Publications

4467

Spartium witches'-broom (SpaWB), buckthorn witches'-broom (BWB) and allocasuarina yellows (AlloY) are witches'-broom and yellows diseases of Spartium junceum (Spanish broom), Rhamnus catharticus (buckthorn) and Allocasuarina muelleriana (Slaty she-oak), respectively. These diseases are associated with distinct phytoplasmas. The SpaWB, BWB and AlloY phytoplasmas share <97·5 % 16S rDNA sequence similarity with each other and with other known phytoplasmas, including the closely related phytoplasmas of the apple proliferation group. Also, the SpaWB, BWB and AlloY phytoplasmas each have a different natural plant host. Based on their unique properties, it is proposed to designate the mentioned phytoplasmas as novel ‘Candidatus’ species under the names ‘Candidatus Phytoplasma spartii’, ‘Candidatus Phytoplasma rhamni’ and ‘Candidatus Phytoplasma allocasuarinae’, respectively.

The symbioses between cellulose-degrading flagellates and bacteria are one of the most fascinating phenomena in the complex micro-ecosystem found in the hindgut of lower termites. However, little is known about the identity of the symbionts. One example is the epibiotic bacteria colonizing the surface of hypermastigote protists of the genusStaurojoenina. By using scanning electron microscopy, it was shown that the whole surface ofStaurojoeninasp. from the termiteNeotermes cubanusis densely covered with long rod-shaped bacteria of uniform size and morphology. PCR amplification of 16S rRNA genes from isolated protozoa and subsequent cloning yielded a uniform collection of clones with virtually identical sequences. Phylogenetic analysis placed them as a new lineage among theBacteroidales, only distantly related to other uncultivated bacteria in the hindgut of other termites, including an epibiont of the flagellateMixotricha paradoxa. The closest cultivated relative wasTannerella forsythensis(<85 % sequence identity). Fluorescencein situhybridization with a newly designed clone-specific oligonucleotide probe confirmed that these sequences belong to the rod-shaped epibionts ofStaurojoeninasp. Transmission electron microscopy confirmed the presence of a Gram-negative cell wall and revealed special attachment sites for the symbionts on the cell envelope of the flagellate host. Based on the isolated phylogenetic position and the specific association with the surface ofStaurojoeninasp., we propose to classify this new taxon ofBacteroidalesunder the provisional name ‘CandidatusVestibaculum illigatum’.

Five strains with strong chitinolytic activity were isolated from a soil sample collected from southern Taiwan. The strains shared more than 92 % DNA–DNA similarity, indicating membership of the same genospecies. This close relationship was supported by high similarities in fatty acid composition and biochemical characteristics. A 16S rRNA gene sequence analysis indicated that the isolates were members of the class ‘Betaproteobacteria’, in which they formed an individual subline of descent that was distantly related (<94 % similarity) to lineages defined by Formivibrio citricus DSM 6150T and Iodobacter fluviatilis DSM 3764T. On the basis of the phylogenetic and phenotypic distinctness of these novel chitin-degrading organisms, a new genus, Chitinibacter, is proposed, with Chitinibacter tainanensis (type strain, S1T=BCRC 17254T=DSM 15459T) as the type species.

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’.

Aster yellows (AY) group (16SrI) phytoplasmas are associated with over 100 economically important diseases worldwide and represent the most diverse and widespread phytoplasma group. Strains that belong to the AY group form a phylogenetically discrete subclade within the phytoplasma clade and are related most closely to the stolbur phytoplasma subclade, based on analysis of 16S rRNA gene sequences. AY subclade strains are related more closely to their culturable relatives, Acholeplasma spp., than any other phytoplasmas known. Within the AY subclade, six distinct phylogenetic lineages were revealed. Congruent phylogenies obtained by analyses of tuf gene and ribosomal protein (rp) operon gene sequences further resolved the diversity among AY group phytoplasmas. Distinct phylogenetic lineages were identified by RFLP analysis of 16S rRNA, tuf or rp gene sequences. Ten subgroups were differentiated, based on analysis of rp gene sequences. It is proposed that AY group phytoplasmas represent at least one novel taxon. Strain OAY, which is a member of subgroups 16SrI-B, rpI-B and tufI-B and is associated with evening primrose (Oenothera hookeri) virescence in Michigan, USA, was selected as the reference strain for the novel taxon ‘Candidatus Phytoplasma asteris’. A comprehensive database of diverse AY phytoplasma strains and their geographical distribution is presented.

Summary ‘ Aegyptianella ranarum ’ (order Rickettsiales), an ultrastructurally defined small, Gram‐negative rod, is known to replicate in the red blood cells of frogs. Heretofore, this bacterium has not been characterized genetically. We cloned and sequenced the 16S rRNA (1310 bp) and gyrB (718 bp) genes of ‘ A. ranarum ’ from a Canadian frog blood specimen. In situ hybridization (with an ‘ A. ranarum ’ 16S rRNA gene polymerase chain reaction product as probe) and electron microscopy confirmed that ‘ A. ranarum ’ forms cytoplasmic inclusions in frog erythrocytes. blast comparisons with GenBank 16S rRNA and gyrB sequences showed that both ‘ A. ranarum ’ genes were most similar (91% and 67% identity) to those of Chryseobacterium meningosepticum , a bacterium in the family Flavobacteriaceae . In contrast, ‘ A. ranarum ’ 16S rRNA shared only 61% identity with Aegyptianella pullorum . Phylogenetic analyses of these genes using phylip supported ‘ A. ranarum ’ as a member of Flavobacteriaceae, but suggested that its cladistic sibling may be Bergeyella zoohelcum or Weeksella virosa , rather than C. meningosepticum. We propose to classify ‘ Aegyptianella ranarum ’ as ‘ Candidatus Hemobacterium ranarum’ in the family Flavobacteriaceae . Our results provide a starting point for studies of related intraerythrocytic bacterial infections in frogs.

Summary Two phylogenetically distinct marine strains producing transparent exopolymers (TEP), designated HTCC2155 T and HTCC2160, were cultivated from Oregon coast seawater by dilution to extinction in a high throughput culturing format. When cultured in low‐nutrient seawater media, these strains copiously produced Alcian Blue‐stainable viscous TEP. Growing cells were attached to each other by the TEP in a three dimensional network. Polymerase chain reaction employing 16S rDNA primers specific for the novel isolates indicated that they are indigenous to the water column of the Atlantic and Pacific oceans. The abundance of the isolates as determined by 16S rRNA dot blots, however, indicated that they are less than 1% of the total bacterial community. In phylogenetic analyses, the strains consistently formed a new phylum‐level lineage within the domain Bacteria , together with members of the candidate phylum VadinBE97, which consists of Victivallis, the first cultured genus in the candidate phylum, and 16S rRNA gene clones from DNA extracted from marine or anaerobic terrestrial habitats. Five putative subgroups were delineated within this phylum‐level lineage, including a marine group and an anaerobic group. The isolates are Gram negative, strictly aerobic, chemoheterotrophic, and facultatively oligotrophic sphere‐shaped bacteria. The DNA G+C content of strain HTCC2155 T was 48.3 mol% and the genome size was 2.9 mb. It is proposed from these observations that the strains be placed into a new genus and a new species named Lentisphaera araneosa (type strain HTCC2155 T  = ATCC BAA‐859 T  = KCTC 12141 T ) gen. nov., sp. nov., the cultured marine representative of the Lentisphaerae phyl. nov., and the phylum be divided into two novel orders named the Lentisphaerales ord. nov. and the Victivallales ord. nov.