Association of ‘Candidatus Phytoplasma trifolii’ related strain with white willow proliferation in Iran
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A phytoplasma was identified in white willow trees exhibiting symptoms of proliferation, growing in urban areas in Hamedan province of Iran. The association of a phytoplasma with this disease was confirmed by nested PCR of the 16S rRNA gene using universal phytoplasma-specific primer pair P1/P7 and R16mF2n/R2. Results of nested-PCR amplified a product of expected size (1.4 Kb). Comparison of the sequencing results with those available in GenBank and phylogenetic analysis allowed classification of the phytoplasma into ‘Candidatus phytoplasma trifolii’ (16SrVI) taxonomic group. To the best of our knowledge, this is the first report on the molecular detection and identification of a clover proliferation group phytoplasma in willow trees in Iran.
KeywordsProliferation ‘Candidatus phytoplasma trifolii’ White willow
The present study was initiated to determine the group affiliations of Hamedan province, Iran white willow proliferation-associated phytoplasmas. Knowledge of group affiliation is significant in order to facilitate the future identification of their insect vectors and alternate plant hosts.
Leaf samples from eight white willow trees with proliferation symptoms were collected in 2016 from Hamedan province, Iran. Asymptomatic samples of white willow and positive samples of lime witches broom disease were also included in the assay as negative and positive controls, respectively. Total DNA was extracted as described Doyle and Doyle (1990). A nested-PCR was performed using the universal phytoplasma primers P1/P7 (Deng and Hiruki 1991) followed by R16MF2n/R16MR2 (Gundersen and Lee 1996). Direct sequencing was carried out in both directions from two of eight positive samples (SPH1 and SPH2). Sequences were aligned using the clustalX software (Thompson et al. 1997) and used for searching against the database of National Center for Biotechnology Information (NCBI) by BLASTn. Published phytoplasma sequences were retrieved from GenBank; 29 sequences of 16S rRNA gene from different groups. Phylogenetic trees were built by maximum parsimony (16S rRNA) methods using the neighbor joining algorithm, with a 100-replicate bootstrap search using MEGA6.
Salix spp. are found throughout the world and are associated with phytoplasmas belonging to different taxonomic groups including ‘Ca. P. asteris’ (16SrI) in China (Mou et al. 2014), clover proliferation group in Canada (Khadhair and Hiruki 1995), stolbur phytoplasma (Alfaro-Fernandez et al. 2011) and ‘Ca. P. phoenicum’ and ‘Ca. P. solani’ in Iran (Ghayeb Zamharir 2017; Ghayeb Zamharir and Taheri 2017). However, Clover proliferation group were reported from Salix bebbiana Sarg., S. discolor Muhl., S. exigua Nutt, and S. petiolaris in Canada, but there are no sequence data to compare Canadian isolates with Iranian ones. Also it appears that isolates related to group 16SrVI of phytoplasmas are associated with disease in different Salix species in Iran and Canada. To the best of our knowledge this is the first report of a ‘Ca. P. trifolii’-related strain associated with Salix alba in Iran.
I would like to express my very great appreciation to Prof. Zahra Tanha Maafi for collecting samples.
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