSs protein by DAC-ELISA. This antibody reacted positively to crude sap from MYSV-infected melon and cucumber leaf tissue by nitrocellulose ELISA (NCM-ELISA), and not to crude sap of symptomatic plants infected with Watermelon silver mottle virus (WSMoV), Capsicum chlorosis virus (CaCV) and Tomato necrotic ring spot virus (TNRV) isolates from Thailand. However, crude sap of MYSV-infected melon from fields did not react with PAb-MYSV-NSs in the DAC-ELISA conditions used in this study.

Twenty-eight isolates of tospoviruses associated with tomato, pepper, cucurbits, peanut, and Physalis plants collected from fields in different regions of Thailand were characterized. On the basis of N gene and protein sequence relationships, three tospoviruses were identified, namely Watermelon silver mottle virus (WSMoV), Capsicum chlorosis virus (CaCV), and Melon yellow spot virus (MYSV). CLUSTAL analysis of selected N protein sequences showed different isolates of CaCV in three distinct clades. Based on necrosis symptoms on tomato and their 93% identity to CaCV isolates in the other two clades, CaCV-TD8, CaCV-AIT and CaCV-KS16-Thailand tomato tospovirus were designated as CaCV-tomato necrosis strain. A phylogenetic tree based on the 413-amino-acid Gc fragment of the CaCV-Pkk isolate supported the existence of three distinct CaCV clades. Vigna unguiculata produced concentric rings useful for discriminating the Thai CaCV peanut isolates from tomato or pepper isolates. By using reverse transcription polymerase chain reaction with species-specific primers, the three tospoviruses could be detected in mixed infections in watermelon and Physalis, as well as in the bodies of thrips vectors, Thrips palmi and Scirtothrips dorsalis, collected from fields.