Phytoplasma classification and phylogeny based on in silico and in vitro RFLP analysis of cpn60 universal target sequences.
Phytoplasmas are unculturable, phytopathogenic bacteria that cause economic losses worldwide. As unculturable micro-organisms, phytoplasma taxonomy has been based on the use of the 16S rRNA-encoding gene to establish 16Sr groups and subgroups based on the restriction fragment length polymorphism (RFLP) pattern resulting from the digestion of amplicon (in vitro) or sequence (in silico) with seventeen restriction enzymes. Problems such as heterogeneity of the ribosomal operon and the inability to differentiate closely related phytoplasma strains has motivated the search for additional markers capable of providing finer differentiation of phytoplasma strains. In this study we developed and validated a scheme to classify phytoplasmas based on the use of cpn60 universal target (cpn60 UT) sequences. Ninety-six cpn60 UT sequences from strains belonging to 19 16Sr subgroups were subjected to in silico RFLP using pDRAW32 software, resulting in 25 distinctive RFLP profiles. Based on these results we delineated cpn60 UT groups and subgroups, and established a threshold similarity coefficient for groups and subgroups classifying all the strains analysed in this study. The nucleotide identity among the reference strains, the correspondence between in vitro and in silico RFLP, and the phylogenetic relationships of phytoplasma strains based on cpn60 UT sequences are also discussed.
Instituto de Biotecnología y Ecología Aplicada (INBIOTECA), Universidad Veracruzana, Avenida de Las Culturas Veracruzanas Xalapa, Veracruz, México.
Edel
E
Pérez-López
Agriculture and Agri-Food Canada, London Research and Development Centre, London, Ontario, Canada.
Chrystel Y
CY
Olivier
Laboratorio de Alta Tecnología de Xalapa - DGI, Universidad Veracruzana, Médicos 5, Unidad del Bosque Xalapa, Veracruz, México.
Mauricio
M
Luna-Rodríguez
Department of Veterinary Microbiology, University of Saskatchewan, Saskatoon, Saskatchewan, Canada.|Agriculture and Agri-Food Canada, Saskatoon Research and Development Centre, Saskatoon, Saskatchewan, Canada.
Tim J
TJ
Dumonceaux
D015373:Bacterial Typing Techniques; D018834:Chaperonin 60 / Q000235:genetics; D004262:DNA Restriction Enzymes / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D009876:Operon; D010802:Phylogeny; D045582:Phytoplasma / Q000145:classification*; D012150:Polymorphism, Restriction Fragment Length; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D017422:Sequence Analysis, DNA; D012984:Software
D018834:Chaperonin 60; D004269:DNA, Bacterial; D012336:RNA, Ribosomal, 16S; D004262:DNA Restriction Enzymes
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