Insights into the population structure complexity and the interactions with hosts of Candidatus Phytoplasma phoenicium, the etiological agent of almond witches'-broom disease

Introduction. 'Candidatus Phytoplasma phoenicium' is the etiological agents of a lethal devastating disease of almond trees (almond witches’-broom, AlmWB) in Lebanon and in Iran. AlmWB was also identified on peach and nectarine. Due to complex ecology of 'Ca. P. phoenicium', it is necessary to evaluate its genetic diversity and to investigate the interactions with the hosts. Objective. The aim of this study was to obtain a draft genome of 'Ca. P. phoenicium' to identify genes suitable for distinguishing closely related strains and to acquire information on its metabolism and mechanisms of interaction with hosts. Materials and Methods. During the spring season in 2012, samples from almond, peach, and nectarine plants showing AlmWB symptoms were collected in Lebanon. 16S rDNA PCR-based amplification and nucleotide sequence analysis were utilized to identify phytoplasmas. DNA extracted from almond infected by 'Ca. P. phoenicium' strain SA213 was used for Illumina sequencing. Bioinformatics tools were employed to assembly and annotate the draft genome, and to predict the presence of transmembrane and secreted proteins. TufB, groEL and inmp genes were selected for investigating the genetic diversity among 20 'Ca. P. phoenicium' strains identified in examined plants. Results. Sequence-based typing and phylogenetic analysis of the gene inmp, coding an integral membrane protein, distinguished AlmWB phytoplasmas from diverse host plants, whereas their sequences of 16S rRNA, tufB and groEL genes were identical. Moreover, dN/dS analysis indicated a positive selection acting on inmp gene. Draft genome analyses suggest a parasitism based on the import of glycerol-3-phosphate, a critical mobile inducer of plant systemic immunity. Moreover, the identification of a putative inhibitor of apoptosis-promoting Bax factor suggested its potential role as a phytoplasma fitness-increasing factor by modification of the host-defense response. Conclusion. Even the incompleteness of the draft genome, the results obtained revealed important insights into both the gene repertoire of the pathogen and its population structure, revealing useful information with regard to the candidate determinants of pathogenicity and highlighting genetic diversity among 'Ca. P. phoenicium' strains associated with the disease.