Differentiation of ‘Candidatus Phytoplasma cynodontis’ Based on 16S rRNA and groEL Genes and Identification of a New Subgroup, 16SrXIV-C

Abstract

‘Candidatus Phytoplasma cynodontis’ is widespread in bermudagrass and has only been found in monocotyledonous plants. Molecular studies carried out on strains collected in Italy, Serbia, and Albania enabled verification of molecular variability in the 16S ribosomal RNA (rRNA) gene. Based on restriction fragment length polymorphism and sequence analyses, the strains from Serbia were clearly differentiated from all others and assigned to a new ribosomal DNA (rDNA) subgroup designated as 16SrXIV-C. A system for amplification of fragments containing the ‘Ca. P. cynodontis’ groEL gene was developed to enable study of its variability in related strains belonging to different 16SrXIV subgroups. Despite the fact that the groEL gene exhibited a greater sequence variation than 16S rRNA, the phylogenetic tree based on groEL gene sequence analysis was highly congruent with the 16S rDNA-based tree. The groEL gene analyses supported differentiation of the Serbian strains and definition of the new subgroup 16SrXIV-C. Phylogenetic analyses of both genes confirmed distinct phylogenetic lineages for strains belonging to 16SrXIV subgroups. Furthermore, groEL is the only nonribosomal marker developed for characterization of ‘Ca. P. cynodontis’ thus far, and its application in molecular surveys should provide better insight into the relationships among these phytoplasmas and correlation between strain differentiation and their geographical distribution.

Phytoplasma classification and phylogeny based on in silico and in vitro RFLP analysis of cpn60 universal target sequences

“…The present study confirms previously published work (Dumonceaux et al , 2014; Mitrović et al , 2011, 2015) showing the capability of cpn60 UT sequences to act as an additional marker to differentiate phytoplasmas. Strains that are closely related based on 16S rRNA gene sequence classification were differentiated as members of new subgroups, contributing to a better identification of the strains.…”

“…Altogether, after trimming the cpn60 UT sequence from the five completely sequenced phytoplasma genomes (Andersen et al , 2013; Bai et al , 2006; Kube et al , 2008; Oshima et al , 2013; Tran-Nguyen et al , 2008), from the draft genome belonging to the group 16SrII-A, strain PnWB (Chung et al , 2013) and 16SrIX-B strain SA213 (Quaglino et al , 2015), from the cpn60 sequences reported by Mitrović et al (2011) for members of the group 16SrI, and members of the group 16SrXIV (Mitrović et al , 2015), from the 3.6 kb DNA fragments obtained by Kakizawa et al (2006), and the sequences previously obtained by our group, we had 96 cpn60 UT sequences in this study.…”

“…All these genes have been used to achieve a finer differentiation of phytoplasmas belonging to different species and/or RFLP groups. Another gene used to improve the resolution of phytoplasmas classification is the groEL gene, also known as chaperonin 60 ( cpn 60) (Dumonceaux et al , 2014; Mitrović et al , 2011,2015). All the genes mentioned above have also been used to differentiate other bacterial species.…”

Molecular detection of ‘Candidatus Phytoplasma australasia’ and ‘Ca. P. cynodontis’ in Iraq

“…Kenya (Obura et al 2010) Italy, Albania and Serbia (Mitrović et al 2015) and Saudi Arabia (Omar 2016) .…”

Association of ‘CandidatusPhytoplasma cynodontis’ with Bermuda grass white leaf disease and its new hosts in Qassim province, Saudi Arabia

“…Bermuda grass white leaf (BGWL), first described in Taiwan, is a destructive phytoplasmal disease of Bermuda grass caused by the phytoplasma group (16SrXIV) and characterized by whitening of the leaves and shortening of the stolons (Chen et al 1972;Obura et al 2010). BGWL disease is reported in many countries including Singapore (Koh et al 2008), Italy (Marcone & Ragozzino 1997), Iran (Salehi et al 2009), Cuba (Arocha et al 2005), Australia (Padovan et al 1999;Tran-Nguyen et al 2000), Turkey (Çağlar et al 2013), Sudan (Dafalla & Cousin 1988), Thailand (Sunpapao 2014), India (Rao et al 2007;Snehi et al 2008;Kumar et al 2015), Malaysia (Nejat et al 2009;Naderali et al 2013), Myanmar (Win et al 2013), Kenya, Tanzania and Uganda (Asudi et al 2015), and Serbia and Albania (Mitrović et al 2015). Phytoplasmas, wall-less prokaryotes, classified in the Class Mollicutes, Order Acholeplasmatales and genus Candidatus are uncultivable (Xiaodong et al 2006).…”