Field observations and laboratory tests were carried out in Harrow to evaluate the sanitary status of the
Clonal Genebank collection of stone fruit. The presence of viruses and viroids was determined by ELISA, tissueprinting
hybridization and GF305 woody indexing. A total of 645 trees (197 peach and nectarine, 183 sweet and sour
cherries, 106 plum, 106 apricot, and 53 other cherries) were tested by ELISA for the presence of Plum pox virus
(PPV), Prunus necrotic ring spot virus (PNRSV) and Prune dwarf virus (PDV). No evidence of PPV infection was
found in the collection. PNRSV and PDV were frequently detected in single and mixed infections. The overall average
of virus infection rate was 20.3%. A total of 336 trees (116 peach and nectarine, 84 sweet and sour cherries, 54 plum,
44 apricot, and 38 other cherries) were tested by tissue printing hybridization for the presence of Peach latent mosaic
viroid (PLMVd) and Hop stunt viroid (HSVd). Thirty samples were infected, 28 peaches and nectarines with PLMVd
and 2 apricots with HSVd. This is the first report to date, of HSVd presence in Canada. Finally, 114 (38.4%)
out of 297 tested accessions were found infected with at least one virus and/or viroid

Michelutti, R.

Myrta, A.

Pallás, V.

R. Michelutti

A. Myrta

V. Pallás

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71Phytopathol. Mediterr. (2005) 44, 71–74IntroductionPlum pox virus (PPV) is the agent of Sharkadisease,which causes heavy losses in peach, nec-tarine, apricot, cherry, and plum around theworld. The virus was identified in Canada for thefirst time in 2000 (Thompson et al., 2001), spe-cifically in Ontario and Nova Scotia, during asurvey conducted by the Canadian Food Inspec-A preliminary account on the sanitary status of stone fruitsat the Clonal Genebank in Harrow, CanadaROBERTO MICHELUTTI1, ARBEN MYRTA2 and VICENTE PALLÁS31Greenhouse and Processing Crops Research Centre, Agriculture and Agri-Food Canada,2585 County Road 20, Harrow, Ontario N0R 1G0, Canada2Istituto Agronomico Mediterraneo, Via Ceglie 9, 70010 Valenzano, Bari, Italy 3Instituto de Biologia Molecular y Celular de Plantas, Universidad Politecnica de Valencia-CSIC,Avenida de los Naranjos s/n, 46022 Valencia, SpainSummary. Field observations and laboratory tests were carried out in Harrow to evaluate the sanitary status of theClonal Genebank collection of stone fruit. The presence of viruses and viroids was determined by ELISA, tissue-printing hybridization and GF305 woody indexing.  A total of 645 trees (197 peach and nectarine, 183 sweet and sourcherries, 106 plum, 106 apricot, and 53 other cherries) were tested by ELISA for the presence of Plum pox virus(PPV), Prunus necrotic ring spot virus (PNRSV) and Prune dwarf virus (PDV). No evidence of PPV infection wasfound in the collection. PNRSV and PDV were frequently detected in single and mixed infections. The overall averageof virus infection rate was 20.3%. A total of 336 trees (116 peach and nectarine, 84 sweet and sour cherries, 54 plum,44 apricot, and 38 other cherries) were tested by tissue printing hybridization for the presence of Peach latent mosaicviroid (PLMVd) and Hop stunt viroid (HSVd). Thirty samples were infected, 28 peaches and nectarines with PLMVdand 2 apricots with HSVd. This is the first report to date, of HSVd presence in Canada. Finally, 114 (38.4%)out of 297 tested accessions were found infected with at least one virus and/or viroid.Key words: PPV, ilarviruses, stone fruit viroids, ELISA, tissue printing hybridization.Corresponding  author: R. MicheluttiFax: +1 519 738 2929E-mail: micheluttir@agr.gc.cation Agency. The Clonal Genebank located inHarrow, Ontario, is the repository for tree fruitand small fruit species in Canada. Subsequentto the discovery and spread of PPV in Ontario, aproject was devised to screen all Genebank ma-terial for the presence of PPV by ELISA and byindexing on GF305. Furthermore all GenebankPrunus accessions were screened for two pollen-transmitted viruses, Prunus necrotic ring spotvirus (PNRSV) and Prune dwarf virus (PDV), byELISA; and two viroids, Peach latent mosaic vi-roid (PLMVd) and Hop stunt viroid (HSVd), bymolecular hybridization. In this paper are report-ed the results of this screening.SHORT NOTES72 Phytopathologia MediterraneaR. Michelutti et al.Materials and methodsClonal GenebankThe Clonal Genebank in Harrow holds approx-imately 300 accessions of the following stone fruitspecies: apricot (Prunus armeniaca L.), cherry plum(P. besseyi  P. hortulana), Chinese bush cherry(P. tomentosa Thumb.), chokecherry (P. virginianaL.), Mongolian cherry (P. fruticosa Pallas), peachand nectarine (P. persica (L.) Batsch), plum (P. do-mestica L.), sand cherry (P. besseyi Bailey), sweetcherry (P. avium L.) and sour cherry (P. cerasusL.). The Genebank holds accessions of mostly Ca-nadian origin (native, cultivars, selections and lan-draces). These are grown as trees in the open field(7-year-old orchards) and/or as potted plants un-der screen.Field observations and sample collectionField observations were carried out duringspring 2002 and 2003, for symptoms associatedwith virus and virus-like diseases. A total of 645trees (197 peach and nectarine, 183 sweet and sourcherries, 106 plum, 106 apricot, and 53 other cher-ry species) were sampled in May for ELISA test-ing. Tests were performed on young, fully-expand-ed leaves taken from the entire tree canopy in thefield and from the main branches of potted plants.In November, old leaves with petioles were sam-pled from 336 selected trees (116 peach and nec-tarine, 84 sweet and sour cherries, 54 plum, 44apricot, and 38 of other cherries) for tissue-print-ing hybridization.ELISAAll trees were tested by DAS-ELISA (Clark andAdams, 1977) for the presence of PNRSV and PDV,and by DASI-ELISA (Cambra et al., 1994) for PPV.Serological reagents for PNRSV and PDV werecommercial kits (Agdia, Elkart, Indiana, USA) andfor PPV from (Durviz, Valencia, Spain).Tissue printing and molecular hybridizationThe presence of PLMVd and HSVd was checkedby tissue-printing hybridization. Petioles of leaveswere cut and printed on a nylon membrane in trip-licate for each sample. The membranes were air-dried and submitted by mail to the analysis facili-ty (Pallás et al., 2003). The digoxigenin-labelledriboprobes used for hybridisation were obtained byT7 RNA polymerase transcription of the linearizedplasmids pHSVd and pPLMVd, described by Astrucet al. (1996) and Badenes and Llacer (2001) respec-tively. Pre-hybridisation and hybridisation werecarried out at 68°C essentially as described by Pal-lás et al. (1998).RT-PCRRT-PCR was performed as described by Astrucet al. (1996) and Ambrós et al. (1998) to confirm allpositive samples in the hybridization test.Woody indexing on GF 305Biological indexing was done for 297 accessions(one tree per accession) onto three seedlings of P.persica cv. GF 305. The indexed trees were select-ed from ELISA-negative material (where possible),in order to test for the presence of other graft-trans-missible pathogens. Indicator plants were doublechip budded with accession donors and maintainedin a temperature-controlled greenhouse at 18–22°Cfor three-four months for symptom development.ResultsField surveysAll trees and potted plants were routinely in-spected during field and greenhouse surveys.  Ingeneral, early symptoms of viruses and virus-likesymptoms are easily confounded with mild miner-al deficiencies and/or pesticide damage. Conse-quently it was difficult to identify virus and virus-like diseases by visual inspection and laboratorytesting was essential.ELISAELISA testing demonstrated that 131 (20.3%)of the tested trees were infected by at least onevirus (Table 1). A total of 23 plums, 9 peaches andnectarines, 64 sweet and sour cherries, 22 apricots,and 13 other cherry species were found to be in-fected. The infection rates of the different specieswere: plum (21.6%), peach and nectarine (4.5%),sweet and sour cherry (34.9%), apricot (20.7%), andother cherry species (24.5%). PPV was not detect-ed in any of the tested trees. The detected viruseswere PNRSV and PDV, in single and mixed infec-tions, both considered to be globally distributed(Diekmann and Putter, 1996). PNRSV was the pre-vailing virus, accounting for 74% of the infectionsin all tested trees, with the highest rate in apricot73Vol. 44, No. 1, April 2005Viruses and viroids of stone fruit genebank in Harrow, Canada(90.9%). PDV was predominant in sweet and sourcherries, and plum, representing 64 and 60% of theinfected trees respectively.Molecular hybridisation and RT-PCRThirty stone fruit samples out of 336 were in-fected by viroids (Table 2). PLMVd occurred in 28peach and nectarine samples (24.1%) of the follow-ing cultivars: Erlyvee, Harblaze, Hardired, Harko,Harbelle, Harken, Harland, Harrow Beauty, Har-row Rubirose, HW264, Redhaven, Silver Gold, Sun-cling, V68101, Vanity, Veeglo, Velvet, Vesper, VillaDoria and Vulcan. HSVd occurred in 2 apricot sam-ples (4.5%) of the cultivars Bulida and Velkopav-lovicka. These samples, determined to be positiveby tissue-printing hybridization, were also positiveby RT-PCR (data not shown).HSVd has not been previously reported in NorthAmerica (Singh et al., 2003) and this is the firstreport of its presence in the Canada. ‘Bulida’ is aSpanish cultivar which was previously shown tohave a very high infection rate (75.3%) for HSVdin Southeastern Spain (Cañizares et al., 1998). InCanada (British Columbia) PLMVd was previous-ly reported by Hadidi et al. (1997) and in Ontarioby Torres et al. (2004). Our data on PLMVd inci-dence are consistent with those obtained previouslyby Torres et al. (2004), who tested a small numberof trees of the same stone-fruit collection.Four cases of mixed infections trees with virus-es and viroids were found in nectarine: one in cv.Harblaze (PLMVd and PNRSV), two in cv. Hardired(PLMVd and PDV) and one in cv. Harko (PLMVd,PDV and PNRSV)Indexing on GF 305No viruses other than PDV were recovered fromgraft-transmission tests on GF305. PDV-infectedplants were stunted showing a striking rosette ofleaves. Only one plum accession induced abnormalproliferation of young shoots, small, leathery and pit-ted leaves. Work to identify the graft-transmissiblepathogen related to these symptoms is ongoing.DiscussionPPV was not detected in the collection, but oth-er viruses and viroids, considered to be minor path-ogens for the Prunus industry, were identified. Inthis initial study, 114 out of 297 accessions (38.4%)Table 1. Viruses detected by ELISA in Clonal Genebank Prunus accessions (Harrow, Canada).No. of trees Detected virusesSpeciesTested Infected PPV PNRSV PDV PDV+PNRSVPeach and nectarine 197  09 0  4   5   0Sweet and sour cherry 183 064 0 23 15 26Plum 106 023 0 09 10   4Apricot 106 022 0 19   2   1Other cherry species 053 013 0   9   2   2Total  (20.3%) 645 131 0 64 34 33Table 2. Viroids detected by molecular hybridisation in Clonal Genebank Prunus accessions (Harrow, Canada).No. of samples Detected viroids                   SpeciesTested Infected HSVd PLMVdPeach and nectarine 116 28 0 28Sweet and sour cherry 84 0 0 0Plum 54 0 0 0Apricot 44 2 2 0Other cherry species 38 0 0 0Total 336  30 2 2874 Phytopathologia MediterraneaR. Michelutti et al.were infected with at least one of these viruses and/or viroids (Table 3). The advent of sensitive andspecific assay methods such as ELISA and molec-ular hybridization has provided the tools to accu-rately and efficiently detect viruses and viroids forwhich detection was previously difficult. Serologi-cal assays for viruses and molecular tests for vi-roids make it feasible to survey and monitor largenumbers of specimens.  They can also be used toenhance and maintain the sanitary status of col-lections such as the Clonal Genebank.AcknowledgementsThis work was conducted as a component of theAAFC/CFIA Plum Pox Initiative. The authorsthank Dan Thompson for his PPV positive sam-ples and for RT-PCR confirmation of doubtful sam-ples; Margie Luffman for consultation regardingthe Genebank Collection; Maher Al Rwahnih, Hec-tor Torres and Gustavo Gomez for providing tech-nical support and expertise in viroid detection andLorna Woodrow for reviewing the manuscript.Literature citedAmbrós S., C. Hernández, J.C. Desvignes and R. Flores,1998. Genomic structure of three phenotypically differ-ent isolates of peach latent mosaic viroid: implicationsof the existence of constraints limiting the heterogene-ity of viroid quasi-species. Journal of Virology 72, 7397–7406.Astruc N., J.F. Marcos, G. Macquaire, T. Candresse and V.Pallás, 1996. Studies on the diagnosis of hop stunt vi-roid in fruit trees: identification of new hosts and ap-plication of a nucleic acid extraction procedure basedon non-organic solvents. European Journal of PlantPathology 102, 837–846.Badenes M.L. and G. Llácer, 2001. Occurrence of peach la-tent mosaic viroid in American peach and nectarinecultivars. Acta Horticulturae 472, 565–570.Cambra M., M. Asensio, M.T. Gorris, M.T. Perez, E. Ca-marassa, J.A. Garcia, J.J. Moya, D. Lopez-Abella andA. Sanz, 1994. Detection of plum pox potyvirus usingmonoclonal antibodies to structural and non-structur-al proteins. EPPO Bulletin 24, 569–577.Cañizares M.C., J.F. Marcos and V. Pallás, 1998. Studieson the incidence of hop stunt viroid in apricot trees (Pru-nus armeniaca) by using an easy and short extractionmethod to analyze a large number of samples. ActaHorticulturae 472, 581–585.Clark M.F. and A.N. Adams, 1977. Characteristics of themicroplate method of enzyme linked immunosorbentassay for the detection of plant viruses. Journal of Gen-eral Virology 34, 475–483.Diekmann M. and C.A.J. Putter, 1996. Stone Fruits. FAO/IPGRI Technical Guidelines for the safe Movements ofGermplasm No. 16., FAO, Rome, Italy.Hadidi A., L. Giunchedi, A.M. Shamloul, C. Poggi-Polliniand M.A. Amer, 1997. Occurrence of peach latent mo-saic viroid in stone fruits and its transmission with con-taminated blades. Plant Disease 81, 154–158.Pallás V., J.A. Sanchez-Navarro, P. Mas, M.C. Cañizares,F. Aparicio and J.F. Marcos, 1998. Molecular diagnosisof plant viruses and viroids and its possible use in stonefruits certification schemes. In: Stone Fruits Viruses andCertification in the Mediterranean Countries: Problemsand Prospects. Options Méditerranéennes, Série B/No.19. (B. Di Terlizzi, A. Myrta, V. Savino, ed.), Bari, Italy,191–208.Pallás V., H. Torres, A. Myrta and G. Gomez, 2003. Valida-tion of the ‘tissue-printing’ technique for detecting stonefruit viroids. In: Virus and Virus-like Diseases of StoneFruits, with Particular Reference to the MediterraneanRegion. Options Méditerranéennes, Série. B/No. 45 (A.Myrta, B. Di Terlizzi, V. Savino, ed.), Bari, Italy, 135–137.Singh R.P., K.F.M. Ready and A. Hadidi, 2003. Viroids inNorth America and global distribution of viroid diseas-es. In: Viroids. (A. Hadidi, R. Flores, J.W. Randles, Se-mancik, ed.), CSIRO Publishing, Collingwood, Austral-ia, 255–264.Thompson D., M. McCann, M. MacLeod, D. Lye, M. Greenand D. James, 2001. First report of Plum pox potyvirusin Ontario, Canada. Plant Disease 85, 97 (abstract).Torres H., G. Gomez, B. Stamo, A. Shalaby, B. Aouane, I.Gavriel, P. Kominek, K. Caglayan, M. Sipahioglu,  R.Michelutti, A. Myrta, N. Fiore and V. Pallás, 2004. De-tection by tissue printing of stone fruit viroids, fromEurope, the Mediterranean and North and South Ameri-ca. Acta Horticulturae 657, 379–383.Accepted for publication: February 11, 2005Table 3. Sanitary status of Clonal Genebank Prunusaccessions (Harrow, Canada) as determined by ELISAand molecular hybridizationNo. of accessions Infection            Species  rateTested Infected (%)Peach and nectarine 81 26 32.1Sweet and sour cherry 83 33 39.8Plum 56 21 37.5Apricot 41 19 46.3Other cherry species 36 15 41.7Total 297 114 38.4

A Preliminary Account on the Sanitary Status of Stone Fruits at the Clonal Genebank in Harrow, Canada

Firenze University Press: E-Journals

71
Phytopathol. Mediterr. (2005) 44, 71–74
Introduction
Plum pox virus (PPV) is the agent of Sharka
disease,which causes heavy losses in peach, nec-
tarine, apricot, cherry, and plum around the
world. The virus was identified in Canada for the
first time in 2000 (Thompson et al., 2001), spe-
cifically in Ontario and Nova Scotia, during a
survey conducted by the Canadian Food Inspec-
A preliminary account on the sanitary status of stone fruits
at the Clonal Genebank in Harrow, Canada
ROBERTO MICHELUTTI1, ARBEN MYRTA2 and VICENTE PALLÁS3
1Greenhouse and Processing Crops Research Centre, Agriculture and Agri-Food Canada,
2585 County Road 20, Harrow, Ontario N0R 1G0, Canada
2Istituto Agronomico Mediterraneo, Via Ceglie 9, 70010 Valenzano, Bari, Italy
 3Instituto de Biologia Molecular y Celular de Plantas, Universidad Politecnica de Valencia-CSIC,
Avenida de los Naranjos s/n, 46022 Valencia, Spain
Summary. Field observations and laboratory tests were carried out in Harrow to evaluate the sanitary status of the
Clonal Genebank collection of stone fruit. The presence of viruses and viroids was determined by ELISA, tissue-
printing hybridization and GF305 woody indexing.  A total of 645 trees (197 peach and nectarine, 183 sweet and sour
cherries, 106 plum, 106 apricot, and 53 other cherries) were tested by ELISA for the presence of Plum pox virus
(PPV), Prunus necrotic ring spot virus (PNRSV) and Prune dwarf virus (PDV). No evidence of PPV infection was
found in the collection. PNRSV and PDV were frequently detected in single and mixed infections. The overall average
of virus infection rate was 20.3%. A total of 336 trees (116 peach and nectarine, 84 sweet and sour cherries, 54 plum,
44 apricot, and 38 other cherries) were tested by tissue printing hybridization for the presence of Peach latent mosaic
viroid (PLMVd) and Hop stunt viroid (HSVd). Thirty samples were infected, 28 peaches and nectarines with PLMVd
and 2 apricots with HSVd. This is the first report to date, of HSVd presence in Canada. Finally, 114 (38.4%)
out of 297 tested accessions were found infected with at least one virus and/or viroid.
Key words: PPV, ilarviruses, stone fruit viroids, ELISA, tissue printing hybridization.
Corresponding  author: R. Michelutti
Fax: +1 519 738 2929
E-mail: micheluttir@agr.gc.ca
tion Agency. The Clonal Genebank located in
Harrow, Ontario, is the repository for tree fruit
and small fruit species in Canada. Subsequent
to the discovery and spread of PPV in Ontario, a
project was devised to screen all Genebank ma-
terial for the presence of PPV by ELISA and by
indexing on GF305. Furthermore all Genebank
Prunus accessions were screened for two pollen-
transmitted viruses, Prunus necrotic ring spot
virus (PNRSV) and Prune dwarf virus (PDV), by
ELISA; and two viroids, Peach latent mosaic vi-
roid (PLMVd) and Hop stunt viroid (HSVd), by
molecular hybridization. In this paper are report-
ed the results of this screening.
SHORT NOTES
72 Phytopathologia Mediterranea
R. Michelutti et al.
Materials and methods
Clonal Genebank
The Clonal Genebank in Harrow holds approx-
imately 300 accessions of the following stone fruit
species: apricot (Prunus armeniaca L.), cherry plum
(P. besseyi  P. hortulana), Chinese bush cherry
(P. tomentosa Thumb.), chokecherry (P. virginiana
L.), Mongolian cherry (P. fruticosa Pallas), peach
and nectarine (P. persica (L.) Batsch), plum (P. do-
mestica L.), sand cherry (P. besseyi Bailey), sweet
cherry (P. avium L.) and sour cherry (P. cerasus
L.). The Genebank holds accessions of mostly Ca-
nadian origin (native, cultivars, selections and lan-
draces). These are grown as trees in the open field
(7-year-old orchards) and/or as potted plants un-
der screen.
Field observations and sample collection
Field observations were carried out during
spring 2002 and 2003, for symptoms associated
with virus and virus-like diseases. A total of 645
trees (197 peach and nectarine, 183 sweet and sour
cherries, 106 plum, 106 apricot, and 53 other cher-
ry species) were sampled in May for ELISA test-
ing. Tests were performed on young, fully-expand-
ed leaves taken from the entire tree canopy in the
field and from the main branches of potted plants.
In November, old leaves with petioles were sam-
pled from 336 selected trees (116 peach and nec-
tarine, 84 sweet and sour cherries, 54 plum, 44
apricot, and 38 of other cherries) for tissue-print-
ing hybridization.
ELISA
All trees were tested by DAS-ELISA (Clark and
Adams, 1977) for the presence of PNRSV and PDV,
and by DASI-ELISA (Cambra et al., 1994) for PPV.
Serological reagents for PNRSV and PDV were
commercial kits (Agdia, Elkart, Indiana, USA) and
for PPV from (Durviz, Valencia, Spain).
Tissue printing and molecular hybridization
The presence of PLMVd and HSVd was checked
by tissue-printing hybridization. Petioles of leaves
were cut and printed on a nylon membrane in trip-
licate for each sample. The membranes were air-
dried and submitted by mail to the analysis facili-
ty (Pallás et al., 2003). The digoxigenin-labelled
riboprobes used for hybridisation were obtained by
T7 RNA polymerase transcription of the linearized
plasmids pHSVd and pPLMVd, described by Astruc
et al. (1996) and Badenes and Llacer (2001) respec-
tively. Pre-hybridisation and hybridisation were
carried out at 68°C essentially as described by Pal-
lás et al. (1998).
RT-PCR
RT-PCR was performed as described by Astruc
et al. (1996) and Ambrós et al. (1998) to confirm all
positive samples in the hybridization test.
Woody indexing on GF 305
Biological indexing was done for 297 accessions
(one tree per accession) onto three seedlings of P.
persica cv. GF 305. The indexed trees were select-
ed from ELISA-negative material (where possible),
in order to test for the presence of other graft-trans-
missible pathogens. Indicator plants were double
chip budded with accession donors and maintained
in a temperature-controlled greenhouse at 18–22°C
for three-four months for symptom development.
Results
Field surveys
All trees and potted plants were routinely in-
spected during field and greenhouse surveys.  In
general, early symptoms of viruses and virus-like
symptoms are easily confounded with mild miner-
al deficiencies and/or pesticide damage. Conse-
quently it was difficult to identify virus and virus-
like diseases by visual inspection and laboratory
testing was essential.
ELISA
ELISA testing demonstrated that 131 (20.3%)
of the tested trees were infected by at least one
virus (Table 1). A total of 23 plums, 9 peaches and
nectarines, 64 sweet and sour cherries, 22 apricots,
and 13 other cherry species were found to be in-
fected. The infection rates of the different species
were: plum (21.6%), peach and nectarine (4.5%),
sweet and sour cherry (34.9%), apricot (20.7%), and
other cherry species (24.5%). PPV was not detect-
ed in any of the tested trees. The detected viruses
were PNRSV and PDV, in single and mixed infec-
tions, both considered to be globally distributed
(Diekmann and Putter, 1996). PNRSV was the pre-
vailing virus, accounting for 74% of the infections
in all tested trees, with the highest rate in apricot
73Vol. 44, No. 1, April 2005
Viruses and viroids of stone fruit genebank in Harrow, Canada
(90.9%). PDV was predominant in sweet and sour
cherries, and plum, representing 64 and 60% of the
infected trees respectively.
Molecular hybridisation and RT-PCR
Thirty stone fruit samples out of 336 were in-
fected by viroids (Table 2). PLMVd occurred in 28
peach and nectarine samples (24.1%) of the follow-
ing cultivars: Erlyvee, Harblaze, Hardired, Harko,
Harbelle, Harken, Harland, Harrow Beauty, Har-
row Rubirose, HW264, Redhaven, Silver Gold, Sun-
cling, V68101, Vanity, Veeglo, Velvet, Vesper, Villa
Doria and Vulcan. HSVd occurred in 2 apricot sam-
ples (4.5%) of the cultivars Bulida and Velkopav-
lovicka. These samples, determined to be positive
by tissue-printing hybridization, were also positive
by RT-PCR (data not shown).
HSVd has not been previously reported in North
America (Singh et al., 2003) and this is the first
report of its presence in the Canada. ‘Bulida’ is a
Spanish cultivar which was previously shown to
have a very high infection rate (75.3%) for HSVd
in Southeastern Spain (Cañizares et al., 1998). In
Canada (British Columbia) PLMVd was previous-
ly reported by Hadidi et al. (1997) and in Ontario
by Torres et al. (2004). Our data on PLMVd inci-
dence are consistent with those obtained previously
by Torres et al. (2004), who tested a small number
of trees of the same stone-fruit collection.
Four cases of mixed infections trees with virus-
es and viroids were found in nectarine: one in cv.
Harblaze (PLMVd and PNRSV), two in cv. Hardired
(PLMVd and PDV) and one in cv. Harko (PLMVd,
PDV and PNRSV)
Indexing on GF 305
No viruses other than PDV were recovered from
graft-transmission tests on GF305. PDV-infected
plants were stunted showing a striking rosette of
leaves. Only one plum accession induced abnormal
proliferation of young shoots, small, leathery and pit-
ted leaves. Work to identify the graft-transmissible
pathogen related to these symptoms is ongoing.
Discussion
PPV was not detected in the collection, but oth-
er viruses and viroids, considered to be minor path-
ogens for the Prunus industry, were identified. In
this initial study, 114 out of 297 accessions (38.4%)
Table 1. Viruses detected by ELISA in Clonal Genebank Prunus accessions (Harrow, Canada).
No. of trees Detected viruses
Species
Tested Infected PPV PNRSV PDV PDV+PNRSV
Peach and nectarine 197  09 0  4   5   0
Sweet and sour cherry 183 064 0 23 15 26
Plum 106 023 0 09 10   4
Apricot 106 022 0 19   2   1
Other cherry species 053 013 0   9   2   2
Total  (20.3%) 645 131 0 64 34 33
Table 2. Viroids detected by molecular hybridisation in Clonal Genebank Prunus accessions (Harrow, Canada).
No. of samples Detected viroids
                   Species
Tested Infected HSVd PLMVd
Peach and nectarine 116 28 0 28
Sweet and sour cherry 84 0 0 0
Plum 54 0 0 0
Apricot 44 2 2 0
Other cherry species 38 0 0 0
Total 336  30 2 28
74 Phytopathologia Mediterranea
R. Michelutti et al.
were infected with at least one of these viruses and/
or viroids (Table 3). The advent of sensitive and
specific assay methods such as ELISA and molec-
ular hybridization has provided the tools to accu-
rately and efficiently detect viruses and viroids for
which detection was previously difficult. Serologi-
cal assays for viruses and molecular tests for vi-
roids make it feasible to survey and monitor large
numbers of specimens.  They can also be used to
enhance and maintain the sanitary status of col-
lections such as the Clonal Genebank.
Acknowledgements
This work was conducted as a component of the
AAFC/CFIA Plum Pox Initiative. The authors
thank Dan Thompson for his PPV positive sam-
ples and for RT-PCR confirmation of doubtful sam-
ples; Margie Luffman for consultation regarding
the Genebank Collection; Maher Al Rwahnih, Hec-
tor Torres and Gustavo Gomez for providing tech-
nical support and expertise in viroid detection and
Lorna Woodrow for reviewing the manuscript.
Literature cited
Ambrós S., C. Hernández, J.C. Desvignes and R. Flores,
1998. Genomic structure of three phenotypically differ-
ent isolates of peach latent mosaic viroid: implications
of the existence of constraints limiting the heterogene-
ity of viroid quasi-species. Journal of Virology 72, 7397–
7406.
Astruc N., J.F. Marcos, G. Macquaire, T. Candresse and V.
Pallás, 1996. Studies on the diagnosis of hop stunt vi-
roid in fruit trees: identification of new hosts and ap-
plication of a nucleic acid extraction procedure based
on non-organic solvents. European Journal of Plant
Pathology 102, 837–846.
Badenes M.L. and G. Llácer, 2001. Occurrence of peach la-
tent mosaic viroid in American peach and nectarine
cultivars. Acta Horticulturae 472, 565–570.
Cambra M., M. Asensio, M.T. Gorris, M.T. Perez, E. Ca-
marassa, J.A. Garcia, J.J. Moya, D. Lopez-Abella and
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Accepted for publication: February 11, 2005
Table 3. Sanitary status of Clonal Genebank Prunus
accessions (Harrow, Canada) as determined by ELISA
and molecular hybridization
No. of accessions Infection
            Species  rate
Tested Infected (%)
Peach and nectarine 81 26 32.1
Sweet and sour cherry 83 33 39.8
Plum 56 21 37.5
Apricot 41 19 46.3
Other cherry species 36 15 41.7
Total 297 114 38.4


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A Preliminary Account on the Sanitary Status of Stone Fruits at the Clonal Genebank in Harrow, Canada

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