Detection of Strawberry crinkle virus in plants and aphids by RT‐PCR using conserved L gene sequences

• Published in: Plant pathology Vol. 51; no. 3; pp. 266 - 274
• Main Authors: Posthuma, K. I., Adams, A. N., Hong, Y., Kirby, M. J.
• Format: Journal Article
• Language: English
• Published: Oxford, UK Blackwell Science Ltd 01.06.2002; Blackwell; Wiley Subscription Services, Inc
• Subjects: Biological and medical sciences; Fragaria; Fundamental and applied biological sciences. Psychology; Generalities. Techniques. Transmission, epidemiology, ecology. Antiviral substances, control; Microbiology; Mononegavirales; Phytopathology. Animal pests. Plant and forest protection; Plant viruses and viroids; Rhabdoviridae; RNA‐dependent RNA polymerase; strawberry; Strawberry crinkle virus; Techniques used in virology; Virology; Plant pathology; Horticulture; Plant pathogen; RNA; Entomology; Small fruits; Insecta; Rosaceae; Transmission; Conserved sequence; Pest; Cytorhabdovirus; Homoptera; Gene; Rhabdoviridae; Dicotyledones; Host plant; Angiospermae; Aphididae; Strawberry crinkle virus; Solanaceae; Reverse transcription polymerase chain reaction; Sequencing; Vector; Nucleic acid; Method study; Chaetosiphon fragaefolii; Aphidoidea; Fragaria; Enzyme; Physalis; Transferases; Experimental study; RNA-directed RNA polymerase; Virology; Infection; Virus; Mononegavirales; Nucleotidyltransferases; Analysis method; Complementary DNA; Fruit crop; Arthropoda; Viral disease; Spermatophyta; Molecular biology; Invertebrata; Detection
• ISSN: 0032-0862; 1365-3059
• DOI: 10.1046/j.1365-3059.2002.00725.x

About 10% of the large (L) protein gene of Strawberry crinkle virus (SCV) was sequenced after amplification with degenerate primers designed to conserved regions of the rhabdovirus L protein. The virus sequence was extended to 1362 nucleotides through rapid amplification of cDNA ends. One pair of degenerate L gene primers amplified a 683‐bp fragment from four different isolates of SCV cultured in the experimental host Physalis pubescens; the nucleotide sequences of these fragments differed by < 1% to 10% indicating the suitability of this region as a diagnostic target. This information enabled the development of a reverse transcription polymerase chain reaction (RT‐PCR) detection method for SCV using primers designed to the L gene sequence. SCV was amplified from infected P. pubescens (573 bp fragment) and from infected Chaetosiphon fragaefolii aphids (770 bp fragment). SCV was also detected by RT‐PCR in total RNA extracts from three strawberry plants showing symptoms typical of SCV infection but failed when the intensity of the disease symptoms decreased. However, both SCV positive‐sense RNA, and negative‐sense genomic RNA, were detected by nested PCR in chronically infected strawberry plants sampled in September.