Multigene sequence data and genetic diversity among ‘Candidatus Phytoplasma ulmi’ strains infecting Ulmus spp. in Serbia

Abstract

Elm yellows phytoplasmas (EY) belonging to the 16SrV-A subgroup were recently proposed as a new candidate species 'Candidatus Phytoplasma ulmi'. These pathogens infect elm trees, causing leaf yellowing and premature drying. In this study, 25 isolates originating from localities in northeast, east and southwest Serbia were characterized by means of RFLP analysis and DNA sequencing of four genomic loci: 16S rRNA, ribosomal protein rpl22-rps3, secY and map. In total, five different genotypes were identified based on collective sequencing of all four genes. Four of these genotypes showed significant nucleotide changes compared with the EY1 T reference strain. Phylogeny based on parsimony analyses of ribosomal protein, secY and map genetic loci indicated a single monophyletic origin of EY1 T and the new 'Ca. Phytoplasma ulmi' strains. Unlike phylogenetic clustering, DNA sequence comparison of EY1 T and the novel strains revealed mutations in oligonucleotide signature sequences for all three genes (16S, rpl22-rps3 and secY) used for the characterization and assignment of 16SrV-A phytoplasmas to the 'Ca. Phytoplasma ulmi' species in the original description. Based on their high degree of genetic variability, the Serbian strains were assigned to four different subtypes of 'Ca. Phytoplasma ulmi' (EY-S1, EY-S2, EY-S3 and EY-S4). New diagnostic enzymes for practical use in 'Ca. Phytoplasma ulmi' identification are proposed for the 16S rRNA, ribosomal protein and secY genes. The implications of genetic variability within signature sequences for taxonomy and identification of 'Ca. Phytoplasma' species, as well as the importance of geographic variability and number of strains characterized for species description, are discussed.

Scientific Opinion on the pest categorisation of Elm phloem necrosis mycoplasm

“…Its identification has also been confirmed by multilocus sequence analysis (Arnaud et al, 2007;Jović et al, 2011 …”

“…Other phytoplasmas are found in other woody perennial hosts and cause diseases known as Alder yellows, flavescence dorée, rubus stunt, cherry lethal yellows or jujube witches' broom (Jović et al, 2011).…”

“…Identification can also be achieved by the use of A B C D E restriction fragment length polymorphism (RFLP) or through multilocus sequence analysis (Arnaud et al, 2007;Jović et al, 2011), targeting other genes such as secY, rpV or FD9.…”

Elm yellows: A phytoplasma disease of concern in forest and landscape ecosystems

“…P. ziziphi" (the jujube witches' broom agent) by less than 2.5% in 16S rDNA sequence similarity, the threshold for assigning species rank to phytoplasmas under the provisional status "Candidatus" (IRPCM Phytoplasma/Spiroplasma Working Team -Phytoplasma taxonomy group, 2004). P. ulmi" strains infecting elm trees in Serbia and Croatia (Jović et al, 2011;Katanić et al, 2016), which differ from the "Ca. Nucleotide sequence comparisons revealed that four strains of "Ca.…”

“…P. rubi" (the rubus stunt agent) and "Ca. However, typing of additional European strains based on RFLP and sequence analyses of 16S rRNA, rplV, rpsC, secY and map genes showed genetic diversity within this pathogen (Arnaud et al, 2007;Jović et al, 2008Jović et al, , 2011Katanić et al, 2016;Quaglino, Casati, Eccher, & Bianco, 2005). However, supporting data for separating the above taxa at the putative species level were obtained by examining other molecular markers and considering biological properties, such as host range and insect vector specificity (Jung et al, 2003;Lee et al, 2004;Malembic-Maher et al, 2011;Martini, Marcone, Lee, & Firrao, 2014).…”