Abstract
Red clover (Trifolium pratense) and Ladino clover (Trifolium repens) plants showing phytoplasma-associated symptoms (yellowing/reddening, virescence and phyllody) have been recovered in Friuli-Venezia Giulia, Italy. Using AluI RFLP analysis of PCR amplified 16S rDNA we showed that the disease can be caused independently by two phylogenetically distinct phytoplasmas. One of them showed the very typical 16S rDNA RFLP pattern of the agent of Clover Phyllody in Canada (CCPh). The 16S rDNA of the other phytoplasma (Italian Clover Phyllody phytoplasma, ICPhp) has been PCR amplified, cloned and sequenced. The sequence revealed high similarity (>98%) with phytoplasmas belonging to the X disease cluster, which includes organisms not reported to cause phyllody on their hosts. The analysis by AluI RFLP of the PCR amplified pathogen 16S rDNA from other herbaceous plants (Crepis biennis, Taraxacum officinale, Leucanthemum vulgare) collected nearby with phytoplasma-associated symptoms showed similar patterns. Southern blot hybridization of their EcoRI digested total DNA revealed identical RFLP patterns, suggesting that the causative agent may be the same organism.
Article PDF
Similar content being viewed by others
Avoid common mistakes on your manuscript.
Abbreviations
- PCR:
-
Polymerase Chain Reaction
- rDNA:
-
gene for the small subunit ribosomal RNA
- RFLP:
-
Restriction Fragment Length Polymorphism
References
Ausubel FA, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA and Struhl K (1992) Current Protocols in Molecular Biology. Greene Publishing and Wiley-Interscience, New York
Carraro L, Osler R, Refatti E and Poggi Pollini C (1988) Transmission of the possible agent of apple proliferation toVinca rosea by dodder. Riv Pat Veg, S IV 26: 43–52
Chen MH and Hiruki C (1975) Electron microscopy of mycoplasma-like bodies associated with clover proliferation disease. Proc Am Phytopathol Soc 2: 52
Chiykowski LN (1962) Clover phyllody virus in Canada and its transmission. Can J Bot 40: 397–404
Chiykowski LN (1981) Association of mycoplasma-like organisms with clover yellow edge disease. Can J Plant Pathol 3: 139–144
Chiykowski LN and Sinha RC (1982) Herbaceous host plants of peach eastern X-disease agent. Can J PLant Pathol 4: 8–15
Chiykowski LN and Sinha RC (1990) Differentiation of MLO diseases by means of symptomatology and vector transmission. In: Stanek G, Cassell G, Tully JG and Whitcomb RF (eds) Recent Advances in Mycoplasmology. Proceedings of the 7th Congress of the International Organization for Mycoplasmology (pp. 280–287) Gustav Fisher Verlag, Stuttgart
Davis RE, Lee I-M, Dally EL, Dewitt N and Douglas SM (1988) Cloned nucleic acid hybridization probes in detection and classification of mycoplasmalike organisms (MLOs). Acta Hort 234: 115–121
Deng S and Hiruki C (1991) Amplification of 16S rRNA genes from culturable and nonculturableMollicutes. J Microbiol Meth 14: 53–61
Devereux J, Haeberli P and Smithies O (1984) A comprehensive set of sequence analysis programs for the VAX. Release 7. Nucleic Acid Res 12: 387–395
Doyle JJ and Doyle JL (1990) Isolation of plant DNA from fresh tissue. Focus 12(1): 13–15
Firrao G, Gobbi E and Locci R (1993) Production of oligonucleotide probes for mycoplasma-like organisms using the polymerase chain reaction. Phytopathology 83: 602–607
Firrao G and Locci R (1993) Rapid preparation of DNA from phytopathogenic mycoplasma-like organisms for polymerase chain reaction analysis. Letters in Appl Microbiol 17: 280–281
Griffiths HM, Gundersen DE, Sinclair WA, Lee IM and Davis RE (1994) Mycoplasmalike organisms from milkweed, goldenrod and spirea represent two new 16S rRNA subgroups and three new strain subclusters related to Peach X disease MLO. Can J Plant Pathol 16:255–260
Gundersen DE, Lee IM, Rehner SA, Davis RE and Kingsbury DT (1994) Phylogeny of mycoplasmalike organisms (phytoplasmas) — a basis for their classification. J Bacteriol 176: 5244–5254
Gundersen DE, Lee IM, Scharf DA, Harrison NA, Chang CJ, Davis RE and Kingsbury DT (1996) Genomic diversity and differentiation among phytoplasma strains in 16S rRNA groups I (Aster Yellows and related phytoplasmas) and III (X-disease and related phytoplasmas). Int J Syst Bacteriol 46: 64–75
Kirkpatrick BC (1991) Mycoplasma-like organisms: Plant and invertebrate pathogens 4050-4067, In: Balows A, Trüper HG, Dworkin M, Harder W and Schliefer KH (eds) The Prokaryotes. 2nd ed. Springer-Verlag Press, New York
Kirkpatrick B, Smart C, Gardner S, Gao J-L, Ahrens U, Maurer R, Schneider B, Lorenz K-H, Seemüller E, Harrison N, Namba S and Daire X (1994) Phylogenetic relathionships of plant pathogenic MLOs established by 16/23S rDNA spacer sequences. IOM Letters 3: 228–229
Kirkpatrick BC, Stenger DC, Morris TJ and Purcell AH (1987) Cloning and detection of DNA from a nonculturable plant pathogenic mycoplasma-like organism. Science 238: 197–200
Kuske CR and Kirkpatrick BC (1992) Phylogenetic relationships between the western aster yellows mycoplasmalike organism and other prokaryotes established by 16S rRNA gene sequence. Internat J Systematic Bacteriol 42: 226–233
Krczal H (1960) A virosis transmissible from white clover to Fragaria vesca. Z Pflkrankh 67: 599–602
Lee IM, Davis RE, Chen TA, Chiykowski LN, Fletcher J, Hiruki C and Schaff DA (1992a) A genotype based system for the identification and classification of mycoplasmalike organisms (MLOs) in the aster yellows MLO strain cluster. Phytopathology 82: 977–986
Lee IM, Gundersen DE, Davis RE and Chiykowski LN (1992) Identification and analysis of a genomic strain cluster of mycoplasmalike organisms associated with Canadian peach (eastern) X-disease western X-disease and clover yellow edge. J Bacteriol 174: 6694–6698
Lee I-M, Hammond RW, Davis RE and Gundersen DE (1993) Universal amplification and analysis of pathogen 16S rDNA for classification and identification of mycoplasma-like organisms. Phytopathology 83: 834–842
Lim P-O and Sears BB (1989) 16S rRNA sequence indicates that plant-pathogenic mycoplasmalike organisms are evolutionarily distinct from animal mycoplasmas. J Bacteriol 171: 5901–5906
Marwitz R (1990): Diversity of yellows disease agents in plant infections In: Stanek G, Cassell G, Tully JG and Whitcomb RF (eds) Recent Advances in Mycoplasmology. Proceedings of the 7th Congress of the International Organization for Mycoplasmology (pp. 431–434) Gustav Fisher Verlag, Stuttgart
McCoy RE, Cauldwell A, Chang CJ, Chen TA, Chiykowski LN, Cousin MT, Dale JL, de Leeuw GTN, Golino DA, Hackett KJ, Kirkpatrick BC, Marwitz R, Petzold H, Sinha RC, Sugiura M, Whitcomb RF, Yong IL, Zhu BM and Seemüller E (1989) Plant diseases associated with mycoplasma-like organisms. In: Whitcomb RF and Tully JG (eds) The Mycoplasmas, Vol V (pp. 545–640) Academic Press, New York
Namba S, Oyaizu H, Kalo S, Iwanami S and Tsuchizaki T (1993) Phylogenetic diversity of phytopathogenic mycoplasmalike organism. Int J Syst Bacteriol 43: 461–467
Osier R, Firrao G, Carraro L, Loi N, Musetti R and Chen TA (1994) Biodiversity of plant MLOs in a well definited ecological area. IOM Letters 3: 286–287
Schneider B, Ahrens U, Kirkpatrick BC and Seemüller E (1993) Classification of plant-pathogenic mycoplasma-like organisms using restriction-site analysis of PCR-amplified 16S rDNA. J Gen Microbiol 139: 519–527
Schneider B and Seemüller E (1994) Studies on taxonomic relationships of mycoplasma-like organisms using Southern blot analysis. J Phytopathol 141: 173–185
Seemüller E, Schneider B, Maurer R, Ahrens U, Daire X, Kison H, Lorenz KH, Firrao G, Avinent L, Sears BB and Stackebrandt E (1994) Phylogenetic classification of phytopathogenic mollicutes by sequence analysis of 16S ribosomal DNA. Int J Syst Bacteriol 44: 440–446
Swofford DL (1991) Phylogenetic Analysis Using Parsimony, Version 3.1. Computer program distributed by the Illinois Natural History Survey, Champaign, Illinois, USA
Tully JC (1993) International Committee on Systematic Bacteriology Subcommittee on the Taxonomy of Mollicutes. Minutes of the interim meetings, 1 and 2 Aug. 1992, Ames, Iowa. Int J Syst Bacteriol 43: 394–397
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Firrao, G., Carraro, L., Gobbi, E. et al. Molecular characterization of a phytoplasma causing Phyllody in Clover and other herbaceous hosts in Northern Italy. Eur J Plant Pathol 102, 817–822 (1996). https://doi.org/10.1007/BF01877050
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF01877050