Articles
USE OF DOUBLE-STRANDED RNA FOR DETECTION AND IDENTIFICATION OF VIRUS DISEASES OF RUBUS SPECIES
Article number
186_7
Pages
51 – 62
Language
Abstract
The yield of double-stranded RNA (ds-RNA) from Rubus spp. depended on the purification procedure used and the size of the ds-RNA. For ds-RNAs less than 4 x 106 daltons the two column procedure of Morris and Dodds (1979) was used except that Tl RNAse and DNAse digestions were carried out between the two chromatography steps.
This procedure could be completed in 1.5 days and gave cleaner ds-RNA preparations in which as little as 5–10 ng of ds-RNA could be detected in agarose gels stained with ethidium bromide.
For larger molecular weight ds-RNAs the cetyltrimethyl ammonium bromide (CTAB) procedure of Murray and Thompson (1980) gave higher yields of ds-RNA. With the CTAB procedure a high molecular weight ds-RNA (>6 x 106 daltons) was found in healthy as well as virus-infected Rubus.
Tomato ringspot virus (TomRSV), raspberry bushy dwarf virus (RBDV) and raspberry leafspot virus (RLSV) were readily detected from infected Rubus spp.
Field samples exhibiting virus-like symptoms usually contained two or more viruses which could be identified from the ds-RNA pattern on agarose gels.
This technique provides an objective method of identifying RLSV which heretofore could be detected only by grafting to indicator plants or by aphid transmissions.
Gel-purified RLSV ds-RNA labelled at the 5′ end with 32P was used as a hybridization probe to detect any relationships with other viruses of raspberry.
This probe hybridized to RLSV samples but not to samples prepared from plants infected with raspberry leaf mottle virus (RLMV), TomRSV, RBDV, black raspberry necrosis virus, rubus yellow net virus, Calico-diseased Rubus, healthy Rubus or to ds-RNA filters of cucumber mosaic virus, potato virus X, or any of four fungal ds-RNAs used as molecular weight markers.
This procedure could be completed in 1.5 days and gave cleaner ds-RNA preparations in which as little as 5–10 ng of ds-RNA could be detected in agarose gels stained with ethidium bromide.
For larger molecular weight ds-RNAs the cetyltrimethyl ammonium bromide (CTAB) procedure of Murray and Thompson (1980) gave higher yields of ds-RNA. With the CTAB procedure a high molecular weight ds-RNA (>6 x 106 daltons) was found in healthy as well as virus-infected Rubus.
Tomato ringspot virus (TomRSV), raspberry bushy dwarf virus (RBDV) and raspberry leafspot virus (RLSV) were readily detected from infected Rubus spp.
Field samples exhibiting virus-like symptoms usually contained two or more viruses which could be identified from the ds-RNA pattern on agarose gels.
This technique provides an objective method of identifying RLSV which heretofore could be detected only by grafting to indicator plants or by aphid transmissions.
Gel-purified RLSV ds-RNA labelled at the 5′ end with 32P was used as a hybridization probe to detect any relationships with other viruses of raspberry.
This probe hybridized to RLSV samples but not to samples prepared from plants infected with raspberry leaf mottle virus (RLMV), TomRSV, RBDV, black raspberry necrosis virus, rubus yellow net virus, Calico-diseased Rubus, healthy Rubus or to ds-RNA filters of cucumber mosaic virus, potato virus X, or any of four fungal ds-RNAs used as molecular weight markers.
Authors
A. Kurppa, R.R. Martin
Keywords
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