Abstract
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.
Highlights
Classification of phytoplasmas is further supported by the 16S rRNA gene through the use of restriction fragment length polymorphism (RFLP) of the 16S rRNA F2nR2 fragment with a set of seventeen endonucleases (Lee et al, 1993, 1998)
The validation of a computer simulated RFLP as an alternative to the actual RFLP, along with the development of the interactive online phytoplasma classification tool iPhyClassifier, increased the accuracy of phytoplasma classification based on 16S rRNA gene sequences (Wei et al, 2007, 2008; Zhao et al, 2009)
The cpn60 universal target (Goh et al, 1996), is a fragment of approximately 550 bp that has been extensively used in the study of microbial communities (Town et al, 2014), and suggested as a molecular barcode for the domain Bacteria (Links et al, 2012)
Summary
Subgroup cpn60 UT I-IA is represented by Brassica spp. phytoplasma strain AY-Ruta The subgroup cpn60 UT I-IB is represented by Linum usitatissimum phytoplasma strain SF1 KJ940013); cpn60 UT I-IIB is represented by Aster yellow phytoplasma strain AY-J Subgroup cpn60 UT I-VB is represented by Iceland poppy yellows phytoplasma strain IPY
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