Abstract
Thirty six isolates of Phoma tracheiphila from Italy, the causal agent of the “mal secco” disease on Citrus species, were characterised by different molecular tools in comparison with representative isolates of other phytopathogenic Phoma species. These included analysis of the distribution of RAPD and microsatellite markers and sequencing of the internal transcribed spacer (ITS) region of the nuclear rRNA genes. The results obtained with 12 RAPD primers (92 markers) and 7 microsatellite primers (56 markers) suggest that Italian isolates of P. tracheiphila are genetically homogeneous, leading to identical patterns upon amplification with all the tested primers. Accordingly, ITSI-5.8S-ITS2 sequences were highly conserved (98–100% identity along a 544-characters alignment) among all the isolates of P. tracheiphila. A neighbor-joining analysis of ITS sequences of P. tracheiphila in comparison with those of other Phoma species, as well as with alignable sequences from anamorphic and teleomorphic taxa retrieved in BLAST searches, revealed a close relationship between P. tracheiphila and Leptosphaeria congesta. A pair of P. tracheiphila-specific primers was designed on the consensus sequence (555 residues) obtained from the alignment of the newly generated P. tracheiphila ITS sequences. A PCR-based specific assay coupled to electrophoretic separation of amplicons made it possible to detect P. tracheiphila in naturally infected Citrus wood tissue collected from both symptomatic and symptomless plants. The limit of detection was 10 pg of genomic DNA and 5 fg of the ITS target sequence.
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References
SM Aljanabi I Martinez (1997) ArticleTitleUniversal and rapid salt-extraction of high quality genomic DNA for PCR-based techniques Nucleic Acids Research 25 4692–4693 Occurrence Handle9358185 Occurrence Handle10.1093/nar/25.22.4692 Occurrence Handle1:CAS:528:DyaK1cXhsFWrsA%3D%3D
SF Altschul TL Madden AA Schäffer J Zhang Z Zhang W Miller DJ Lipman (1997) ArticleTitleGapped BLAST and PSI-BLAST: a new generation of protein database search programs Nucleic Acids Research 25 3389–3402 Occurrence Handle9254694 Occurrence Handle10.1093/nar/25.17.3389 Occurrence Handle1:CAS:528:DyaK2sXlvFyhu7w%3D
Baldacci E (1950) Caratteri colturali delle razze di Bakerophoma tracheiphila. Ricerca Scientifica 18: 5–6 (in Italian)
Baldacci E and Garofalo F (1948) Conoscenze e ricerche sul “Mal secco” degli Agrumi. Humus 4: 21–24 (in Italian)
Balmas V, Scherm B, Di Primo P, Rau D, Marcello A and Migheli Q (in press) Molecular characterization of vegetative compatibility groups in Fusarium oxysporum f. sp. radicis-lycopersici and f. sp. lycopersici by random amplification of polymorphic DNA and microsatellite-primed PCR. European Journal of Plant Pathology.
Cacciola SO, Perrotta G, Graniti A and Magnano di San Lio G (1986) Esame preliminare di ceppi di Phoma tracheiphila (Petri) Kane. et Ghick. mediante elettroforesi. In: Il recente contributo della ricerca allo sviluppo dell’Agrumicoltura Italiana (pp. 687–691) Carlo Delfino, Sassari (in Italian)
MPS Câmara ME Palm P Berkum Particlevan NR O’Neill (2002) ArticleTitleMolecular phylogeny of Leptosphaeria and Phaemphaeria Mycologia 94 630–640 Occurrence Handle10.2307/3761714
Ciccarone A (1971) Il fungo del “mal secco” degli Agrumi. Phytopathologia Mediterranea 10: 68–75 (in Italian)
De Cicco V and Luisi N (1977) Variabilità della patogenicità e di alcuni caratteri colturali in isolati di Phoma tracheiphila, provenienti da aree e ospiti diversi del Mediterraneo. Annali della Facoltà di Agraria dell”Università di Bari 29: 565–573 (in Italian)
ZN Deng A Gentile F Domina E Nicolosi E Tribulato (1995) ArticleTitleSelecting lemon protoplasts for insensitivity to Phoma tracheiphila toxin and regenerating tolerant plants Journal of the American Society for Horticultural Science 120 902–905
UJ Desai PK Pfaffle (1995) ArticleTitleSingle step purification of a thermostable DNA polymerase expressed in Escherichia coli BioTechniques 19 780–784 Occurrence Handle8588916 Occurrence Handle1:CAS:528:DyaK2MXpt1eqsrg%3D
V Fogliano A Marchese A Scaloni A Ritieni A Visconti G Randazzo A Graniti (1998) ArticleTitleCharacterization of a 60 kDa phytotoxic glycoprotein produced by Phoma tracheiphila and its relation to malseccin Physiological and Molecular Plant Pathology 53 149–161 Occurrence Handle10.1006/pmpp.1998.0172 Occurrence Handle1:CAS:528:DyaK1MXjtVWmtA%3D%3D
H Forster JE Adaskaveg (1999) ArticleTitleIdentification of subpopulations of Colletotrichum acutatum and epidemiology of almond anthracnose in California Phytopathology 89 1056–1065 Occurrence Handle10.1094/PHYTO.1999.89.11.1056 Occurrence Handle1:CAS:528:DyaK1MXnsVGqsbo%3D
A Gentile E Tribulato ZN Deng E Galun R Fluhr A Vardi (1993) ArticleTitleNucellar callus of Femminello lemon, selected for tolerance to Phoma tracheiphila toxin, shows enhanced release of chitinase and glucanase into the culture medium Theoretical and Applied Genetics 86 527–532 Occurrence Handle10.1007/BF00838705 Occurrence Handle1:CAS:528:DyaK2cXht12ksbk%3D
Gentile A, Deng ZN, Tribulato E, Albanese G, Grimaldi V, Catara A and Vardi A (2000) Evaluation of lemon somaclones for tolerance to mal secco disease by artificial inoculation. In: Goren R and Goldschmidt E (eds) Proceedings 1st International Symposium on Citrus Biotechnology. Acta Horticulturae 535: 259–263
JM Henson R French (1993) ArticleTitleThe polymerase chain reaction and plant-disease diagnosis Annual Review of Phytopathology 31 81–109 Occurrence Handle18643762 Occurrence Handle10.1146/annurev.py.31.090193.000501 Occurrence Handle1:STN:280:DC%2BD1cvms1ersg%3D%3D
G di San Lio Magnano SO Cacciola A Pane S Grasso (1992) ArticleTitleRelationship between xylem colonization and symptom expression in mal secco infected sour orange seedlings Proceedings International Society of Citriculture 2 873–876
T Maniatis EF Fritsch J Sam brook (1982) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Cold Spring Harbor USA
A Nachmias I Barash V Buchner Z Solel GA Strobel (1977) ArticleTitleA phytotoxic glycopeptide from lemon leaves infected with Phoma tracheiphila Physiological Plant Pathology 14 135–140 Occurrence Handle10.1016/0048-4059(79)90001-8
A Nachmias I Barash Z Solel GA Strobel (1979) ArticleTitlePurification and characterization of a phytotoxin produced by Phoma tracheiphila, the causal agent of mal secco disease of citrus Physiological Plant Pathology 10 147–157 Occurrence Handle10.1016/0048-4059(77)90018-2
M Nei WH Li (1979) ArticleTitleMathematical model for studying genetic variation in terms of restriction endonucleases Proceedings of the National Academy of Sciences USA 76 5269–5273 Occurrence Handle10.1073/pnas.76.10.5269 Occurrence Handle1:CAS:528:DyaL3cXitVWn
Nicholas KB and Nicholas HB Jr (1997) GeneDoc: analysis and visualization of genetic variation. http://www.psc.edu/ biomed/genedoc/.
G Perrotta A Graniti (1988) Phoma tracheiphila (Petri) Kanchaveli & Gikashvili. In: Smith IM J Dunez RA Lelliott DH Phillips SA Archer (Eds) European Handbook of Plant Diseases Blackwell Scientific Publications Oxford, UK 396–398
E Punithalingam P Holliday (1973) Deuterophoma tracheiphila. CMI Descriptions of Pathogenic Fungi and Bacteria No. 399. Commonweath Mycological Institute Kew UK
PV Reddy R Patel JF White (1998) ArticleTitlePhylogenetic and developmental evidence supporting reclassification of cruciferous pathogens Phoma lingam and Phoma wasabiae in Plenodomus Canadian Journal of Botany 76 1916–1922 Occurrence Handle10.1139/cjb-76-11-1916
Recupero G Reforgiato A Gentile MP Russo F Domina (1997) ArticleTitleGenetic analysis of resistance to Phoma tracheiphila in three Citrus and Poncirus progenies Plant Breeding 116 198–200 Occurrence Handle10.1111/j.1439-0523.1997.tb02180.x
F Rollo A Amici F Foresi Silvestro I Di (1987) ArticleTitleConstruction and characterization of a cloned probe for the detection of Phoma tracheiphila in plant tissues Applied Microbiology and Biotechnology 26 352–357 Occurrence Handle10.1007/BF00256668 Occurrence Handle1:CAS:528:DyaL2sXkvFWhtLg%3D
F. Salvi R Rollo P Torchia (1990) ArticleTitleHighly sensitive and fast detection of Phoma tracheiphila by polymerase chain reaction Applied Microbiology and Biotechnology 32 572–576 Occurrence Handle1366441
B Rosciglione A Burgio A Bottalico C Laviola (1991) ArticleTitleRelationship between phytotoxicity of metabolites produced in vitro by Phoma tracheiphila (Petri) Kane et Ghik. and their virulence Journal of Phytopathology 133 23–28 Occurrence Handle10.1111/j.1439-0434.1991.tb00133.x
Rozen S and Skaletsky HJ (1998) Primer3. Code available at http://frodo.wi.mit.edu/primer3/primer3_code.html.
Salerno M and Perrotta G (1966) Ricerche sul mal secco degli agrumi (Deuterophoma tracheiphila Petri). Virulenza e caratteri colturali del fungo in Sicilia. Rivista di Patologia Vegetale Ser. 4° 2: 203–312 (in Italian)
M Salerno Z Solel (1988) Mal Secco JO Whiteside SM Garnsey LW Timmer (Eds) Compendium of Citrus Diseases APS Press St Paul, USA 18–20
Scrivani P (1954) Patogenesi, riproduzione sperimentale del mal secco da Deuterophoma tracheiphila Petri e ricerche sulla formazione di metaboliti tossici in coltura. Phytopathologische Zeitschrift 22: 83–100 (in Italian)
D Simioniuc R Uptmoor W Friedt F Ordon (2002) ArticleTitleGenetic diversity and relationships among pea cultivars revealed by RAPDs and AFLPs Plant Breeding 121 429–435 Occurrence Handle10.1046/j.1439-0523.2002.733320.x Occurrence Handle1:CAS:528:DC%2BD38Xpslegsb4%3D
Z Solel (1976) ArticleTitleEpidemiology of mal secco disease of lemons Phytopathologische Zeitschrift 85 90–92 Occurrence Handle10.1111/j.1439-0434.1976.tb04804.x
GL Sun O Diaz B Salomon Bothmer R von (1999) ArticleTitleGenetic diversity in Elymus caninus as revealed by isozyme, RAPD, and microsatellite markers Genome 42 420–431 Occurrence Handle10382290 Occurrence Handle10.1139/gen-42-3-420 Occurrence Handle1:CAS:528:DyaK1MXkt1Chtbk%3D
G Surico V Cicco ParticleDe NS Iacobellis (1981) ArticleTitleOsservazioni sulla patogenicità di Phoma tracheiphila (Petri) Kanc et Ghik. in relazione alla produzione in vitro di metaboliti fitotossici Phytopathologia Mediterranea 20 17–21
BC Sutton (1980) The Coelomycetes. Fungi Imperfecti with Pycnidia Acervuli and Stromata. Commonwealth Mycological Institute Kew, Surrey, UK
DL Swofford (2000) PAUP* phylogenetic analysis using parsimony (*and other methods) Ver. 4. Sinauer Associates Sunderland, MA, USA
N Tusa del Bosco S Fatta F Nigro A Ippolito (2000) ArticleTitleResponse of cybrids and a somatic hybrid of lemon to Phoma tracheiphila infections HortScience 35 125–127
Y Peer ParticleVan de R Wachter ParticleDe (1994) ArticleTitleTREECON for Windows: a software package for the construction and drawing of evolutionary trees for the Microsoft Windows environment Computer Applications in the Biosciences 10 569–570 Occurrence Handle7828077
TJ White T Bruns S Lee JW Taylor (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics MA Innis DH Gelfand J Sninsky TJ White (Eds) PCR Protocols: A Guide to Methods and Applications Academic Press New York, USA 315–322
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Balmas, V., Scherm, B., Ghignone, S. et al. Characterisation of Phoma tracheiphila by RAPD-PCR, microsatellite-primed PCR and ITS rDNA sequencing and development of specific primers for in planta PCR detection. Eur J Plant Pathol 111, 235–247 (2005). https://doi.org/10.1007/s10658-004-4173-x
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DOI: https://doi.org/10.1007/s10658-004-4173-x