Characterization Of Three Emerging Viruses On Vegetables In The United States And The Development Of Detection Methods

Viral diseases is a major limiting factor of vegetable industry in the United States,resulting in reduction of vegetable crops and serious economic losses.Tomato mottle mosaic virus(ToMMV),Tomato chlorotic spot orthotospovirus(TCSV)and Cucumber green mottle mosaic virus(CGMMV)are three of the emerging viruses on tomato plants and cucurbit crops in the U.S.Here,the studies on these three viruses were conducted as follows:Tomato mottle mosaic virus(ToMMV)was first identified in 2013 as a novel tobamovirus infecting tomatoes in Mexico.In this study,we collected two new ToMMV isolates in the U.S.,named ToMMV SC13-05 and ToMMV CA16-01 and their complete genome sequences were obtained(GenBank Accession no.KX898033 and KX898034).Comparative sequence and phylogenetic analysis revealed a close genetic relationship among all the ToMMV isolates(99.4-99.6%)and a closer genetic relationship to ToMV(84.3%)than to several other tobamoviruses.To distinguish three tomato-infecting tobamoviruses,a sensitive multiplex RT-PCR was developed to allow for species-specific detection and identification of Tobacco mosaic virus(TMV),Tomato mosaic virus(ToMV)and ToMMV.In host range study,ToMMV could systematically infect several plant species in the Solanaceae family,including Nicotiana tabacum ’Xanthi nc’ and Datura stramonium.Thus,ToMMV has a broader host range than that of ToMV.In the pathogenicity test,we observed that a small proportion(≈10%)of cultivar ’B’ plants showed symptoms after inoculated with ToMMV MX5.Through sequence analysis of Tm2 gene and Genotyping-by-sequencing(GBS),we confirmed both resistant and susceptible cv.’B’ plants were homozygous Tm-22/Tm-22 plants and no single nucleotide polymorphism(SNP)was identified in association with the susceptibility.In transcriptom analysis,genes in the tomato-pathogen interaction pathway including Pathogenesis-related protein lb、WRKY transcription factor and NAD(P)H-quinone oxidoreductase subunit 1 were up-regulated in the susceptible cv.’B’ plants with necrosis when comparing to that of the resistant cv.’B’ plants.Therefore,the producing of necrotic symptoms was probably in association with the resistant reaction of tomato cv.’B’ plants.Moreover,ToMV V13-07 and ToMMV MX5 could induce the resistant reaction and the presence of systemic necrosis on cv.’B’ plants at higher temperature.TCSV was first identified to infect tomato in Florida of U.S.at 2012.In this study,a preliminary investigation was conducted to understand the occurrence of orthotospoviruses in Long Island,New York.From the samples collected,we confirmed V16-150 was positive to TCSV,this is the first report of TCSV in New York.In order to fit the requirement of further investigation,a rapid,simple and sensitive one-step reverse transcription loop-mediated isothermal amplification(RT-LAMP)assay was developed to achieve species-specific detection for TCSV.A preliminary survey was operated to investigate the occurrence of TCSV in Florida by using RT-LAMP.Among the 40 samples tested,30 were positive to TCSV.In 2013,CGMMV was detected from the symptomatic melon,cucumber and watermelon plants in seed production fields in California.In the present study,we obtained two new CGMMV isolates from the CGMMV positive control of Agdia ELISA kit,named CGMMV EU1 and CGMMV EU2 and their complete genome sequences were obtained(KY910828 and KY910829).Comparative sequence and phylogenetic analysis revealed that CGMMV EU1 and EU2 has a close genetic relationship with CGMMV isolates from Russia,but has distant relationship with CGMMV isolate ABCA13-01 from Canada.In the host range study,CGMMV ABCA13-01 mainly infected watermelon,melon and some other plant species in the Cucurbitaceae family.Moreover,CGMMV ABCA13-01 could systemically infect Lactuca sativa and Glebionis coronaria in the Asteraceae family and Catharanthus roseus in the Apocynaceae family,indicating this isolate has broader host range than that of the other CGMMV isolates.At last,we conducted comparative evaluation of four CGMMV detection methods in seed health testing,including enzyme-linker immunosorbent assay(ELISA),Immuno Stripe(?),real-time RT-PCR and RT-LAMP.ELISA from a new antibody was more sensitive,reliable and stable for the CGMMV detection in seed health assay.Based on the occurrence of emerging vegetable viral diseases in the U.S.,we determined the molecular and biological properties of ToMMV and the occurrence of ToMMV in the U.S.for the first time.A survey was conducted on the occurrence of TCSV in Long Island,New York for the first time.The molecular and biological properties of CGMMV were systematically studied.Finally,the species-specific detection assays were developed to identify each of the three viruses respectively.This study provides a theoretical basis for the prevention and control of these three emerging viral diseases and provides an effective detection method for each of the three viruses.