ABSTRACT
Cucurbit chlorotic yellows virus (CCYV) of the genus Crinivirus within the family Closteroviridae is an emerging infectious agent of cucurbits leading to severe disease and significant economic losses. Effective detection and identification methods for this virus are urgently required. In this study, a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed to detect CCYV from its vector Bemisia tabaci. LAMP primer sets to detect CCYV were evaluated for their sensitivity and specificity, and a primer set designed from the HSP70h gene with corresponding loop primers were selected. The RT-LAMP assay was applied to detect CCYV from viruliferous B. tabaci trapped on sticky traps. A simple extraction procedure using RNAsecure™ was developed for template preparation. CCYV was detected in all of the B. tabaci 0, 1, 7 and 14 days after they were trapped. Although the rise of turbidity was delayed in reactions using RNA from B. tabaci trapped for 7 and 14 days compared with those from 0 and 1 day, the DNA amplification was sufficient to detect CCYV in all of the samples. These findings therefore present a simple template preparation method and an effective RT-LAMP assay, which can be easily and rapidly performed to monitor CCYV-viruliferous B. tabaci in the field.
Subject(s)
Crinivirus/isolation & purification , Hemiptera/virology , Insect Vectors , Nucleic Acid Amplification Techniques/methods , Reverse Transcription , Animals , DNA Primers/genetics , Sensitivity and Specificity , TemperatureABSTRACT
Cucurbit chlorotic yellows virus (CCYV) (family Closteroviridae, genus Crinivirus) is an emerging virus which causes severe diseases on melon (Cucumis melo) plants. CCYV-infected melon plants display yellowing, mottling, chlorosis, or chlorotic spots on leaves. To develop a new control strategy, the potential for 1,2,3-benzothiadiazole-7-thiocarboxylic acid-S-methyl-ester (ASM) to suppress CCYV infection was evaluated. ASM treatment on melon plants greatly increased the expression levels of pathogenesis-related 1a gene, a marker gene for systemic acquired resistance. ASM treatment on melon plants before inoculation of CCYV suppressed systemic symptoms and decreased CCYV accumulation. ASM treatment on melon even after inoculation of CCYV reduced disease severity and accumulation levels of CCYV. The results show the potential for ASM treatment on attenuation of the CCYV disease symptoms.
Subject(s)
Crinivirus/drug effects , Cucumis melo/drug effects , Disease Resistance/drug effects , Plant Diseases/immunology , Plant Proteins/genetics , Thiadiazoles/pharmacology , Crinivirus/genetics , Crinivirus/physiology , Cucumis melo/genetics , Cucumis melo/immunology , Cucumis melo/virology , Plant Diseases/virology , RNA, Plant/genetics , RNA, Plant/metabolism , RNA, Viral/genetics , RNA, Viral/metabolism , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
Cucurbit chlorotic yellows virus (CCYV) causes chlorotic yellows on cucumber (Cucumis sativus) and melon (Cucumis melo) and is transmitted by Bemisia tabaci biotype B and Q whiteflies. To characterize the host range of CCYV, 21 cucurbitaceous and 12 other plant species were inoculated using whitefly vectors. All tested Cucumis spp. except Cucumis anguria and Cucumis zeyheri were systemically infected with CCYV, although infection rates varied among species. Citrullus lanatus, Cucurbita pepo, and Luffa cylindrica were susceptible to CCYV; however, the infection rates were low and symptoms were unclear. In addition to the cucurbitaceous plants, Beta vulgaris, Chenopodium amaranticolor, Chenopodium quinoa, Spinacia oleracea, Lactuca sativa, Datura stramonium, and Nicotiana benthamiana were also systemically infected by CCYV. Complete RNA1 and RNA2 were reverse-transcribed, cloned, and sequenced. CCYV RNA1 was found to be 8,607 nucleotides (nt) long and contained four open reading frames (ORFs). The first ORF spanned methyltransferase and RNA helicase domains followed by an RNA-dependent RNA polymerase domain, presumably translated by a +1 ribosomal frameshift. CCYV RNA2 was found to be 8,041 nt long and contained eight ORFs, including the hallmark gene array of the family Closteroviridae. Phylogenetic analysis demonstrated that CCYV was genetically close to Lettuce chlorosis virus, Bean yellow disorder virus, and Cucurbit yellow stunting disorder virus. Amino acid sequence similarities of representative proteins with these viruses indicated that CCYV should be classified as a distinct crinivirus species.
Subject(s)
Crinivirus/genetics , Cucumis/virology , Genome, Viral , Host-Pathogen Interactions , Base Sequence , Molecular Sequence Data , Phylogeny , Plant Diseases/virology , Sequence Analysis, RNAABSTRACT
Analysis of antibiosis resistance to common cutworm (Spodoptera litura Fabricius) in soybean (Glycine max (L.) Merr.) has progressed significantly, but the immediate cause remains unknown. We performed quantitative trait loci (QTL) analysis of pubescence density and plant development stage because these factors are assumed to be the immediate cause of resistance to cutworm. The QTLs for pubescence appeared to be identical to the previously detected the Pd1 and Ps loci controlling pubescence density. We found no candidate loci for flowering time QTLs, although one could be identical to the gene governing the long-juvenile trait or to the E6 loci controlling maturity. None of the QTLs overlapped with the QTLs for antibiosis resistance.
