A simple and reliable protocol for the detection of apple stem grooving virus by RT–PCR and in a multiplex PCR assay
Introduction
Apple stem grooving virus (ASGV) is widely distributed in apple trees (Nemeth, 1986) and has been associated with tree decline and graft union necrosis in sensitive combinations of scion and rootstock (Yanase et al., 1990). The virus has also been detected in other species such as pear (Sawamura et al., 1988) and apricot (Takahashi et al., 1990). ASGV is an important member of the group of latent viruses infecting apple (Yanase et al., 1990). It is a target for detection and control in all programs involved in the maintenance and distribution of virus-free Malus germplasm (Nemeth, 1986, Howell et al., 1996). To achieve control of this important virus, reliable diagnostic techniques capable of detecting all isolates are required. Virginia Crab and Pyronia veitchii are woody indicator plants that have been used for the detection of ASGV (Welsh and van der Meer, 1989), but there are difficulties associated with this approach (Howell et al., 1996). Indexing is time consuming and the results may be unreliable. Recently, Howell et al. (1996) identified three Malus clones that are rapid and highly sensitive for the detection of ASGV. Serological techniques have been used for the detection of ASGV but the virus may be undetectable because of low concentrations (Kinard et al., 1996), and useful ASGV antiserum is difficult to produce. Consequently antiserum of a virus related serologically, citrus tatter leaf virus, has been used for ASGV detection (Kawai et al., 1991).
ASGV is the type member of the capillovirus genus (Murphy et al., 1995) and the entire genome of the virus has been sequenced (Yoshikawa et al., 1992). The availability of this sequence information has facilitated the design of oligonucleotide primers that are specific for the detection of ASGV by RT–PCR. Nucleic acid-based diagnostic techniques such as RT–PCR are prohibitively expensive, require specialised equipment and skills, and are not yet adaptable for routine use in plant disease diagnosis (Putnam, 1995). RT–PCR techniques have been described for the detection of ASGV but involve difficult and complex RNA extractions (Kinard et al., 1996, Marinho et al., 1998). In this study, attempts were made to develop and identify RT–PCR protocols that were simple, reliable, adaptable for routine diagnosis of ASGV, and with reduced costs.
Section snippets
Virus source
Apple stem grooving virus (ASGV) isolates 1041-05, 1162-05 (IR2, 111-4), and 1422-03 in apple (Malus domestica), and 1288-08 in pear (Pyrus communis) were maintained at the Centre for Plant Health, Sidney, British Columbia. Isolate 1162-05 was mechanically sap transmitted to the herbaceous hosts Chenopodium quinoa and Nicotiana occidentalis. Leaf and budwood samples from plants infected with a range of ASGV isolates from around the world were kindly provided by Bill Howell, NRSP5/IR2, Prosser
RT–PCR detection
The primers ASGV-U and ASGV-2 amplified specifically a 499 bp product when used to assay plants infected with ASGV. The virus was detected by RT–PCR in the herbaceous hosts C. quinoa, N. occidentalis, and in Pyrus and Malus hosts. In woody hosts reliable detection was obtained when using either infected leaves or dormant budwood. The total RNA extracted from all uninfected controls gave negative results when tested by RT–PCR.
IC/RT–PCR detection
Buffer F was the most effective sample grinding buffer for the
Discussion
In this study the oligonucleotide primers ASGV-U and ASGV-2 were used reliably to detect various Malus and Pyrus isolates of ASGV from a wide range of sources suggesting that the primer sequences are highly conserved. ASGV was detected in Malus, Pyrus, C. quinoa, and N. occidentalis using RT–PCR and IC/RT–PCR, and by TC/RT–PCR in Malus, C. quinoa and N. occidentalis. The virus was detectable in leaf tissue or in the bark tissue of dormant wood. Storage at −80°C over 4 months did not affect the
Acknowledgements
I wish to thank Claire Brehelin, Sharon Godkin, and Meghan MacLeod for their technical assistance; Bill Howell and Dan Thompson for providing the ASGV isolates; and Don MacKenzie for his assistance with the ASGV primer design.
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