Quantitation of cucurbit yellow stunting disorder virus in Bemisia tabaci (Genn.) using digoxigenin-labelled hybridisation probes.
A cost-efficient hybridisation assay was developed to estimate the amount of cucurbit yellow stunting disorder virus (CYSDV) in Bemisia tabaci (Gennadius) whiteflies infesting protected cucumber crops. cDNA from the coat protein (cp) gene and the hsp70 homologue protein gene from CYSDV were obtained by reverse transcriptase-PCR from viruliferous whiteflies and cloned into plasmids. Digoxigenin (DIG)-labelled cDNA probes reacted with extracts from these whiteflies applied on nylon membranes. Precision and linear ranges were established in a hybridisation analysis using known concentrations of unlabelled homologue cDNA. Extracts from non-viruliferous B. tabaci showed a concentration-dependent effect on the assay with cp-specific probes but not with hsp70-specific probes. The hsp70 probe was used to evaluate natural B. tabaci populations in commercial cucumber crops, and the estimated amounts of CYSDV per whitefly were found ranging from 5.6 fg to approximately 2.5 pg of corresponding hsp70-cDNA.
Ruiz
Leticia
L
Centro de Investigacion y Formacion Agraria, Unidad de Virologija, Apartado de Correos 91, 04700-El Ejido, Almeria, Spain.
Janssen
Dirk
D
Velasco
Leonardo
L
Segundo
Eduardo
E
Cuadrado
Isabel M
IM
D000818:Animals; D029383:Crinivirus / Q000235:genetics / Q000302:isolation & purification*; D018553:Cucumis sativus / Q000821:virology*; D015342:DNA Probes; D004279:DNA, Viral / Q000032:analysis*; D004076:Digoxigenin / Q000737:chemistry*; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D006430:Hemiptera / Q000821:virology*; D008163:Luminescent Measurements; D009693:Nucleic Acid Hybridization / Q000379:methods; D010957:Plasmids; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012621:Seasons; D014170:Transformation, Genetic
D015342:DNA Probes; D004279:DNA, Viral; D004076:Digoxigenin