Abstract
Phytoplasmas infecting fruit trees are considered quarantine organisms in Europe and North America. Detection often is hampered by their extremely irregular distribution in host plants. A sensitive, specific and quick diagnostic test would be highly desirable for routine detection, mainly to avoid using infected planting material. PCR methods require tedious preparation of DNA; also, the available primers are highly specific and exhibit some homology to chloroplast and plastid DNA. To address these problems, we compared several DNA preparation protocols for purity of DNA, cost and time required. We also developed new primers using rDNA sequence information from an Austrian isolate of European Stone Fruit Yellows (ESFY). These primers operate at high annealing temperatures and, thus, increase the specificity and decrease the risk of false positives. The primers could reliably detect the European phytoplasmas (AP, ESFY and PD) within a collection of isolates maintained in micropropagated periwinkle. Thus, they are suitable as general primers for phytoplasma detection. The primers also can be used for strain identification by direct PCR followed by RFLP analysis as demonstrated with micropropagated fruit tree material. Finally, an IC-PCR method that uses the primers for AP detection was found very sensitive and suitable for large-scale testing of apple materialin vivo andin vitro.
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Abbreviations
- ACLSV:
-
apple chlorotic leafspot virus
- A-AY:
-
American aster yellows
- AP:
-
apple proliferation
- ASGV:
-
apple stem grooving virus
- ASHY:
-
ash yellows
- ASPV:
-
apple stem pitting virus
- AT:
-
apple proliferation strain
- BVK:
-
phytoplasma fromPsammotettix cephalotes
- CHRY:
-
chrysanthemum yellows
- DAS-ELISA:
-
double antibody sandwich-enzyme linked immunosorbent assay
- ESFY:
-
European stone fruit yellows
- EY-C:
-
elm yellows
- FBPSA:
-
faba bean phyllody
- GVX:
-
Green Valley X disease
- GSFY:
-
German stone fruit yellows
- IC-PCR:
-
immuno capture-polymerase chain reaction
- KVM:
-
clover phyllody
- LNS:
-
plum leptonecrosis
- LUM:
-
Lucerne virescence
- MOL:
-
Molière disease
References
Batjer LP and Schneider H (1960) Relation of pear decline to rootstocks and sieve-tube necrosis. Proceed Am Soc Hort Sci 76: 85–97.
Bertaccini A, Carraro L, Davies D, Laimer da Câmara Machado M, Martini M, Paltrinieri S and Seemüller E (2000) Micropropagation of a collection of phytoplasma strains in periwinkle and other host plants. In: Proc. XIII Congress of IOM, July 14–19, pp 101, ACROS Fukuoka.
Bertheau Y, Frechon D, Toth I and Hyman LJ (1998) DNA amplification by polymerase chain reaction (PCR) In: Perombelon MCM and van der Wolf JM (eds), Methods for the Detection and Quantification ofErwinia carotovora subsp.atroseptica on Potatoes, pp 39–59, Occasional Publication n 10, Scottish Crop Research Institute.
Braun EJ and Sinclair WA (1978) Translocation of phloem necrosis-diseased American elm seedlings. Phytopathol 68: 1733–1737.
Catlin PB, Olsson EA and Beutel JA (1975) Reduced translocation of carbon and nitrogen from leaves with symptoms of pear curl. J Am Soc Hort Sci 100: 184–187.
Gibb KS, Padovan AC and Mogen BD (1995) Studies on sweet potato little-leaf phytoplasma detected in sweet potato and other plant species in northern Australia. Phytopathol 85: 169–174.
Gundersen DE and Lee I-M (1996) Ultrasensitive detection of phytoplasmas by nested-PCR assays using two universal primer pairs. Phytopathologia Mediterranea 35: 144–151.
Jarausch W, Saillard C and Dosba F (1996) Longterm maintainance of nonculturable apple-proliferation phytoplasmas in their micropropagated natural host plant. Plant Pathol 45: 778–786.
Jarausch W, Saillard C, Helliot B, Garnier M and Dosba F (2000a) Genetic variability of apple proliferation phytoplasmas as determined by PCR-RFLP and sequencing of a non-ribosomal fragment. Mol Cellular Probes 14: 17–24.
Jarausch W, Saillard C, Broquaire J, Garnier M and Dosba F (2000b) PCR-RFLP and sequence analysis of a non-ribosomal fragment for genetic characterization of European stone fruit yellows phytoplasmas infecting variousPrunus species. Mol Cellular Probes 14: 171–179.
Kartte S and Seemüller E (1991) Histopathology of apple proliferation inMalus taxa and hybrids of different susceptibility. J Phytopathol 131: 149–160.
Kison H, Kirkpatrick BC and Seemüller E (1997) Genetic comparison of the peach yellows leaf froll agent with European fruit tree phytoplasmas of the apple proliferation group. Plant Pathol 46: 1–7.
Kobayashi N, Horikoshi T, Katsumaya H, Handa T and Takayanagi K (1998) A simple and efficient DNA extraction method for plants, especially woody plants. Plant Tissue Culture and Biotech 4: 76–80.
Laimer da Câmara Machado M, Paltrinieri S, Hanzer V, Arthofer W, Strommer S, Martini M, Pondrelli M and Bertaccini A (2001) Presence of European stone fruit (ESFY or 16SrX-B) phytoplasmas in apricots in Austria. Plant Pathol 50(1): 130–135.
Lee I-M, Gundersen DE, Hammond RW and Davis RE (1994) Use of mycoplasmalike organism (MLO) group-specific oligonucleotide primers for nested-PCR assays to detect mixed-MLO infections in a single host plant. Phytopathol 84: 559–566.
Lee I-M, Bertaccini A, Vibio M and Gundersen DE (1995) Detection of multiple phytoplasmas in perennial fruit trees with decline symptoms in Italy. Phytopathol 85: 728–735.
Lee I-M, Gundersen-Rindal DE, Davis RE and Bartoszyki IM (1998) Revised classification scheme of phytoplasmas based on RFLP analyses of 16S rRNA and ribosomal protein gene sequences. Intl J of Systematic Bacteriol 48: 1153–1169.
Lepka P, Stitt M, Moll E and Seemüller E (1999) Effect of phytoplasmal infection on concentration and translocation of carbohydrates and amino acids in periwinkle and tobacco. Physiol and Mol Plant Pathol 55: 59–68.
Loi N, Ermacora P, Carraro L, Osler R and Chen TA (1998) Apple proliferation detection using monoclonal antibodies. In: Proc XII Congress of IOM, Sidney, July 22–28, pp 73–74.
Lorenz K-H, Schneider B, Ahrens U and Seemüller E (1995) Detection of the apple proliferation and pear decline phytoplasmas by PCR amplification of ribosomal and nonribosomal DNA. Phytopathol 85: 771–776.
Musetti R, Favali M and Pressacco L (2000) Histopathology and polyphenol content in plants infected by phytoplasmas. Cytobios 102: 133–147.
Smart C, Schneider B, Blomquist C, Guerra L, Harrison N, Ahrens U, Lorenz K, Seemüller E and Kirkpatrick B (1996) Phytoplasma-specific PCR primers based on sequences of the 16S–23S rRNA spacer region. Appl Environ Microbiol 62: 2988–2993.
Schneider B, Seemüller E, Smart CD and Kirkpatrick BC (1995) Phylogenetic classification of plant pathogenic mycoplasma-like organisms or phytoplasmas. In: Razin R and Tully JG (eds), Molecular and Diagnostic Procedures in Mycoplasmology, vol 1, pp. 369–380, Academic Press, San Diego.
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Heinrich, M., Botti, S., Caprara, L. et al. Improved detection methods for fruit tree phytoplasmas. Plant Mol Biol Rep 19, 169–179 (2001). https://doi.org/10.1007/BF02772160
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DOI: https://doi.org/10.1007/BF02772160