J. Mitrovic

Current View on Phytoplasma Genomes and Encoded Metabolism

Phytoplasmas are specialised bacteria that are obligate parasites of plant phloem tissue and insects. These bacteria have resisted all attempts of cell-free cultivation. Genome research is of particular importance to analyse the genetic endowment of such bacteria. Here we review the gene content of the four completely sequenced ‘Candidatus Phytoplasma genomes that include those of ‘Ca. P. asteris strains OY-M and AY-WB, ‘Ca. P. australiense, and ‘Ca. P. mali. These genomes are characterized by chromosome condensation resulting in sizes below 900u2009kb and a G + C content of less than 28%. Evolutionary adaption of the phytoplasmas to nutrient-rich environments resulted in losses of genetic modules and increased host dependency highlighted by the transport systems and limited metabolic repertoire. On the other hand, duplication and integration events enlarged the chromosomes and contribute to genome instability. Present differences in the content of membrane and secreted proteins reflect the host adaptation in the phytoplasma strains. General differences are obvious between different phylogenetic subgroups. ‘Ca. P. mali is separated from the other strains by its deviating chromosome organization, the genetic repertoire for recombination and excision repair of nucleotides or the loss of the complete energy-yielding part of the glycolysis. Apart from these differences, comparative analysis exemplified that all four phytoplasmas are likely to encode an alternative pathway to generate pyruvate and ATP.

'Candidatus Phytoplasma convolvuli', a new phytoplasma taxon associated with bindweed yellows in four European countries.

Plants of Convolvulus arvensis exhibiting symptoms of undersized leaves, shoot proliferation and yellowing, collectively defined as bindweed yellows, were sampled in different regions of Europe and assessed for phytoplasma infection by PCR amplification using phytoplasma universal rRNA operon primer pairs. Positive results were obtained for all diseased plants. RFLP analysis of amplicons comprising the16S rRNA gene alone or the16S rRNA gene and 16-23S intergenic spacer region indicated that the detected phytoplasmas were distinguishable from all other previously described rRNA gene sequences. Analysis of 16S rRNA gene sequences derived from seven selected phytoplasma strains (BY-S57/11, BY-S62/11, BY-I1015, BY-I1016, BY-BH1, BY-BH2 and BY-G) showed that they were nearly identical (99.9-100% gene sequence similarity) but shared less than 97.5% similarity with comparable sequences of other phytoplasmas. Thus, BY phytoplasmas represent a new taxon whose closest relatives are stolbur phytoplasma strains and Candidatus Phytoplasma fragariae with which they share 97.2% and 97.1% 16S rRNA gene sequence similarity, respectively. Phylogenetic analysis of 16S rRNA gene sequences confirmed that bindweed yellows phytoplasma strains collectively represent a distinct lineage within the phytoplasma clade and share a common ancestor with previously published or proposed Candidatus Phytoplasma taxa within a major branch including aster yellows and stolbur phytoplasmas. On the basis of unique 16S rRNA gene sequences and biological properties that include a single host plant species and a geographical distribution limited to parts of Europe, the bindweed yellows (BY) phytoplasmas represent a coherent but discrete taxon, Candidatus Phytoplasma convolvuli, with strain BY-S57/11 (GenBank accession no. JN833705) as the reference strain.

Analysis of the Complete Genomes of Acholeplasma brassicae , A. palmae and A. laidlawii and Their Comparison to the Obligate Parasites from ' Candidatus Phytoplasma'

Analysis of the completely determined genomes of the plant-derived Acholeplasma brassicae strain O502 and A. palmae strain J233 revealed that the circular chromosomes are 1,877,792 and 1,554,229 bp in size, have a G + C content of 36 and 29%, and encode 1,690 and 1,439 proteins, respectively. Comparative analysis of these sequences and previously published genomes of A. laidlawii strain PG-8, ‘Candidatus Phytoplasma asteris strains, ‘Ca. P. australiense and ‘Ca. P. mali show a limited shared basic genetic repertoire. The acholeplasma genomes are characterized by a low number of rearrangements, duplication and integration events. Exceptions are the unusual duplication of rRNA operons in A. brassicae and an independently introduced second gene for a single-stranded binding protein in both genera. In contrast to phytoplasmas, the acholeplasma genomes differ by encoding the cell division protein FtsZ, a wide variety of ABC transporters, the F₀F1 ATP synthase, the Rnf-complex, SecG of the Sec-dependent secretion system, a richly equipped repertoire for carbohydrate metabolism, fatty acid, isoprenoid and partial amino acid metabolism. Conserved metabolic proteins encoded in phytoplasma genomes such as the malate dehydrogenase SfcA, several transporters and proteins involved in host-interaction, and virulence-associated effectors were not predicted for the acholeplasmas.

Generation and Analysis of Draft Sequences of 'Stolbur' Phytoplasma from Multiple Displacement Amplification Templates

Phytoplasma-associated diseases are reported for more than 1,000 plant species worldwide. Only a few genome sequences are available in contrast to the economical importance of these bacterial pathogens. A new strategy was used to retrieve phytoplasma strain-specific genome data. Multiple displacement amplification was performed on DNA obtained from <3 g of plant tissue from tobacco and parsley samples infected with ‘stolbur strains. Random hexamers and Phi29 polymerase were evaluated with and without supplementation by group-assigned oligonucleotides providing templates for Illuminas sequencing approach. Metagenomic drafts derived from individual and pooled strain-specific de novo assemblies were analyzed. Supplementation of the Phi29 reaction with the group-assigned oligonucleotides resulted in an about 2-fold enrichment of the percentage of phytoplasma-assigned reads and thereby improved assembly results. The obtained genomic drafts represent the largest datasets available from ‘stolbur phytoplasmas. Sequences of the two strains (558 kb, 448 proteins and 516 kb, 346 proteins, respectively) were annotated allowing the identification of prominent membrane proteins and reconstruction of core pathways. Analysis of a putative truncated sucrose phosphorylase provides hints on sugar degradation. Furthermore, it is shown that drafts obtained from repetitive-rich genomes allow only limited analysis on multicopy regions and genome completeness.

Differentiation of ‘Candidatus Phytoplasma cynodontis’ Based on 16S rRNA and groEL Genes and Identification of a New Subgroup, 16SrXIV-C

Candidatus Phytoplasma cynodontis is widespread in bermudagrass and has only been found in monocotyledonous plants. Molecular studies carried out on strains collected in Italy, Serbia, and Albania enabled verification of molecular variability in the 16S ribosomal RNA (rRNA) gene. Based on restriction fragment length polymorphism and sequence analyses, the strains from Serbia were clearly differentiated from all others and assigned to a new ribosomal DNA (rDNA) subgroup designated as 16SrXIV-C. A system for amplification of fragments containing the Ca. P. cynodontis groEL gene was developed to enable study of its variability in related strains belonging to different 16SrXIV subgroups. Despite the fact that the groEL gene exhibited a greater sequence variation than 16S rRNA, the phylogenetic tree based on groEL gene sequence analysis was highly congruent with the 16S rDNA-based tree. The groEL gene analyses supported differentiation of the Serbian strains and definition of the new subgroup 16SrXIV-C. Phylogenetic analyses of both genes confirmed distinct phylogenetic lineages for strains belonging to 16SrXIV subgroups. Furthermore, groEL is the only nonribosomal marker developed for characterization of Ca. P. cynodontis thus far, and its application in molecular surveys should provide better insight into the relationships among these phytoplasmas and correlation between strain differentiation and their geographical distribution.

Leafhoppers and Cixiids in Phytoplasma-infected Carrot Fields: Species Composition and Potential Phytoplasma Vectors

SUMMARY The first molecular analysis of samples collected in southern Backa (Serbia) confirmed the presence of aster yellows (16SrI) and stolbur phytoplasmas (16SrXII) in insects belonging to the family Cicadellidae, as well as in carrot plants where the insects were collected. A correct identification of the phytoplasmas and their vectors is essential to arrange effective control strategies to prevent diseases associated with phytoplasmas from spreading to carrots and other vegetable crops. In order to enhance knowledge about insect vectors of aster yellows and stolbur phytoplasmas in Serbia, Cicadellidae and Cixiidae (Homoptera Auchenorrhyncha), the most common vectors of these phytoplasmas, were monitored in southern Backa during 2008. Adults leaf- and planthoppers were collected and identified at species level using standard entomological methods, and tested for phytoplasma presence by means of PCR/RFLP. A total of 13 insect species of Cicadellidae were identified, as follows: a) three species of the subfamily Agallinae: Anaceratagallia ribauti (Ossiannilsson), Anaceratagallia venosa (Fourcroy), and Anaceratagallia laevis (Ribaut); b) seven species of the subfamily Deltocephalinae: Psammotettix confinis (Dahlbom), Psammotettix striatus (Linnaues) Psammottettix alienus (Dahlbom), Macrosteles sexnotatus (Fallen), Ophiola decumana (Kontkanen), Errastunus ocellaris Fallen, and Scaphoideus titanus Ball; c) three species of the subfamily Typhlocibinae: Eupteryx atropunctata (Goeze), Eupteryx mellissae Curtis, Zyginidia pullula (Boheman). Female specimens of the genus Euscelis (Deltocephalinae) were also collected,

Ralstonia solanacearum - a New Threat to Potato Production in Serbia

SuMMARY A survey of ware potatoes (a total of 1127 samples) from localities in Serbia during two consecutive years resulted in detection and identification of R. solanacearum in 17 tuber samples. The monitoring detected the causal agent of bacterial wilt and brown rot of potato in three districts of Vojvodina province. In 2011, the infection by R. solanacearum was confirmed in 7 samples of ware potato tubers (varieties – Saturna, Pirol, Hermes, Panda) in West Backa and South Backa Districts. In 2012, the infection by R. solanacearum was confirmed in 10 potato tuber samples (Lady Claire, Desiree, Panda, Red Fantasy and Vineta varieties) from two districts: South Backa and Central Banat. Bacterial strains obtained from positive samples were identified as R. solanacearum biovar 2 using PCR/RFLP analysis, pathogenicity test on tomato transplants, and nutritional, enzymatic and biovar determination tests. To our best knowledge, these are the only findings of R. solanacearum infection in ware potatoes in Serbia. R. solanacearum was not detected in tomato or any other host plant tested in this study. Furthermore, the bacterium was not found in any of the water samples tested, including those originating from areas in which the bacterium was found in ware potato samples.