A simple and reliable protocol for the detection of apple stem grooving virus by RT–PCR and in a multiplex PCR assay

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Abstract

Primers were identified which amplify specifically a 499 bp fragment in the coat protein coding region of apple stem grooving virus (ASGV) genome. These primers were used in various RT–polymerase chain reaction (PCR) analyses for the detection of ASGV in Chenopodium quinoa, Nicotiana occidentalis, and in species of Malus and Pyrus. Isolates of ASGV in Malus and Pyrus from locations in Canada, China, Israel, Japan, Nepal, Pakistan, South Africa, and the U.S.A. were reliably detected in leaf and bark (budwood) tissue. Storage of the tissues at −80°C for more than 4 months did not affect the reliability of detection by immunocapture (IC) RT–PCR. Triton-X was not necessary for the detection of ASGV by IC/RT–PCR, and it was also possible to combine the antibody incubation and virus sap incubation into a single step without any obvious loss in sensitivity. A Tube Capture (TC) RT–PCR procedure was developed that eliminated the need for antibody binding of the virus. Phosphate buffered saline with 2% PVP was identified as the most effective sample-grinding buffer for the detection of ASGV by IC/RT–PCR and TC/RT–PCR. TC/RT–PCR facilitated the simultaneous detection (multiplex PCR) of ASGV and cherry mottle leaf virus.

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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