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Introduction

  1. Top of page
  2. Specific scope
  3. Specific approval and amendment
  4. Introduction
  5. Identity
  6. Detection
  7. Identification
  8. Conclusions
  9. Reporting and documentation
  10. Further information
  11. Acknowledgements
  12. References
  13. Appendices

Apple is the main host of Phytoplasma mali (EPPO/CABI, 1996). Cultivars vary in reaction but most, including seedlings, appear to be susceptible. The disease can be observed either on cultivars or on rootstocks, as well as on wild and ornamental Malus. It is commonly found in young orchards and nurseries.

Identity

  1. Top of page
  2. Specific scope
  3. Specific approval and amendment
  4. Introduction
  5. Identity
  6. Detection
  7. Identification
  8. Conclusions
  9. Reporting and documentation
  10. Further information
  11. Acknowledgements
  12. References
  13. Appendices

Name: Candidatus Phytoplasma mali.

Synonyms: Apple proliferation phytoplasma.

Taxonomic position: Bacteria, Firmicutes, Mollicutes, Acholeplasmatales, Acholeplasmataceae. P. mali is in the 16SrX phytoplasma group. It was proposed as a ‘Candidatus’ by Seemüller & Schneider (2004).

EPPO code: PHYPMA.

Phytosanitary categorization: EPPO A2 list no. 87; EU Annex designation I/A2.

Detection

  1. Top of page
  2. Specific scope
  3. Specific approval and amendment
  4. Introduction
  5. Identity
  6. Detection
  7. Identification
  8. Conclusions
  9. Reporting and documentation
  10. Further information
  11. Acknowledgements
  12. References
  13. Appendices

Disease symptoms

The most typical symptom caused by P. mali is witches’ broom at the end of shoots (Web Fig. 1). On diseased trees, leaves are generally smaller and more dented, with unusually enlarged stipules (Web Fig. 2). Fruits are smaller and flattened, and peduncles longer (Web Fig. 3). Early leaf reddening is a good indication of the presence of the disease. The presence of a fine hairy root system on nursery plants during winter may be another indication.

The distribution of P. mali in the tree is uneven and is not constant over the year. It may vary from one year to the next, as in some years symptoms may not be observed. The distribution pattern in the tree is also dependent on temperature. In winter, the content of P. mali in the tree declines due to sieve-tube degeneration, and the phytoplasma concentrates more in the roots. During spring, at the beginning of sap flow (e.g. April to May), P. mali reinvades the stem and canopy from the roots and reaches a peak in late summer or early autumn. P. mali is detected in phloem tissues in shoots from mid summer to the end of sap flow. However, as temperature may influence concentration of the pest, detection may be unreliable at both low and too high temperatures. Detection on roots is possible throughout the year, although uneven distribution also applies here (Schaper & Seemüller, 1982; Seemüller et al., 1984).

Identification

  1. Top of page
  2. Specific scope
  3. Specific approval and amendment
  4. Introduction
  5. Identity
  6. Detection
  7. Identification
  8. Conclusions
  9. Reporting and documentation
  10. Further information
  11. Acknowledgements
  12. References
  13. Appendices

Since phytoplasmas cannot be readily cultivated and purified, only inoculation to woody indicators was used until recently for detection on fruit trees. However, this method is time-consuming and costly. Genetic amplification methods developed by several teams have greatly improved the situation and have proved rapid, sensitive and less costly. At the end of the growing period, phytoplasmas move from the apical part of the plant to roots where they overwinter. If analyses have to be performed in winter time, roots should be sampled. Samples of fresh plant material should not be stored at −20°C for no longer than 6 months.

Woody indicators

Tests can be done in the field or in the glasshouse. In the glasshouse, test plants are grafted (by chip-budding or grafting of stem or root cuttings), onto the indicator apple Malus ×domestica cv. ‘Charden’, ‘Golden Delicious’ or ‘Rode Schone van Boskoop’. This cultivar can express symptoms after a period of dormancy. Following in vitro propagation and growth in pots of the indicators, the test is carried out in spring on actively growing plants. 2 chips per indicator and 5 replicates are used. When the graft union is formed (usually after 2 weeks), the plants are put in a cold room at 5°C ± 4°C for 60–70 days of forced dormancy. Plants are then moved back to the glasshouse. Plants are pruned, leaving 1 or 2 buds above the graft, in order to concentrate P. mali in the young and vigorous shoots after regrowth. One or two months after pruning, the first symptoms may be observed (enlarged stipules, presence of witches’ brooms). Total duration of the test is about 4 months. The success rate of this method is variable, depending on test conditions, but it may reach 80%. In the field, indicators are inoculated at the end of summer or in autumn. The same indicators are used with 5 replicates and at least 2 years of observation.

DAPI staining

Thin sections of young tissues (petioles of young leaves, or phloem tissues of shoots, branches and roots), are stained with 1 µg/mL DAPI solution (4′6 diamidino-2-phenylindole). Sections are observed under a fluorescence microscope. A bluish fluorescence (at 460 nm) in the sieve tubes indicates the presence of phytoplasmas (Seemüller, 1976). This method, previously the only one available, requires good experience of observing slides and is not always sufficiently sensitive. The advantages of this method include rapidity and low cost, but it is not specific. For each sample, sections of young tissues should be taken from different parts of the plant, because of the uneven distribution of the phytoplasma.

ELISA

The availability of monoclonal antibodies specific for P. mali (Loi et al., 2002) allows direct ELISA to be used, which is particularly useful when a large number of samples has to be checked. The most reliable results can be obtained when leaf midribs or stems collected from late spring to end of summer (June – end of September) are tested. Leaf samples should be collected randomly all around the plant, because of the uneven distribution of P. mali in the foliage. ELISA should be performed according to manufacturer's instructions (Bioreba). In cooler climates and in case of latent infections, in Northern and Western Europe, ELISA may not be sensitive enough to detect the relatively low concentrations, so testing may be unreliable.

Molecular techniques

Molecular techniques which are both sensitive and specific are available. DNA is extracted from P. mali following Ahrens & Seemüller (1992) or a simplified version (e.g. Ministère de l’Agriculture, 2005), using apple shoots or roots, and the extract is amplified by PCR. Different types of universal primers are able to amplify phytoplasma DNA extracted from phloem. The most frequently used are the ones described by Lorenz et al. (1995) and Lee et al. (1998). Both are able to amplify a product by PCR from any phytoplasma, including P. mali.

If universal primers fU5/rU3 (Lorenz et al., 1995) or R16F2n/R16R2 (Lee et al., 1998) are used, the amplification product may be digested by restriction enzyme AluI to ensure that the phytoplasma belongs to group AP (Seemüller et al., 1998) or to group 16SrX (subgroup A) (Lee et al., 1998).

If AP-or 16SrX-group specific primers fO1/rO1 (Lorenz et al., 1995) are used, the amplification product may be digested by the restriction enzymes SspI and SfeI (Lorenz et al., 1995) for differentiation of P. mali from P. pyri and P. prunorum.

If a set of P. mali-specific primers AP5/4 (Jarausch et al., 1994, 1995) is used following the same protocol, then RFLP analysis is not required. However, with these specific primers, the test has a reduced sensitivity and some isolates may not be detected.

DNA extraction for PCR

Shoots, roots, leaves or petioles may be used. Shoots and roots are debarked, and a sample of phloem is removed with a sterile blade. For leaves, only the midrib is taken. Any effective grinding method may be used (Appendix I). The best results are obtained if DNA is extracted from leaf midribs or stems collected from late spring to end of summer (June – end of September).

For PCR, it is advisable to include a phytoplasma enrichment procedure as described in Appendix 1, because low concentrations may escape detection. The conditions for PCR amplification are given in Appendix 2. Electrophoresis is done on a 1% agarose gel, under stable 90 V, in TBE buffer. A sample is positive if a band appears at the level of the expected number of base pairs.

Characterization of the phytoplasma using RFLP

Amplified DNA (10 µL) is added to 10 µL of digestion solution (sterile distilled water, enzyme-specific buffer 1× , 2 units enzyme and amplified DNA). The proportion (10 + 10) may vary depending on the concentration of the amplicons. The mixture is incubated for at least 2 h at 37°C, and then subjected to electrophoresis on 2% agarose gel. Further details on RFLP analysis are given in Appendix 2.

Conclusions

  1. Top of page
  2. Specific scope
  3. Specific approval and amendment
  4. Introduction
  5. Identity
  6. Detection
  7. Identification
  8. Conclusions
  9. Reporting and documentation
  10. Further information
  11. Acknowledgements
  12. References
  13. Appendices

The presence of clear symptoms (witches’ broom and enlarged stipules) gives strong presumptive evidence of identification. Laboratory confirmation can be obtained by PCR (or ELISA). This may be followed by RFLP or PCR using a different set of primers if additional confirmation is required (e.g. in the absence of symptoms). A test on an indicator plant may be performed if necessary (e.g. in case of dispute).

Further information

  1. Top of page
  2. Specific scope
  3. Specific approval and amendment
  4. Introduction
  5. Identity
  6. Detection
  7. Identification
  8. Conclusions
  9. Reporting and documentation
  10. Further information
  11. Acknowledgements
  12. References
  13. Appendices

Further information on this organism can be obtained from:

Service de la Protection des Végétaux, Unité de virologie des ligneux, BP 81, 33883 Villenave d’Ornon Cedex (France)

Laboratoire de biologie cellulaire et moléculaire, INRA–UMR 1090, BP81, 33883 Villenave d’Ornon Cedex (France)

B. Schneider, Biologische Bundesanstalt für Land- und Forstwirtschaft, Institut für Pflanzenschutz im Obstbau, Schwabenheimer Str. 101, D-69221 Dossenheim (Germany)

A. Bertaccini, UCI-STAA Patologia vegetale, Universita di Bologna, Italy

N. Loi, Università di Udine, Dipartimento di Biologia Applicata alla Difesa delle Piante, Udine (Italy)

W. Jarausch, Institut für molekulare und angewandte Pflanzenforschung Rheinland-Pfalz, RLP AgroScience GmbH, Breitenweg 71, 67435 Neustadt-an-der-Weinstrasse (Germany).

Footnotes
  • 1

    The figures in this Standard marked ‘Web Fig.’ are published on the EPPO website http://www.eppo.org.

Acknowledgements

  1. Top of page
  2. Specific scope
  3. Specific approval and amendment
  4. Introduction
  5. Identity
  6. Detection
  7. Identification
  8. Conclusions
  9. Reporting and documentation
  10. Further information
  11. Acknowledgements
  12. References
  13. Appendices

This protocol was originally drafted for EPPO by F. Costard, Ministère de l’Agriculture, Service de la Protection des Végétaux, Unité de virologie des ligneux, Villenave d’Ornon (France).

References

  1. Top of page
  2. Specific scope
  3. Specific approval and amendment
  4. Introduction
  5. Identity
  6. Detection
  7. Identification
  8. Conclusions
  9. Reporting and documentation
  10. Further information
  11. Acknowledgements
  12. References
  13. Appendices
  • Ahrens U & Seemüller E (1992) Detection of DNA of plant pathogenic mycoplasma-like organisms by a polymerase chain reaction that amplifies a sequence of the 16S RNA gene. Phytopathology 82, 828832.
  • EPPO/CABI (1996) Apple proliferation phytoplasma. In: Quarantine Pests for Europe, 2nd edn, pp. 959962. CAB International, Wallingford (GB).
  • Jarausch W, Saillard C, Dosba F & Bové JM (1994) Differentiation of mycoplasma-like organims (MLOs) in European fruit trees by PCR using specific primers derived from the sequence of a chromosomal fragment of apple proliferation, MLO. Applied and Environmental Microbiology 60, 29162923.
  • Jarausch W, Saillard C, Dosba F & Bové JM (1995) Specific detection of mycoplasma-like organisms in European fruit trees by PCR. Bulletin OEPP/EPPO Bulletin 25, 219225.
  • Lee IM, Gundersen-Rindal DE, Davis RE & Bartoszyk IM (1998) Revised classification scheme of phytoplasmas based on RFLP analyses of 16S rRNA and ribosomal protein gene sequences. International Journal of Systematic Bacteriology 48, 11531169.
  • Lim PO & Sears BB (1989) 16S rDNA sequence indicates that plant-pathogenic mycoplasma-like organisms are evolutionarily distinct from animal mycoplasmas. Journal of Bacteriology 171, 59015906.
  • Loi N, Ermacora P, Carraro L, Osler R & Chen TA (2002) Production of monoclonal antibodies against apple proliferation phytoplasma and their use in serological detection. European Journal of Plant Pathology 108, 8186.
  • Lorenz KH, Schneider B, Ahrens U & Seemüller E (1995) Detection of the apple proliferation and pear decline phytoplasmas by PCR amplification of ribosomal and non ribosomal DNA. Phytopathology 85, 771776.
  • Menzel W, Jelkman W & Mais E (2002) Detection of four apple viruses by multiplex RT-PCR assays with coamplification of plant mRNA as internal control. Journal of Virological Methods 99, 8192.
  • Ministère de l’Agriculture (2005) Méthode VL/05/12 version a: détection de l’enroulement chlorotique de l’abricotier, de la prolifération du pommier et du dépérissement du poirier sur rameaux et racines par la technique d’amplification génique PCR (Polymerase Chain Reaction). Journal Officiel de la République Française, 2005-06-02.
  • Sambrook J, Fritsch EF & Maniatis T (1989) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor (US).
  • Schaper U & Seemüller E (1982) Condition of the phloem and the persistence of mycoplasma-like organisms associated with apple proliferation and pear decline. Phytopathology 72, 736742.
  • Schneider B, Seemüller E, Smart CD & Kirkpatrick BC (1995) Phylogenetic classification of plant pathogenic mycoplasma-like organisms or phytoplasmas. In: Molecular and Diagnostic Procedures in Mycoplasmology (Ed. Razin, S), pp. 369380. Academic Press, San Diego (US).
  • Seemüller E (1976) Investigations to demonstrate mycoplasma-like organisms in diseased plants by fluorescence microscopy. Acta Horticulturae 67, 109111.
  • Seemüller E, Marcone C, Lauer U, Ragozzino A & Göschl M (1998) Current status of molecular classification of the phytoplasmas. Journal of Plant Pathology 80, 326.
  • Seemüller E, Schaper U & Zimbelmann R (1984) Seasonal variation in the colonization patterns of mycoplasma-like organisms associated with apple proliferation and pear decline. Zeitschrift für Pflanzenkrankenheiten und Pflanzenschutz 91, 371382.
  • Seemüller E & Schneider B (2004) Candidatus Phytoplasma mali, Candidatus Phytoplasma pyri and Candidatus Phytoplasma prunorum, the causal agents of apple proliferation, pear decline and European stone fruit yellows, respectively. International Journal of Systematic and Evolutionary Microbiology 54, 12171226.

Appendices

  1. Top of page
  2. Specific scope
  3. Specific approval and amendment
  4. Introduction
  5. Identity
  6. Detection
  7. Identification
  8. Conclusions
  9. Reporting and documentation
  10. Further information
  11. Acknowledgements
  12. References
  13. Appendices

Appendix I DNA extraction

DNA extraction

The extraction buffer contains: 2% CTAB (soluble at approximately 50°C), 1.4 m NaCl, 20 mm EDTA, 100 mm Tris-HCl pH 8. Small rigid plastic bags containing the sample and extraction buffer (5 mL for 1 g of phloem) are triturated using a ball mill. Transfer 1–2 mL extract to 2 mL microtubes. Incubate microtubes, if possible with shaking, for at least 30 min at 65°C, then centrifuge at 2000 g for 2 min in a microcentrifuge. Put 1 mL of supernatant in a microtube and add 1 mL of chloroform-octanol solution (24 : 1). Mix the two phases to obtain an emulsion. Centrifuge at 13 000 g for 5 min. Transfer the supernatant to a new microtube, and add approximately 800 µL of cold isopropanol. Mix and centrifuge at 15000 g for 10 min. Remove the supernatant. Add 500 µL of 70% ethanol and centrifuge at 15 000 g for 5 min. Empty microtubes and dry residues. Add 100 µL of distilled water and shake with a vortex to help dissolution.

Alternatively, commercial kits (e.g. DNeasy, Qiagen) or other described methods, e.g. extraction with a silica-suspension as described by Menzel et al. (2002), can be used for DNA extraction. The extracts can be stored in a deep-freeze (−80°C) for a year.

Phytoplasma enrichment procedure

The extraction buffer contains (per 100 mL): K2HPO4 anhydrous 1.67 g; KH2PO4 0.41 g; sucrose 10 g; bovine serum albumen (frac V) 0.15 g; polyvinylpyrrolidone P.M. 10.000 2 g; ascorbic acid 0.53 g, adjusted to pH 7.6 with drops of KOH. Grind 1.5 g of fresh midveins in a sterile cold mortar and pestle with 7–8 mL of freshly prepared buffer and 50 mg of sterile quartz sand (Sigma, cod. S9887). Incubate 10–15 min in ice. Add another 5 mL of the same buffer and homogenize thoroughly. Transfer into 15 mL tubes (Corex) and centrifuge in a precooled rotor (Beckman JA 20) at 5000 rev min−1 for 5 min (4°C). Transfer the supernatant to a cold clean Corex tube. Centrifuge at 19 000 rev min−1 for 20 min (Beckman JA 20). Dry the pellet and re-suspend with 2 mL of 2% CTAB buffer, prewarmed at 60°C. Incubate 10–20 min at 60°C with gentle agitation. Transfer 1 mL to a clean 2 mL microtube and proceed with the DNA extraction procedure described above, as from the chloroform-octanol extraction.

Appendix II PCR amplification and RFLP analysis

Universal PCR (Lorenz et al., 1995)

The forward and reverse PCR primers are fU5 (5′-CGG CAA TGG AGG AAA CT-3′) and rU3 (5′-TTC AGC TAC TCT TTG TAA CA-3′), respectively, amplifying a 876-bp fragment of 16S rDNA. Primers fU5 and rU3 correspond to nucleotides 369-386 and 1251-1231 of the 16S rDNA sequence of aster yellows strain OAY (Oenothera hookeri MLO, Lim & Sears, 1989).

PCR

The 40 µL reaction mixture is composed as follows: 0.5 µm of each primer, 100 µm dNTPs, 0.2 Units of Taq DNA polymerase (e.g. Goldstar polymerase, Eurogentec) and 5 µL DNA extract in the reaction buffer supplied by the manufacturer of the Taq DNA polymerase. The PCR is performed in 0.2 mL reaction tubes in a thermocycler (e.g. GeneAmp PCR system 9600, Applied Biosystems) with the following parameters: 2 min at 94°C, 40 cycles of 20 s at 94°C, 20 s at 55°C, and 1 min at 72°C, followed by a final extension for 4 min at 72°C, and cooled to 4°C. After amplification, 10 µL of the PCR products is subjected to electrophoresis on 1% agarose gel under stable 90 V, in TBE buffer according to standard procedures (Sambrook et al., 1989) along with a DNA ladder (e.g. 100 bp-ladder, Fermentas) to size fragments. PCR products are viewed and photographed under UV light.

RFLP analysis

PCR products are analysed by digestion with restriction enzyme AluI. Each 20-µL reaction mixture is composed as follows: 2 Units restriction enzyme and 10 µL PCR product in the reaction buffer supplied by the manufacturer of the enzyme. Reactions are incubated at 37°C for at least 2 h. Digested PCR products are subjected to electrophoresis on 2% agarose gel along with a DNA ladder (e.g. 100 bp-ladder, Fermentas) to size fragments. PCR products are viewed and photographed under UV light.

Interpretation of band patterns

The phytoplasma is identified as a member of the AP or 16SrX group if the PCR product is digested with AluI to give 476, 189, 149 and 56 bp fragments.

Universal PCR (Lee et al., 1998)

The forward and reverse PCR primers are R16F2n (5′-GAA ACG ACT GCT AAG ACT GG-3′) and R16R2 (5′-TGA CGG GCG GTG TGT ACA AAC CCC g-3′), respectively, amplifying a 1239-bp fragment of 16S rDNA.

PCR

The 40-µL reaction mixture is composed as follows: 0.4 µm of each primer, 200 µm dNTPs, 1 Unit of Taq DNA polymerase (e.g. AmpliTaq, Applied Biosystems) and 5 µL DNA extract in the reaction buffer supplied by the manufacturer of the Taq DNA polymerase. PCR parameters for a DNA Thermal Cycler 480 (Applied Biosystems) are: 2 min at 94°C, 35 cycles of 1 min at 94°C, 2 min at 60°C, and 3 min at 72°C, followed by a final extension for 10 min at 72°C and cooled to 4°C. When using a faster thermocycler (e.g. GeneAmp PCR System 9600/9700, Applied Biosystems), cycle times should be decreased. After amplification, 5–10 µL of the PCR products is subjected to electrophoresis on 1% agarose gel under stable 90 V, in TBE buffer according to standard procedures (Sambrook et al., 1989) along with a DNA ladder (e.g. 100 bp-ladder, Fermentas) to size fragments. PCR products are viewed and photographed under UV light.

RFLP analysis

As above.

Interpretation of band patterns

The phytoplasma is identified as a member of the AP or 16SrX group if the PCR product is digested with AluI to give 476, 229, 189, 150, 139 and 56 bp fragments.

AP- or 16SrX-group specific PCR (Lorenz et al., 1995)

The forward and reverse PCR primers are fO1 (5′-CGG AAA CTT TTA GTT TCA GT-3′) and rO1 (5′-AAG TGC CCA ACT AAA TGA T-3′), respectively, amplifying a 1071 bp-fragment of 16S rDNA. Primers fO1 and rO1 correspond to nucleotides 65-91 and 1135-1115 of the 16Sr DNA sequence of aster yellows strain OAY (Oenothera hookeri MLO, Lim & Sears, 1989).

PCR

The 40-µL reaction mixture is composed as follows: 0.5 µm of each primer, 100 µm dNTPs, 0.2 Units of Taq DNA polymerase (e.g. Goldstar polymerase, Eurogentec), 5 µL DNA extract in the reaction buffer supplied by the manufacturer of the Taq DNA polymerase. The PCR is performed in 0.2 mL reaction tubes in a thermocycler (e.g. GeneAmp PCR system 9600, Applied Biosystems) with the following parameters: 2 min at 94°C, 40 cycles of 20 s at 95°C, 20 s at 55°C, and 1 min at 72°C, followed by a final extension for 4 min at 72°C and cooled to 4°C. After amplification, 10 µL of the PCR products is subjected to electrophoresis on a 1% agarose gel under stable 90 V, in TBE buffer according to standard procedures (Sambrook et al., 1989) along with a DNA ladder (e.g. 100 bp-ladder, Fermentas) to size fragments. PCR products are viewed and photographed under UV light.

RFLP analysis

As above, except that restriction enzymes SspI and SfeI are used in separate reactions.

Interpretation of band patterns

The phytoplasma is identified as P. mali if the PCR product is digested with SspI at position 419, and with SfeI at position 998.

AP-specific PCR (Jarausch et al., 1994, 1995)

The forward and reverse PCR primers are AP5 (5′-TCT TTT AAT CTT CAA CCA TGG C-3′) and AP4 (5′-CCA ATG TGT GAA ATC TGT AG-3′), respectively, amplifying a 483-bp fragment of a nitroreductase-like protein gene. Primer AP5 and AP4 correspond to nucleotides 560-581 and 1042-1023, respectively, of a cloned 1.8 kb fragment of P. mali strain AT deposited under accession code L22217 in the GenBank data library.

PCR

The 40-µL reaction mixture is composed as follows: 0.5 µm of each primer, 125 µm dNTPs, 0.5 Units Taq DNA polymerase (e.g. Replitherm polymerase, Epicentre), 5 µL DNA in the reaction buffer supplied by the manufacturer of the Taq DNA polymerase. The PCR is performed in thin-walled 0.2 mL reaction tubes in a thermocycler (e.g. GeneAmp PCR system 9600 (Applied Biosystems) with the following parameters: 1 min at 95 °C, 40 cycles of 10 s at 95°C, 15 s at 58°C, and 45 s at 72°C, followed by a final extension for 4 min at 72°C and cooled to 4°C. After amplification, 10 µL of the PCR products is subjected to electrophoresis on 1% agarose gel under stable 90 V, in TBE buffer according to standard procedures (Sambrook et al., 1989) along with a DNA ladder (e.g. 100 bp-ladder, Fermentas) to size fragments. PCR products are viewed and photographed under UV light.