Šeruga Musić, Martina

Phytoplasmas (‘Candidatus Phytoplasma’) are intracellular pleomorphic plant pathogens belonging to the class Mollicutes. They colonize both plant hosts and insect vectors in their life cycle. Apple proliferation (AP) is one of the most important phytoplasmoses present in Europe, causing significant economic losses in apple production. The causal agent, ‘Ca. P. mali’, was identified in apple and Cacopsylla picta samples using both real-time PCR and nested PCR based on the amplification of 16S rDNA. The objective of this study was to gain deeper insights into the epidemiology of apple proliferation in Croatia. Variability of genetic markers other than 16S rRNA was used for characterization of strains. Four molecular markers differing in level of conservation, aceF, pnp, imp, and secY, were selected in line with previously typed fruit tree phytoplasmas. New genotypes were discerned for each genetic marker, and 20 different sequence types were revealed in the Croatian strains of ‘Ca. P. mali’. On the basis of this comprehensive analysis, the founder sequence type ST1 (A13–P10–S12–I21) can be proposed. This is the first extensive research and multigene typing performed on Croatian ‘Ca. P. mali’ strains. Obtained results reveal considerable genetic diversity of epidemiological relevance limited to only two locations in north-western Croatia. Additionally, novel primers were constructed to amplify fragments larger than the entire coding region for all four genes in order to further expand the phytoplasma multi-locus sequence typing scheme.

Phytoplasmas ('Candidatus Phytoplasma') are intracellular pleomorphic plant pathogens belonging to the class Mollicutes. They colonize both plant hosts and insect vectors in their life cycle. Apple proliferation (AP) is one of the most important phytoplasmoses present in Europe causing significant economic losses in apple production. The causal agent, ‘Ca. P. mali', was identified in apple and Cacopsylla picta samples using both real-time PCR and nested PCR based on the amplification of 16S rDNA. The objective of this study was to gain deeper insights into the epidemiology of apple proliferation in Croatia. Variability of genetic markers other than 16S rRNA was used for characterization of strains. Four molecular markers differing in level of conservation, aceF, pnp, imp and secY, were selected in line with previously typed fruit tree phytoplasmas. New genotypes were discerned for each genetic marker and 20 different sequence types were revealed in the Croatian isolates of ‘Ca. P. mali’. On the basis of this comprehensive analysis, the founder sequence type ST1 (A13-P10-S12-I21) can be proposed. This is the first extensive research and multigene typing performed on Croatian ‘Ca. P. mali’ isolates. Obtained results reveal considerable genetic diversity of epidemiological relevance limited to only two locations in north-western Croatia. Additionally, novel primers were constructed to amplify fragments larger than the entire coding region for all four genes in order to further expand phytoplasma multi locus sequence typing scheme.

Phytoplasmas (genus ‘Candidatus Phytoplasma’) encompass a group of uncultivated bacteria affecting numerous plant species and causing significant damage in agriculture worldwide. They have a dual parasitic cycle, including colonization of both plant phloem and insect cells. Their genomes are small, diverse, repetitive, prone to rearrangements and harbour transposon-like elements known as potential mobile units (PMUs). In the Euro-Mediterranean region, ‘Ca. P. solani’ is an important species due to its broad range of plant hosts and insect vectors. To provide insights into the genomic diversity of this species, particularly the repertoire of putative effectors and PMUs, this study conducted genome sequencing and analyses of two ‘Ca. P. solani’ strains originating from different plants and transmitted by different insects. Based on de novo assembly, we obtained 19 contigs totalling 656 141 bp for strain STOL and 28 contigs totalling 707 036 bp for strain ST19. The prevalence of repetitive sequences and PMUs contributed to the fragmentation of these draft assemblies. The annotation identified 28 and 26 genes that encode putative secreted proteins in these two strains, respectively, including several homologues of previously characterized phytoplasma effectors. Our comparative analyses further identified species- and strain-specific genes. Frequently, genes that encode putative secreted proteins and effectors were found within PMU-like regions in both genomes. Moreover, strain STOL showed characteristics of a more reduced genome, having fewer PMU-like repetitive elements and genome rearrangements, while strain ST19 exhibited a higher level of sequence divergence in its PMU genes. The high levels of genomic diversity among ‘Ca. P. solani’ strains suggested rapid evolution of this species, which may contribute to its wide host range and adaptability potential. This study provides novel data on the diversification of ‘Ca. P. solani’ genomes. These results provide a foundation for future functional studies of putative effectors and their interactions with host targets, which could facilitate deciphering the pathogenicity strategies of this successful and versatile pathogen.

Abstract'Candidatus Phytoplasma solani’ (CPs), a phytoplasma endemic to the Euro-Mediterranean basin is a causative agent of several plant diseases, including the grapevine yellows disease “bois noir” (BN). As different CPs strains have been shown to have different ecological reservoirs and pathways for spread, the genetic characterization of CPs strains is a prerequisite, and better control of BN relies on the identification of reservoir plants. The variability of the phytoplasma genotypes involved in the BN pathosystem in Croatian vineyards was assessed by a multilocus sequence typing (MLST) approach. The genotyping was performed on selected grapevine, wild plants, and insects collected within the eleven years of national survey conducted in all Croatian viticultural regions. The extensive tuf, secY, stamp, and vmp1 genes-based MLST analyses revealed two new genotypes for stamp and vmp1 genes, designated as ST59 and V28, respectively, and overall identified 28 different CPs MLST genotypes. The prevalent MLST genotype in grapevine CPsSqt21 (S6/ST6/V18/tuf-b2) was widespread in nine counties across Uplands, Slavonia, and Danube wine regions and was affiliated to the known vector Hyalesthes obsoletus and to Urtica dioica. The other two most frequent genotypes were the U. dioica-associated CPsSqt28 (S39/ST46/V3/tuf-a) and the C. arvensis-associated CPsSqt2 (S1/ST9/V4/tuf-b1). CPs of different vmp1 genotypes was also detected in Cixius wagneri specimens originating from different parts of Croatia. In addition, CPs was detected in several Dichtyophara europaea insects and in two new potential plant reservoirs Ailanthus altissima and Robinia pseudoacacia. The substantial number of found MLST genotypes indicates the presence of several independent epidemiological cycles and is certainly a consequence of a unique geographical position of Croatia, bridging the different eco-climatic areas of central and south-eastern Europe.