General Medicine

Apple proliferation (AP), pear decline (PD) and European stone fruit yellows (ESFY) are among the most economically important plant diseases that are caused by phytoplasmas. Phylogenetic analyses revealed that the 16S rDNA sequences of strains of each of these pathogens were identical or nearly identical. Differences between the three phytoplasmas ranged from 1·0 to 1·5 % of nucleotide positions and were thus below the recommended threshold of 2·5 % for assigning species rank to phytoplasmas under the provisional status ‘Candidatus’. However, supporting data for distinguishing the AP, PD and ESFY agents at the species level were obtained by examining other molecular markers, including the 16S–23S rDNA spacer region, protein-encoding genes and randomly cloned DNA fragments. The three phytoplasmas also differed in serological comparisons and showed clear differences in vector transmission and host-range specificity. From these results, it can be concluded that the AP, PD and ESFY phytoplasmas are coherent but discrete taxa that can be distinguished at the putative species level, for which the names ‘Candidatus Phytoplasma mali’, ‘Candidatus Phytoplasma pyri’ and ‘Candidatus Phytoplasma prunorum’, respectively, are proposed. Strains AP15R, PD1R and ESFY-G1R were selected as reference strains. Examination of available data on the peach yellow leaf roll (PYLR) phytoplasma, which clusters with the AP, PD and ESFY agents, confirmed previous results showing that it is related most closely to the PD pathogen. The two phytoplasmas share 99·6 % 16S rDNA sequence similarity. Significant differences were only observed in the sequence of a gene that encodes an immunodominant membrane protein. Until more information on this phytoplasma is available, it is proposed that the PYLR phytoplasma should be regarded as a subtype of ‘Candidatus Phytoplasma pyri’.

The trivial name ‘phytoplasma’ has been adopted to collectively name wall-less, non-helical prokaryotes that colonize plant phloem and insects, which were formerly known as mycoplasma-like organisms. Although phytoplasmas have not yet been cultivated in vitro, phylogenetic analyses based on various conserved genes have shown that they represent a distinct, monophyletic clade within the class Mollicutes. It is proposed here to accommodate phytoplasmas within the novel genus ‘Candidatus (Ca.) Phytoplasma’. Given the diversity within ‘Ca. Phytoplasma’, several subtaxa are needed to accommodate organisms that share <97·5 % similarity among their 16S rRNA gene sequences. This report describes the properties of ‘Ca. Phytoplasma’, a taxon that includes the species ‘Ca. Phytoplasma aurantifolia’ (the prokaryote associated with witches'-broom disease of small-fruited acid lime), ‘Ca. Phytoplasma australiense’ (associated with Australian grapevine yellows), ‘Ca. Phytoplasma fraxini’ (associated with ash yellows), ‘Ca. Phytoplasma japonicum’ (associated with Japanese hydrangea phyllody), ‘Ca. Phytoplasma brasiliense’ (associated with hibiscus witches'-broom in Brazil), ‘Ca. Phytoplasma castaneae’ (associated with chestnut witches'-broom in Korea), ‘Ca. Phytoplasma asteris' (associated with aster yellows), ‘Ca. Phytoplasma mali’ (associated with apple proliferation), ‘Ca. Phytoplasma phoenicium’ (associated with almond lethal disease), ‘Ca. Phytoplasma trifolii’ (associated with clover proliferation), ‘Ca. Phytoplasma cynodontis' (associated with Bermuda grass white leaf), ‘Ca. Phytoplasma ziziphi’ (associated with jujube witches'-broom), ‘Ca. Phytoplasma oryzae’ (associated with rice yellow dwarf) and six species-level taxa for which the Candidatus species designation has not yet been formally proposed (for the phytoplasmas associated with X-disease of peach, grapevine flavescence dorée, Central American coconut lethal yellows, Tanzanian lethal decline of coconut, Nigerian lethal decline of coconut and loofah witches'-broom, respectively). Additional species are needed to accommodate organisms that, despite their 16S rRNA gene sequence being >97·5 % similar to those of other ‘Ca. Phytoplasma’ species, are characterized by distinctive biological, phytopathological and genetic properties. These include ‘Ca. Phytoplasma pyri’ (associated with pear decline), ‘Ca. Phytoplasma prunorum’ (associated with European stone fruit yellows), ‘Ca. Phytoplasma spartii’ (associated with spartium witches'-broom), ‘Ca. Phytoplasma rhamni’ (associated with buckthorn witches'-broom), ‘Ca. Phytoplasma allocasuarinae’ (associated with allocasuarina yellows), ‘Ca. Phytoplasma ulmi’ (associated with elm yellows) and an additional taxon for the stolbur phytoplasma. Conversely, some organisms, despite their 16S rRNA gene sequence being <97·5 % similar to that of any other ‘Ca. Phytoplasma’ species, are not presently described as Candidatus species, due to their poor overall characterization.

Previously, analysis of 16S rDNA sequences placed a newly discovered lineage of bacterial symbionts of arthropods in the ‘Bacteroidetes’. This symbiont lineage is associated with a number of diverse host reproductive manipulations, including induction of parthenogenesis in severalEncarsiaparasitoid wasps (Hymenoptera: Aphelinidae). In this study, electron microscopy and phylogenetic analysis of the 16S rRNA andgyrBgenes of symbionts fromEncarsia hispidaandEncarsia pergandiellaare used to describe and further characterize these bacteria. Phylogenetic analyses based on these two genes showed that theEncarsiasymbionts are allied with theCytophaga aurantiacalineage within the ‘Bacteroidetes’, with their closest described relative being the acanthamoeba symbiont ‘CandidatusAmoebophilus asiaticus’. TheEncarsiasymbionts share 97 % 16S rDNA sequence similarity withBrevipalpusmite andIxodestick symbionts and 88 % sequence similarity with ‘CandidatusA. asiaticus’. Electron microscopy revealed that many of the bacteria found in the ovaries of the twoEncarsiaspecies contained a regular, brush-like array of microfilament-like structures that appear to be characteristic of the symbiont. Finally, the role of this bacterium in parthenogenesis induction inE. hispidawas confirmed. Based on phylogenetic analyses and electron microscopy, classification of the symbionts fromEncarsiaas ‘CandidatusCardinium hertigii’ is proposed.

Intracellular bacteria were observed in the hepatopancreas of the terrestrial isopod Porcellio scaber. Comparative 16S rRNA gene sequence analysis and electron microscopic observations were used to determine the taxonomic position of these intracellular bacteria. Phylogenetic analysis and a complex developmental cycle affiliate these bacteria to the order Chlamydiales, within which they form a distinctive lineage, close to the family Simkaniaceae. They share <92 % 16S rRNA gene sequence similarity with their closest relative and <88 % similarity with other members of the order Chlamydiales. A specific signature oligonucleotide sequence was identified and used as a probe, enabling the identification of intracellular bacteria in infected hepatopancreatic tissue. According to the distinctive morphology of their elementary bodies, which are rod-shaped rather than spherical and contain translucent oblong structures, their genomic properties and their crustacean host, the name ‘Candidatus Rhabdochlamydia porcellionis' is proposed for intracellular bacteria in the hepatopancreas of P. scaber.

Elm yellows group (16SrV) phytoplasmas, which are associated with devastating diseases in elm, grapevine, blackberry, cherry, peach and several other plant species in America, Europe and Asia, represent one of the most diverse phytoplasma clusters. On the basis of phylogenetic analysis of 16S rDNA sequences, elm yellows group phytoplasmas form a discrete subclade within the phytoplasma clade. Three phylogenetic parameters, namely 16S rRNA, ribosomal protein and secY genes, have been evaluated for their usefulness in differentiating elm yellows group phytoplasmas. RFLP analysis of 16S rRNA sequences differentiated the elm yellows group phytoplasmas into five subgroups. Twelve RFLP subgroups were differentiated on the basis of ribosomal protein and 13 were differentiated using secY gene sequences. Phylogenetic analysis of the ribosomal protein genes and secY gene alone or in combination indicated that the subgroups constitute 12 genetically distinct lineages, each of which appears to have evolved under different ecological constraints such as specific vector or plant hosts. On the basis of unique DNA and biological properties, it is proposed that the elm yellows phytoplasma EY1T represents a novel taxon, ‘Candidatus Phytoplasma ulmi’.