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Comparison of bioassays and laboratory assays for apple stem grooving virus
Journal of Virological Methods 93 (2001) 167– 173 www.elsevier.com/locate/jviromet
Comparison of bioassays and laboratory assays for apple stem grooving virus M.J. Kirby, C.M. Guise, A.N. Adams * Horticulture Research International, East Malling, West Malling, Kent ME19 6BJ, UK Received 5 October 2000; received in revised form 24 January 2001; accepted 25 January 2001
Abstract The standard field double-budding assay with the indicator Virginia Crab and the glasshouse test with the indicator Malus micromalus, were compared with ELISA and immunocapture PCR for the detection of Apple stem groo6ing 6irus (ASGV) in 102 apple trees and three oriental pear. Twenty-two trees were positive for ASGV by both ELISA and IC-PCR but three of these trees were negative by Virginia Crab, three were negative by M. micromalus and one was negative by both these bioassays. The infected trees were re-tested by IC-PCR and ELISA in a second year; the IC-PCR results were confirmed but two of the 22 infected trees were negative by ELISA. On this evidence, IC-PCR is a more reliable assay for ASGV than the slow and expensive bioassays. © 2001 Elsevier Science B.V. All rights reserved. Keywords: Field assays; Glasshouse assays; ELISA; Polymerase chain reaction; IC-PCR; Apple stem grooving virus
1. Introduction Apple stem groo6ing 6irus (ASGV) is a member of the genus Capillo6irus and is widespread in rosaceous fruit trees, particularly species of Malus and Pyrus (Nemeth, 1986). Although it is latent in most commercial rootstock – scion combinations, ASGV causes a serious disease in Japan in apples growing on Malus sieboldii rootstocks (Yanase, 1983) and the virus is tested for world wide in schemes for producing pathogen-tested planting material. * Corresponding author. Tel.: + 44-1732-843833; fax: +441732-849067. E-mail address: [email protected] (A.N. Adams).
A range of bioassays and laboratory assays is available for the detection of ASGV. Bioassays include the indicator Virginia Crab either in the field for 3 years or in the glasshouse for 6 months (Anonymous, 1998), or Malus micromalus (clone GMAL 273.a) in the glasshouse for 4 weeks (Howell et al., 1996). Laboratory assays include ELISA (Fuchs, 1980) and various forms of PCR (Kinard et al., 1996; Marinho et al., 1998; James, 1999; Crossley et al., 1998). We have compared field and glasshouse bioassays with ELISA and IC-PCR for the detection of ASGV using material from 105 different cultivars of apple and oriental pear, a proportion of which were known to be infected with ASGV. The purpose of these tests was to determine whether the relatively rapid and
0166-0934/01/$ - see front matter © 2001 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 6 - 0 9 3 4 ( 0 1 ) 0 0 2 7 1 - 3
168
M.J. Kirby et al. / Journal of Virological Methods 93 (2001) 167–173
cheap laboratory assays were as effective as bioassays for detecting ASGV.
2. Materials and methods
2.1. Field bioassay M26 rootstocks were double-budded with test material and Malus pumila cv. Virginia Crab indicator buds in August 1996. Three replicate trees were budded in the field for each of the 105 sources of test material. The trees for testing were chosen at random from the variety collection at the UK National Fruit Collection, Faversham, Kent. However, 12 trees were included that were known from previous laboratory assays to be infected with ASGV. Five positive and five negative controls were included with each set of 10 tests. After 3 years the trees were cut at about 30 cm above the graft union and 5 cm below it for final symptom assessment. The shoot pieces were autoclaved and the bark removed to examine the condition of the wood and union.
2.2. Glasshouse bioassay M26 rootstocks (8-mm diameter) were planted in forestry pots, double budded with inoculum and M. micromalus indicator buds in February 2000 and retained in a glasshouse at 22°C with drip irrigation. Four replicates were budded for each of the 105 sources of inoculum and positive and negative controls were included for every 12 tests. Symptom development was recorded for 2 months after the rootstocks had been cut back to the M. micromalus bud.
2.3. ELISA Reagents were obtained from Loewe Phytodiagnostica (Sauerlach, Germany) and used according to their ‘Simultan’ double antibody sandwich protocol. Plates were coated with IgG followed by incubation with a mixture of sample and alkaline phosphatase-labelled IgG. 4-Nitrophenyl phosphate substrate was used. Negative controls (healthy apple extracts) were included in eight
wells of each ELISA plate and samples were considered positive if they reached twice the OD of the average of the controls. The test was specific for ASGV and no cross-reaction was detectable with extracts from herbaceous host plants infected with other apple and pear viruses (Apple stem pitting 6irus — ASPV, Apple chlorotic leaf spot 6irus — ACLSV, Apple mosaic 6irus — ApMV).
2.4. IC-PCR ThermoFast plates (Advanced Biotechnologies) were coated with 100 ml antiserum (Loewe) diluted 1:200 in carbonate-coating buffer. Young unfurled leaf samples were ground in phosphatebuffered saline, pH 7.4, containing 0.05% Tween 20, 2% polyvinyl pyrrolidone, 0.2% ovalbumin and the extracts diluted to 1:400. Extracts were incubated in the plates overnight at 4°C for immunocapture to occur. A two-step RT-PCR, based on Jelkmann and Kein-Konrad (1997) was carried out using primers designed to regions in the coat protein gene. Reverse transcription: 20 ml reaction containing 4 ml of 5× reaction buffer (Promega), 1.25 mM each dNTP, 35 U ribonuclease inhibitor (MBI), 20 pmoles oligo dT primer and 0.5 U AMV-RT (Promega). Reactions were overlaid with mineral oil and incubated at 37°C for 1 h. PCR: 50 ml reaction containing 3 ml of cDNA, 1× PCR buffer (Gibco BRL), 2 mM MgCl2, 0.4 mM each dNTP, 0.4 pmoles of each primer and 2 U Taq DNA polymerase (Gibco BRL). Reactions were overlaid with mineral oil and cycled in an OmniGene PCR machine (Hybaid) 95°C for 90 s followed by 35 cycles of 95°C for 30 s, 60°C for 30 s and 72°C for 60 s. Ten ml of each reaction was analysed by electrophoresis in a 1.5% agarose gel containing 0.5 mg/ml ethidium bromide. The size of the amplified fragment was determined using a 1 kb DNA ladder (Gibco BRL). Primers SGTL4 (5%-GAGAGGATTTAGGTCCCTCT-3%) and SGTL5 (5%-CTCCTAACCCTCCAGTTCCA-3%) were used initially to amplify a 664 bp region (nucleotides 5594 –6257 isolate P209 [Yoshikawa et al., 1992], accession number D14995) from the coat protein coding
M.J. Kirby et al. / Journal of Virological Methods 93 (2001) 167–173
region of 11 different ASGV isolates. The resulting PCR fragments were sequenced directly so sequence data presented is of the most common variant. Despite the high degree of similarity over this coat protein region (96.6 – 99.6% at the nucleic acid level) the European apple isolates clustered together into a cluster that is distinct from P209, the Oriental pear and citrus tatter leaf isolates (data not shown). Diagnostic primers SGTL6 (5%-AAGGTGAAAGCTTTGAAGGCA3%) and SGTL9 (5%-TCAAAAGCTTTGGGCCATTTC-3%) were designed to areas of complete nucleic acid homology between the 11 isolates sequenced although there is one mismatch in each primer with respect to P209 (Fig. 1). An ASGV diagnostic product of 424 bp is amplified by this primer pair (nucleotides 5762 – 6185 isolate P209). No product was amplified with these primers from herbaceous plants infected with other apple and pear viruses (ASPV, ACLSV and ApMV).
3. Results
3.1. Virginia Crab bioassays All negative controls had healthy unions and smooth wood under the bark. All positive controls had typical symptoms of necrosis at the graft union. Eighteen of the 105 trees tested induced necrosis at the graft union of the rootstock with the indicator scion of all three replicates. This symptom is diagnostic for ASGV. The majority of the apple trees were infected with ASPV, which induces pitting in the wood of Virginia Crab, but none of the three oriental pear trees were infected with this virus.
3.2. Malus micromalus glasshouse assay Although the budwood of M. micromalus was of much smaller diameter than the M26 root-
169
stocks there were few failures of inoculum or indicator buds. All negative controls grew strongly and the leaves were green, unblemished and without distortion. The M. micromalus bud shields of positive controls, infected either with ASGV alone or ASGV plus ASPV, ACLSV and Star Crack disease, united, and they remained alive. However, the buds either failed to grow out or grew for a short time and then became necrotic and died. There was no difference between control trees budded with pure ASGV or the multiple infection containing ASGV. These symptoms were typical of the majority of trees budded with ASGV-infected test material. Those shoots that did not die were stunted with epinasty and necrotic spotting on the leaves. Most of the material under test was infected with ASPV, based on the Virginia Crab assay, and some may also have been infected with other latent viruses. Growth of the M. micromalus budded with test material that was not infected with ASGV was clearly inferior to that of the uninfected control trees although not bearing any of the symptoms described by Howell et al., (1996) for ASGV. Budwood from 18 of the 105 trees under test induced symptoms diagnostic for ASGV; all replicates showed diagnostic symptoms except for one test in which two of the four M. micromalus were symptomless. However, different results were obtained for six trees compared with the Virginia Crab field test. Three trees that were positive in the field assay were negative by the glasshouse assay and three different trees were positive by the glasshouse assay and negative in the field. One of the four replicates that were budded with the oriental pear cv. Nijiseiki showed slight epinasty of two lower leaves and a few small necrotic spots. These symptoms were mild and they were only noticed after re-examination of the trees budded with material from sources known to be infected with ASGV. This test was scored negative.
Fig. 1. Sequence comparison of 11 ASGV isolates and the published strain P209 (accession number D14995) over the coat protein region defined by the diagnostic primer pair SGTL6 and 9. Nucleotides identical to P209 are marked with a dash, the non-identical nucleotides are written below. Alignment using Clustal (DNAstar). ASGV isolates from apple cultivars Erwin Bauer (EB); Golden Delicious (GD); Hunter Melba (HM); PJ Bergius (PJB); Maidstone Favourite (MF); Thoday’s Quarrenden (TQ); Mate Denes Dr (MDD); Tewkesbury Baron (TB) and Japanese Pear (-JP) cultivars Chojuro (CHO); Niitaka (NIIT) and Nijiseiki (NIJ).
170
M.J. Kirby et al. / Journal of Virological Methods 93 (2001) 167–173
Fig. 1. (Continued)
M.J. Kirby et al. / Journal of Virological Methods 93 (2001) 167–173 Table 1 ASGV ELISA values (9 S.E.) of paired samples from six fruit buds and six vegetative buds of apple in April 1999 and May 2000 Tissue
1999
2000
Fruit buds Blossom Leaves
0.159 0.10 0.33 9 0.13
0.12 9 0.11 0.62 9 0.28
Vegetati6e buds: Unfurled leaves Opened leaves
1.129 0.24 0.54 9 0.26
0.77 9 0.36 0.36 9 0.18
171
tive buds gave the highest readings (Table 1) and these tissues were therefore used for ELISA and for IC-PCR. The ELISA and PCR results were in complete agreement in 1999 (Table 2). Twenty-two of the 105 trees sampled were positive for ASGV by both PCR and ELISA. The remaining 83 trees were negative by all assays, including the field double-budding. However, four of the 22 trees that were positive by the laboratory assays were not positive by the field tests and an additional three were negative by the M. micromalus test. In May 2000, the laboratory assays were repeated on samples from all the trees that were positive in 1999. All these trees were again positive by PCR but two of them were negative by ELISA. The primer pair SGTL6 and SGTL9 successfully amplified ASGV from a number of different
3.3. ELISA and IC-PCR Preliminary ELISAs were carried out in April 1999 and in May 2000 just prior to conducting laboratory assays on the samples from 105 trees. In both years, young unfurled leaves from vegeta-
Table 2 Origin of plant material infected with ASGV and the tests in which infection was detected (+) or not detected (−)a in assays on 102 cultivars of apple and three oriental pear from the National Fruit Collection, Faversham, UK in 1999 Cultivar and origin
PCR
ELISA
Virginia Crab
M. micromalus
Apple Climax, UK Hunter Melba, USA Hosszufalusi, Hungary Indo, UK Ingall’s Red, UK Just Gyula, Hungary Maidstone Favourite, UK Meri Cretesti, Germany Nobil de Geoagiu, Romania PJ Bergius, UK Priolov Delises, Yugoslavia Ringstad, UK Thoday’s Quarrenden, UK Calville Duquesne, France Tewkesbury Baron, UK Erwin Bauer, Germany Marie Nimitz (false), France Tewkesbury Baron, UK Alton, Netherlands
+ + + + + + + + + + + + + + + + + + +
+ + + + + + + + + + + + + + + + + + +
+ + + + + + + + + + + + + + + − − − −
+ + + + + + + + + + + + + − − + + + −
Oriental pear Chojuro, Japan Niitaka, Japan Nijiseiki, Japan
+ + +
+ + +
+ + +
+ + −
a
Samples from 83 trees were negative by all tests.
172
M.J. Kirby et al. / Journal of Virological Methods 93 (2001) 167–173
apple cultivars from a variety of European countries and the USA (Table 2), as well from Oriental pear, which suggests that they are universally applicable.
4. Discussion The IC-PCR assay described above, based on primers designed to conserved regions from 11 isolates of ASGV, successfully amplified 22 isolates of the virus which came from plants originating over a wide geographic area (USA, Japan, and six countries in Europe) and from Pyrus and Malus. The availability of commercial antisera allows the use of immunocapture, which simplifies the assay as well as improving sensitivity (James, 1999). A one-step RT-PCR protocol, such as that used by Marinho et al. (1998), would be a further refinement, but in our hands has not been as reliable as separating RT from amplification. The disparity in detection results between ELISA and IC-PCR may reflect low virus titres. Although we used unfurled leaves in the spring, to optimise virus concentrations, others have amplified ASGV successfully from dormant bark, fruit and root tissues (Marinho et al., 1998; James, 1999). It is not clear which tissues should be sampled, and in which season, for best IC-PCR results. Bioassays are recognised internationally as being the most reliable means of testing for viruses in fruit trees (Anonymous, 1998). Most schemes for producing healthy planting material require that bioassays are used, at least for establishing the health status of the Nuclear Stock plants. Our results show that some isolates of ASGV are not detected by one or other of the two bioassays employed in this work. It is noteworthy that only 15 trees gave the same results in all four assays and that three trees that were negative by M. micromalus were positive by Virginia Crab and vice versa. James (1999) also found that two of 23 isolates, originating from Pyrus and Malus from eight different countries, were not detected by woody indexing but were detected by IC-PCR. The equivocal results of the M. micromalus assay with cv. Nijiseiki
would have necessitated a re-test and even then may not have given a clear result. Re-tests with laboratory assays are far quicker to conduct and for PCR may only require re-amplification of the cDNA taking a few hours. Virus isolates held in collections, and used to assess the effectiveness of assays, have often been found originally by woody indexing. This automatically biases any comparison of assays in favour of woody indicators. On the evidence of our tests, and those of James (1999), there is no good reason to believe that woody indicators are superior to IC-PCR for the detection of ASGV. It is essential to conduct comparisons of the type reported here with all fruit tree viruses so that the various assays now available can be utilised to their full potential.
Acknowledgements This work was part of a HortLink project funded by the Ministry of Agriculture Fisheries and Food, the East Malling Trust for Horticultural Research, the Horticultural Development Council and the Nuclear Stock Association. We are grateful to Mrs Karen Lower for excellent technical assistance and to the Brogdale Horticultural Trust, Faversham, Kent, for allowing us access to the National Fruit Collection.
References Anonymous, 1998. Recommendations for pathogen detection. Acta Hort. 472 (2), 757– 783. Crossley, S.J., Jacobi, V., Adams, A.N., 1998. IC-PCR amplification for apple stem grooving isolates and comparison of polymerase and coat protein gene sequences. Acta Hort. 472, 113– 118. Fuchs, E., 1980. Serological detection of apple chlorotic leaf spot virus and apple stem grooving virus in apple trees. Acta Phytopathol. 15, 69 – 74. Howell, W.E., Mink, G.I., Hurtt, S.S., Foster, J.A., Postman, J.D., 1996. Select Malus clones for rapid detection of apple stem grooving virus. Plant Disease 80, 1200– 1202. James, D., 1999. A simple and reliable protocol for the detection of apple stem grooving virus by RT-PCR and in a multiplex PCR assay. J. Virol. Methods 83, 1 – 9.
M.J. Kirby et al. / Journal of Virological Methods 93 (2001) 167–173 Jelkmann, W., Kein-Konrad, R., 1997. Immuno-capture polymerase chain reaction and plate-trapped ELISA for the detection of apple stem pitting virus. J. Phytopathol. 145, 499– 503. Kinard, G.R., Scott, S.W., Barnett, O.W., 1996. Detection of apple chlorotic leaf spot and apple stem grooving viruses using RT-PCR. Plant Dis. 80, 616–621. Marinho, V.L.A., Kummert, G., Rufflard, G., Colinet, D., Lepoivre, P., 1998. Detection of apple stem grooving virus in dormant apple trees with crude extracts as templates for one-step RT-PCR. Plant Dis. 82, 785–790.
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Nemeth, M., 1986. Virus, Mycoplasma and Rickettsia Diseases of Fruit Trees. Akademiai Kiado, Budapest, p. 841. Yanase, H., 1983. Back transmission of apple stem grooving virus to apple seedlings and induction of symptoms of apple topworking disease in Mitsuba Kaido (Malus sieboldii ) and Kobano Zumi (Malus sieboldii var. arborescens) rootstocks. Acta Hort. 130, 117– 122. Yoshikawa, N., Sasaki, E., Kato, M., Takahashi, T., 1992. The nucleotide sequence of apple stem grooving capillovirus genome. Virology 191, 98 – 105.
.
Comparison of bioassays and laboratory assays for apple stem grooving virus M.J. Kirby, C.M. Guise, A.N. Adams * Horticulture Research International, East Malling, West Malling, Kent ME19 6BJ, UK Received 5 October 2000; received in revised form 24 January 2001; accepted 25 January 2001
Abstract The standard field double-budding assay with the indicator Virginia Crab and the glasshouse test with the indicator Malus micromalus, were compared with ELISA and immunocapture PCR for the detection of Apple stem groo6ing 6irus (ASGV) in 102 apple trees and three oriental pear. Twenty-two trees were positive for ASGV by both ELISA and IC-PCR but three of these trees were negative by Virginia Crab, three were negative by M. micromalus and one was negative by both these bioassays. The infected trees were re-tested by IC-PCR and ELISA in a second year; the IC-PCR results were confirmed but two of the 22 infected trees were negative by ELISA. On this evidence, IC-PCR is a more reliable assay for ASGV than the slow and expensive bioassays. © 2001 Elsevier Science B.V. All rights reserved. Keywords: Field assays; Glasshouse assays; ELISA; Polymerase chain reaction; IC-PCR; Apple stem grooving virus
1. Introduction Apple stem groo6ing 6irus (ASGV) is a member of the genus Capillo6irus and is widespread in rosaceous fruit trees, particularly species of Malus and Pyrus (Nemeth, 1986). Although it is latent in most commercial rootstock – scion combinations, ASGV causes a serious disease in Japan in apples growing on Malus sieboldii rootstocks (Yanase, 1983) and the virus is tested for world wide in schemes for producing pathogen-tested planting material. * Corresponding author. Tel.: + 44-1732-843833; fax: +441732-849067. E-mail address: [email protected] (A.N. Adams).
A range of bioassays and laboratory assays is available for the detection of ASGV. Bioassays include the indicator Virginia Crab either in the field for 3 years or in the glasshouse for 6 months (Anonymous, 1998), or Malus micromalus (clone GMAL 273.a) in the glasshouse for 4 weeks (Howell et al., 1996). Laboratory assays include ELISA (Fuchs, 1980) and various forms of PCR (Kinard et al., 1996; Marinho et al., 1998; James, 1999; Crossley et al., 1998). We have compared field and glasshouse bioassays with ELISA and IC-PCR for the detection of ASGV using material from 105 different cultivars of apple and oriental pear, a proportion of which were known to be infected with ASGV. The purpose of these tests was to determine whether the relatively rapid and
0166-0934/01/$ - see front matter © 2001 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 6 - 0 9 3 4 ( 0 1 ) 0 0 2 7 1 - 3
168
M.J. Kirby et al. / Journal of Virological Methods 93 (2001) 167–173
cheap laboratory assays were as effective as bioassays for detecting ASGV.
2. Materials and methods
2.1. Field bioassay M26 rootstocks were double-budded with test material and Malus pumila cv. Virginia Crab indicator buds in August 1996. Three replicate trees were budded in the field for each of the 105 sources of test material. The trees for testing were chosen at random from the variety collection at the UK National Fruit Collection, Faversham, Kent. However, 12 trees were included that were known from previous laboratory assays to be infected with ASGV. Five positive and five negative controls were included with each set of 10 tests. After 3 years the trees were cut at about 30 cm above the graft union and 5 cm below it for final symptom assessment. The shoot pieces were autoclaved and the bark removed to examine the condition of the wood and union.
2.2. Glasshouse bioassay M26 rootstocks (8-mm diameter) were planted in forestry pots, double budded with inoculum and M. micromalus indicator buds in February 2000 and retained in a glasshouse at 22°C with drip irrigation. Four replicates were budded for each of the 105 sources of inoculum and positive and negative controls were included for every 12 tests. Symptom development was recorded for 2 months after the rootstocks had been cut back to the M. micromalus bud.
2.3. ELISA Reagents were obtained from Loewe Phytodiagnostica (Sauerlach, Germany) and used according to their ‘Simultan’ double antibody sandwich protocol. Plates were coated with IgG followed by incubation with a mixture of sample and alkaline phosphatase-labelled IgG. 4-Nitrophenyl phosphate substrate was used. Negative controls (healthy apple extracts) were included in eight
wells of each ELISA plate and samples were considered positive if they reached twice the OD of the average of the controls. The test was specific for ASGV and no cross-reaction was detectable with extracts from herbaceous host plants infected with other apple and pear viruses (Apple stem pitting 6irus — ASPV, Apple chlorotic leaf spot 6irus — ACLSV, Apple mosaic 6irus — ApMV).
2.4. IC-PCR ThermoFast plates (Advanced Biotechnologies) were coated with 100 ml antiserum (Loewe) diluted 1:200 in carbonate-coating buffer. Young unfurled leaf samples were ground in phosphatebuffered saline, pH 7.4, containing 0.05% Tween 20, 2% polyvinyl pyrrolidone, 0.2% ovalbumin and the extracts diluted to 1:400. Extracts were incubated in the plates overnight at 4°C for immunocapture to occur. A two-step RT-PCR, based on Jelkmann and Kein-Konrad (1997) was carried out using primers designed to regions in the coat protein gene. Reverse transcription: 20 ml reaction containing 4 ml of 5× reaction buffer (Promega), 1.25 mM each dNTP, 35 U ribonuclease inhibitor (MBI), 20 pmoles oligo dT primer and 0.5 U AMV-RT (Promega). Reactions were overlaid with mineral oil and incubated at 37°C for 1 h. PCR: 50 ml reaction containing 3 ml of cDNA, 1× PCR buffer (Gibco BRL), 2 mM MgCl2, 0.4 mM each dNTP, 0.4 pmoles of each primer and 2 U Taq DNA polymerase (Gibco BRL). Reactions were overlaid with mineral oil and cycled in an OmniGene PCR machine (Hybaid) 95°C for 90 s followed by 35 cycles of 95°C for 30 s, 60°C for 30 s and 72°C for 60 s. Ten ml of each reaction was analysed by electrophoresis in a 1.5% agarose gel containing 0.5 mg/ml ethidium bromide. The size of the amplified fragment was determined using a 1 kb DNA ladder (Gibco BRL). Primers SGTL4 (5%-GAGAGGATTTAGGTCCCTCT-3%) and SGTL5 (5%-CTCCTAACCCTCCAGTTCCA-3%) were used initially to amplify a 664 bp region (nucleotides 5594 –6257 isolate P209 [Yoshikawa et al., 1992], accession number D14995) from the coat protein coding
M.J. Kirby et al. / Journal of Virological Methods 93 (2001) 167–173
region of 11 different ASGV isolates. The resulting PCR fragments were sequenced directly so sequence data presented is of the most common variant. Despite the high degree of similarity over this coat protein region (96.6 – 99.6% at the nucleic acid level) the European apple isolates clustered together into a cluster that is distinct from P209, the Oriental pear and citrus tatter leaf isolates (data not shown). Diagnostic primers SGTL6 (5%-AAGGTGAAAGCTTTGAAGGCA3%) and SGTL9 (5%-TCAAAAGCTTTGGGCCATTTC-3%) were designed to areas of complete nucleic acid homology between the 11 isolates sequenced although there is one mismatch in each primer with respect to P209 (Fig. 1). An ASGV diagnostic product of 424 bp is amplified by this primer pair (nucleotides 5762 – 6185 isolate P209). No product was amplified with these primers from herbaceous plants infected with other apple and pear viruses (ASPV, ACLSV and ApMV).
3. Results
3.1. Virginia Crab bioassays All negative controls had healthy unions and smooth wood under the bark. All positive controls had typical symptoms of necrosis at the graft union. Eighteen of the 105 trees tested induced necrosis at the graft union of the rootstock with the indicator scion of all three replicates. This symptom is diagnostic for ASGV. The majority of the apple trees were infected with ASPV, which induces pitting in the wood of Virginia Crab, but none of the three oriental pear trees were infected with this virus.
3.2. Malus micromalus glasshouse assay Although the budwood of M. micromalus was of much smaller diameter than the M26 root-
169
stocks there were few failures of inoculum or indicator buds. All negative controls grew strongly and the leaves were green, unblemished and without distortion. The M. micromalus bud shields of positive controls, infected either with ASGV alone or ASGV plus ASPV, ACLSV and Star Crack disease, united, and they remained alive. However, the buds either failed to grow out or grew for a short time and then became necrotic and died. There was no difference between control trees budded with pure ASGV or the multiple infection containing ASGV. These symptoms were typical of the majority of trees budded with ASGV-infected test material. Those shoots that did not die were stunted with epinasty and necrotic spotting on the leaves. Most of the material under test was infected with ASPV, based on the Virginia Crab assay, and some may also have been infected with other latent viruses. Growth of the M. micromalus budded with test material that was not infected with ASGV was clearly inferior to that of the uninfected control trees although not bearing any of the symptoms described by Howell et al., (1996) for ASGV. Budwood from 18 of the 105 trees under test induced symptoms diagnostic for ASGV; all replicates showed diagnostic symptoms except for one test in which two of the four M. micromalus were symptomless. However, different results were obtained for six trees compared with the Virginia Crab field test. Three trees that were positive in the field assay were negative by the glasshouse assay and three different trees were positive by the glasshouse assay and negative in the field. One of the four replicates that were budded with the oriental pear cv. Nijiseiki showed slight epinasty of two lower leaves and a few small necrotic spots. These symptoms were mild and they were only noticed after re-examination of the trees budded with material from sources known to be infected with ASGV. This test was scored negative.
Fig. 1. Sequence comparison of 11 ASGV isolates and the published strain P209 (accession number D14995) over the coat protein region defined by the diagnostic primer pair SGTL6 and 9. Nucleotides identical to P209 are marked with a dash, the non-identical nucleotides are written below. Alignment using Clustal (DNAstar). ASGV isolates from apple cultivars Erwin Bauer (EB); Golden Delicious (GD); Hunter Melba (HM); PJ Bergius (PJB); Maidstone Favourite (MF); Thoday’s Quarrenden (TQ); Mate Denes Dr (MDD); Tewkesbury Baron (TB) and Japanese Pear (-JP) cultivars Chojuro (CHO); Niitaka (NIIT) and Nijiseiki (NIJ).
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M.J. Kirby et al. / Journal of Virological Methods 93 (2001) 167–173
Fig. 1. (Continued)
M.J. Kirby et al. / Journal of Virological Methods 93 (2001) 167–173 Table 1 ASGV ELISA values (9 S.E.) of paired samples from six fruit buds and six vegetative buds of apple in April 1999 and May 2000 Tissue
1999
2000
Fruit buds Blossom Leaves
0.159 0.10 0.33 9 0.13
0.12 9 0.11 0.62 9 0.28
Vegetati6e buds: Unfurled leaves Opened leaves
1.129 0.24 0.54 9 0.26
0.77 9 0.36 0.36 9 0.18
171
tive buds gave the highest readings (Table 1) and these tissues were therefore used for ELISA and for IC-PCR. The ELISA and PCR results were in complete agreement in 1999 (Table 2). Twenty-two of the 105 trees sampled were positive for ASGV by both PCR and ELISA. The remaining 83 trees were negative by all assays, including the field double-budding. However, four of the 22 trees that were positive by the laboratory assays were not positive by the field tests and an additional three were negative by the M. micromalus test. In May 2000, the laboratory assays were repeated on samples from all the trees that were positive in 1999. All these trees were again positive by PCR but two of them were negative by ELISA. The primer pair SGTL6 and SGTL9 successfully amplified ASGV from a number of different
3.3. ELISA and IC-PCR Preliminary ELISAs were carried out in April 1999 and in May 2000 just prior to conducting laboratory assays on the samples from 105 trees. In both years, young unfurled leaves from vegeta-
Table 2 Origin of plant material infected with ASGV and the tests in which infection was detected (+) or not detected (−)a in assays on 102 cultivars of apple and three oriental pear from the National Fruit Collection, Faversham, UK in 1999 Cultivar and origin
PCR
ELISA
Virginia Crab
M. micromalus
Apple Climax, UK Hunter Melba, USA Hosszufalusi, Hungary Indo, UK Ingall’s Red, UK Just Gyula, Hungary Maidstone Favourite, UK Meri Cretesti, Germany Nobil de Geoagiu, Romania PJ Bergius, UK Priolov Delises, Yugoslavia Ringstad, UK Thoday’s Quarrenden, UK Calville Duquesne, France Tewkesbury Baron, UK Erwin Bauer, Germany Marie Nimitz (false), France Tewkesbury Baron, UK Alton, Netherlands
+ + + + + + + + + + + + + + + + + + +
+ + + + + + + + + + + + + + + + + + +
+ + + + + + + + + + + + + + + − − − −
+ + + + + + + + + + + + + − − + + + −
Oriental pear Chojuro, Japan Niitaka, Japan Nijiseiki, Japan
+ + +
+ + +
+ + +
+ + −
a
Samples from 83 trees were negative by all tests.
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apple cultivars from a variety of European countries and the USA (Table 2), as well from Oriental pear, which suggests that they are universally applicable.
4. Discussion The IC-PCR assay described above, based on primers designed to conserved regions from 11 isolates of ASGV, successfully amplified 22 isolates of the virus which came from plants originating over a wide geographic area (USA, Japan, and six countries in Europe) and from Pyrus and Malus. The availability of commercial antisera allows the use of immunocapture, which simplifies the assay as well as improving sensitivity (James, 1999). A one-step RT-PCR protocol, such as that used by Marinho et al. (1998), would be a further refinement, but in our hands has not been as reliable as separating RT from amplification. The disparity in detection results between ELISA and IC-PCR may reflect low virus titres. Although we used unfurled leaves in the spring, to optimise virus concentrations, others have amplified ASGV successfully from dormant bark, fruit and root tissues (Marinho et al., 1998; James, 1999). It is not clear which tissues should be sampled, and in which season, for best IC-PCR results. Bioassays are recognised internationally as being the most reliable means of testing for viruses in fruit trees (Anonymous, 1998). Most schemes for producing healthy planting material require that bioassays are used, at least for establishing the health status of the Nuclear Stock plants. Our results show that some isolates of ASGV are not detected by one or other of the two bioassays employed in this work. It is noteworthy that only 15 trees gave the same results in all four assays and that three trees that were negative by M. micromalus were positive by Virginia Crab and vice versa. James (1999) also found that two of 23 isolates, originating from Pyrus and Malus from eight different countries, were not detected by woody indexing but were detected by IC-PCR. The equivocal results of the M. micromalus assay with cv. Nijiseiki
would have necessitated a re-test and even then may not have given a clear result. Re-tests with laboratory assays are far quicker to conduct and for PCR may only require re-amplification of the cDNA taking a few hours. Virus isolates held in collections, and used to assess the effectiveness of assays, have often been found originally by woody indexing. This automatically biases any comparison of assays in favour of woody indicators. On the evidence of our tests, and those of James (1999), there is no good reason to believe that woody indicators are superior to IC-PCR for the detection of ASGV. It is essential to conduct comparisons of the type reported here with all fruit tree viruses so that the various assays now available can be utilised to their full potential.
Acknowledgements This work was part of a HortLink project funded by the Ministry of Agriculture Fisheries and Food, the East Malling Trust for Horticultural Research, the Horticultural Development Council and the Nuclear Stock Association. We are grateful to Mrs Karen Lower for excellent technical assistance and to the Brogdale Horticultural Trust, Faversham, Kent, for allowing us access to the National Fruit Collection.
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