engleski

Non-specific amplification of soil bacteria tuf gene by phytoplasma-specific primers

Although evolutionary conserved 16S rDNA forms a basis for phytoplasma classification, in order to characterize certain isolate and accommodate it into a ribosomal subgroup or a ‘ Candidatus Phytoplasma’ species, it is necessary to analyze regions other than 16S rDNA. For that purpose, additional gene regions such as ribosomal protein genes, secY gene, tuf gene, are commonly used as supplementary phylogenetic markers. A group of phytoplasma isolates from the Croatian grapevines previously assigned to the 16SrXII-A ribosomal subgroup (stolbur/to be described as ’ Ca. P. solani’ ) was chosen to perform molecular typing of tuf gene region and to investigate its variability. To amplify a partial tuf gene sequence of approximately 850 bp, primer pair fTufu/rTufu was employed. All obtained PCR products had a single band of the expected size. However, RFLP analyses revealed that in some samples, along with patterns charateristic of stolbur phytoplasmas, non-specific faint patterns were present. SSCP (single-strand conformation polymorphism) analyses of these PCR fragments also revealed mixed profiles and confirmed the presence of a population of different DNA sequences. Therefore, PCR products from selected samples were cloned and sequenced. In samples that showed RFLP patterns characteristic of phytoplasmas, only phytoplasma tuf gene sequences were confirmed. When BLAST search was performed and tuf sequences showing non-specific patterns were compared to all available sequences in the GenBank, it was confirmed that amplified fragments of the same size are not of phytoplasmal origin. These sequences share 79% identity and 100% coverage with tuf gene reported from Terrimonas ferruginea (previously Flavobacterium ferruginea), a bacterium that lives in the soil. Since tuf fragments were amplified from grapevine tissue that was not rinsed prior to extraction, it is possible that these non-specific amplicons come from a contamination of samples with bacteria from the soil, such as T. ferruginea or closely related. In the future, it would be useful to examine whether this non-specific amplification could be avoided by using other available primers, such as fTufAy/rTufAy, that amplify larger portion of phytoplasma tuf gene.

phytoplasma; PCR; soil bacteria; tufB gene

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