General Medicine

Pinus silvestris and Pinus halepensis trees grown in Germany and Spain, respectively, showing abnormal shoot branching, dwarfed needles and other symptoms were examined for the presence of plant-pathogenic mollicutes (phytoplasmas). While phytoplasmas could not be detected unambiguously with microscopical methods, PCR amplification using universal phytoplasma primers yielded positive results. Samples collected from symptomatic and non-symptomatic plant parts of both symptomatic Pinus silvestris and Pinus halepensis trees tested positive. Also, surrounding non-symptomatic trees proved to be phytoplasma-infected. Comparisons revealed that the 16S rRNA gene sequences of the phytoplasmas identified in Pinus silvestris and Pinus halepensis were nearly identical. However, the pine phytoplasma is only distantly related to other phytoplasmas. The closest relatives are members of the palm lethal yellowing and rice yellow dwarf groups and ‘Candidatus Phytoplasma castaneae’, which share between 94·5 and 96·6 % 16S rRNA gene sequence similarity. From these data it can be concluded that the phytoplasmas identified in the two Pinus species represent a coherent but discrete taxon; it is proposed that this taxon be distinguished at putative species level under the name ‘Candidatus Phytoplasma pini’.

A novel small haemoplasma was detected following cytological examination of blood smears from a splenectomized dog with haemic neoplasia. The 16S rRNA and rnpB genes of the organism were partially sequenced and a phylogenetic tree constructed. The organism was most closely related to the small feline haemoplasma, ‘Candidatus Mycoplasma haemominutum’ (94 % 16S rRNA gene nucleotide sequence identity; 75 % rnpB) and was only distantly related to Mycoplasma haemocanis (78 % 16S rRNA gene nucleotide sequence identity; 65 % rnpB). As this organism has not been cultured in vitro, the candidate species name ‘Candidatus Mycoplasma haematoparvum’ is proposed.

Phylogenetic 16S rRNA gene analysis was used to assign the bacterial leaf-nodulating endosymbionts of two tropical African Psychotria species to the genus Burkholderia. The microsymbionts of the different Psychotria hosts were recognized as distinct and novel species of Burkholderia on the basis of relatively low intersequence similarities and sufficiently large evolutionary distances when compared with each other and their closest validly named neighbours. The obligate endosymbiotic nature of the bacteria prevented their in vitro cultivation and the deposition of type strains to culture collections. Therefore, the provisional status Candidatus is assigned to the bacterial partners of Psychotria calva and Psychotria nigropunctata, with the proposal of the names ‘Candidatus Burkholderia calva’ and ‘Candidatus Burkholderia nigropunctata’, respectively.

While Helicobacter pylori is accepted as the major bacterial agent of gastric disease in humans, some patients and many animals are infected with a larger, tightly helical-shaped bacterium previously referred to as ‘Helicobacter heilmannii’ or ‘Gastrospirillum hominis’. Taxonomic classification of these bacteria has been hampered by the inability to cultivate them in vitro and by the inadequate discriminatory power of 16S rRNA gene sequence analysis. This study describes the detection and phylogenetic analysis of 26 different gastrospirillum isolates from humans and animals, which incorporates sequence data based on the 16S rRNA and urease genes. Fifteen gastrospirilla detected in humans, primates and pigs clustered with ‘Candidatus Helicobacter suis’, thus expanding the host range for this organism. By comparison, based on 16S rRNA data, the remaining 11 gastrospirilla could not be differentiated from Helicobacter felis, Helicobacter bizzozeronii and Helicobacter salomonis. However, urease gene sequence analysis allowed for the discrimination of this latter group into four discrete clusters, three of which contained the above recognized species. The fourth cluster contained isolates from human and feline hosts, and should provisionally be considered a unique bacterial species, for which the name ‘Candidatus Helicobacter heilmannii’ is proposed.

A novel bacterium that infects laboratory rats was isolated from wild Rattus norvegicus rats in Japan. Transmission electron microscopy of the spleen tissue revealed small cocci surrounded by an inner membrane and a thin, rippled outer membrane in a membrane-bound inclusion within the cytoplasm of endothelial cells. Phylogenetic analysis of the 16S rRNA gene sequence of the bacterium found in R. norvegicus rats and Ixodes ovatus ticks in Japan revealed that the organism represents a novel clade in the family Anaplasmataceae, which includes the Schotti variant found in Ixodes ricinus ticks in the Netherlands and the Ehrlichia-like Rattus strain found in R. norvegicus rats from China. The novel clade was confirmed by phylogenetic analysis of groESL sequences found in R. norvegicus rats and Ixodes ovatus ticks in Japan. No serological cross-reactivity was detected between this bacterium and members of the genera Anaplasma, Ehrlichia or Neorickettsia in the family Anaplasmataceae. It is proposed that this new cluster of bacteria should be designated ‘Candidatus Neoehrlichia mikurensis’.

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.

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.

Clover proliferation phytoplasma (CPR) is designated as the reference strain for the CP phylogenetic group or subclade, on the basis of molecular analyses of genomic DNA, the 16S rRNA gene and the 16S–23S spacer region. Other strains related to CPR include alfalfa witches'-broom (AWB), brinjal little leaf (BLL), beet leafhopper-transmitted virescence (BLTV), Illinois elm yellows (ILEY), potato witches'-broom (PWB), potato yellows (PY), tomato big bud in California (TBBc) and phytoplasmas from Fragaria multicipita (FM). Phylogenetic analysis of the 16S rRNA gene sequences of BLL, CPR, FM and ILEY, together with sequences from 16 other phytoplasmas that belong to the ash yellows (AshY), jujube witches'-broom (JWB) and elm yellows (EY) groups that were available in GenBank, produced a tree on which these phytoplasmas clearly clustered as a discrete group. Three subgroups have been classified on the basis of sequence homology and the collective RFLP patterns of amplified 16S rRNA genes. AWB, BLTV, PWB and TBBc are assigned to taxonomic subgroup CP-A, FM belongs to subgroup CP-B and BLL and ILEY are assigned to subgroup CP-C. Genetic heterogeneity between different isolates of AWB, CPR and PWB has been observed from heteroduplex mobility assay analysis of amplified 16S rRNA genes and the 16S–23S spacer region. Two unique signature sequences that can be utilized to distinguish the CP group from others were present. On the basis of unique properties of the DNA from clover proliferation phytoplasma, the name ‘Candidatus Phytoplasma trifolii’ is proposed for the CP group.

Bermuda grass white leaf (BGWL) is a destructive, phytoplasmal disease of Bermuda grass (Cynodon dactylon). The causal pathogen, the BGWL agent, differs from other phytoplasmas that cluster in the same major branch of the phytoplasma phylogenetic clade in <2·5 % of 16S rDNA nucleotide positions, the threshold for assigning species rank to phytoplasmas under the provisional status ‘Candidatus’. Thus, the objective of this work was to examine homogeneity of BGWL isolates and to determine whether there are, in addition to 16S rDNA, other markers that support delineation of the BGWL agent at the putative species level. Phylogenetic analyses revealed that the 16S rDNA sequences of BGWL strains were identical or nearly identical. Clear differences that support separation of the BGWL agent from related phytoplasmas were observed within the 16S–23S rDNA spacer sequence, by serological comparisons, in vector transmission and in host-range specificity. From these results, it can be concluded that the BGWL phytoplasma is a discrete taxon at the putative species level, for which the name ‘Candidatus Phytoplasma cynodontis' is proposed. Strain BGWL-C1 was selected as the reference strain. Phytoplasmas that are associated with brachiaria white leaf, carpet grass white leaf and diseases of date palms showed 16S rDNA and/or 16S–23S rDNA spacer sequences that were identical or nearly identical to those of the BGWL phytoplasmas. However, the data available do not seem to be sufficient for a proper taxonomic assignment of these phytoplasmas.