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MULTILOCUS SEQUENCE TYPING OF CROATIAN ‘CANDIDATUS PHYTOPLASMA MALI’ ISOLATES

MULTILOCUS SEQUENCE TYPING OF CROATIAN ‘CANDIDATUS PHYTOPLASMA MALI’ ISOLATES Križanac, I., Plavec, J., Budinšćak, Ž., Škorić, D., Šeruga Musić, M. Introduction Apple proliferation disease that severely affects apple trees and its causal agent, ‘Candidatus Phytoplasma mali’, are dispersed in Europe’s apple growing regions. The disease is in nature transmitted by psyllid vectors and can be disseminated further by infected planting material. In 2011, ‘Ca. P. mali’ was detected and molecularly characterized in Croatia for the first time. Until that time, the disease had been regarded as present based on the symptoms and electron microscopy imaging of phytoplasma-like cells. The purpose of our study was to assess a variability of ‘Ca. P.mali’ isolates from apple trees and Cacopsylla picta samples collected in Croatian apple orchards from 2011 until 2016 by using multilocus sequence typing approach. Materials and Methods Apple orchards in major fruit growing regions were surveyed and sampled in a four-year period (2011-2014), with additional sampling in 2016. Phytoplasma presence and identification in plant and insect samples was performed by both conventional PCR/RFLP of 16S rRNA gene and real-time PCR (1). Newly designed primers were used to amplify the entire gene sequences (imp, aceF, pnp and secY) in a direct PCR (2). Amplicons were sequenced on both strands, raw nucleotide sequences assembled and edited using the Sequencher™ 4.7 software and aligned with ClustalX 2.0. Phylogenetic analyses were performed with the MEGA 7 software using the maximum parsimony method. Results and Dıscussıon The genetic diversity of European fruit tree phytoplasmas has been previously determined by MLST (3). However, a limited number of ‘Ca. P. mali’ isolates was fully genotyped in the scope of that study and the new data are scarce. In our survey, majority of isolates corresponded to previously described ‘Ca. P. mali’ genotypes with the prevalent sequence type identical to Italian AP15 isolate (comprehensive genotype A13-P10-I23-S12). Nonetheless, new genotypes were revealed for each of the four genes. In agreement with previous results (3), the most variable gene was imp and the most conserved one was pnp, with seven and two newly discovered genotypes, respectively. Based on the MLST results we can once again report higher than expected genetic variability and an interesting blend of already described and new genotypes. This could be attributed to Croatia’s geographic position at the intersection of the trade routes, as well as possible psyllid long distance migratory ways between the east and west. References 1. Baric & Dalla-Via, 2004. Journal of Microbiological Methods 57: 135-145. 2. Križanac et al., 2017. Journal of Plant Pathology 99: 91-97 3. Danet et al., 2011. Microbiology 157: 438-450.

aceF, pnp, secY, imp, variability, phylogeny

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