Mariniplasma anaerobium gen. nov., sp. nov., a novel anaerobic marine mollicute, and proposal of three novel genera to reclassify members of Acholeplasma clusters II-IV.

A novel strictly anaerobic chemoorganotrophic bacterium, designated Mahy22T, was isolated from sulfidic bottom water of a shallow brackish meromictic lake in Japan. Cells of the strain were Gram-stain-negative, non-motile and coccoid in shape with diameters of about 600-800 nm. The temperature range for growth was 15-37 °C, with optimum growth at 30-32 °C. The pH range for growth was pH 6.2-8.9, with optimum growth at pH 7.2-7.4. The strain grew with NaCl concentrations of 5% or below (optimum, 2-3%). Growth of the strain was enhanced by the addition of thiosulfate. The major cellular fatty acids were C16:0 and anteiso-C15:0. Respiratory quinones were not detected. The complete genome sequence of strain Mahy22T possessed a 1 885 846 bp circular chromosome and a 12 782 bp circular genetic element. The G+C content of the genome sequence was 30.1 mol%. Phylogenetic analysis based on the 16S rRNA gene revealed that the novel strain belonged to the family Acholeplasmataceae, class Mollicutes. The closest relative of strain Mahy22T with a validly published name was Acholeplasma palmae J233T with a 16S rRNA gene sequence similarity of 90.5%. Based on the results of polyphasic analysis, the name Mariniplasma anaerobium gen. nov., sp. nov. is proposed to accommodate strain Mahy22T, along with reclassification of some Acholeplasma species into Alteracholeplasma gen. nov., Haploplasma gen. nov. and Paracholeplasma gen. nov.

Molecular and microscopical detection of aster yellows phytoplasma associated with infected parsnip.

Typical phytoplasma yellows symptoms were observed in parsnip (Pastinaca sativa L.) plants grown around Edmonton, Alberta, Canada. Examination of ultrathin sections of leaf midribs by electron microscopy revealed numerous phytoplasma bodies localized in the phloem cells. DNA extracted from the infected leaves was amplified with a 16S rDNA universal primer pair P1/P6 giving the expected PCR product of 1.5 kb. The phytoplasma was confirmed as a member of the aster yellows (AY) group by amplification with the specific primer pair R16(1)/F1/R1 that was designed on the basis of AY phytoplasma 16S rDNA sequences. In the nested PCR assays, the expected DNA fragment of 1.1 kb was amplified with this specific primer set. Similar restriction patterns were found for the 1.1 kb PCR products of the phytoplasma isolated from parsnip and an AY phytoplasma control after digestion with restriction endonucleases AluI, HhaI, KpnI and RsaI. This is the first reported observation of aster yellows in parsnip in Canada.

Detection of aster yellows phytoplasma in false flax based on PCR and RFLP.

False flax (Camelina sativa L.) plants were found to be infected with a yellows-type disease caused by a phytoplasma in experimental plots at the Edmonton Research station. Alberta, Canada. Typical phytoplasmas were detected in the phloem cells in ultrathin sections from leaf midrib tissues examined by electron microscopy. These observations were supported by polymerase chain reaction (PCR) using two primer pairs, R16 F2n/R2 and R16(1)F1/R1, derived from phytoplasma rDNA sequences. Aster yellows (AY) and potato witches'-broom (PWB) phytoplasma DNA samples served as controls and were used to study group relatedness. In a direct PCR assay, DNA amplification with universal primer pair R16F2n/R2 gave the expected PCR products of 1.2 kb. Based on a nested-PCR assay using the latter PCR products as templates, and a specific primer pair, R16(1)F1/R1, designed on the basis of AY phytoplasma rDNA sequences, a PCR product of 1.1 kb was obtained from each phytoplasma-infected false flax and AY sample, but not from PWB phytoplasma and healthy controls. DNA amplification with specific primer pair R16(1)F1/R1 and restriction fragment length polymorphism indicated the presence of AY phytoplasma in the infected false flax sample. This is the first reported characterization of AY phytoplasma in false flax.

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.

'Candidatus Phytoplasma omanense', associated with witches'-broom of Cassia italica (Mill.) Spreng. in Oman.

Samples from plants of Cassia italica exhibiting typical witches'-broom symptoms (Cassia witches'-broom; CWB) were examined for the presence of plant pathogenic phytoplasmas by PCR amplification using universal phytoplasma primers. All affected plants yielded positive results. RFLP analyses of rRNA gene products indicated that the phytoplasmas detected were different from those described previously. Phylogenetic analysis of 16S rRNA gene sequences confirmed that CWB represents a distinct lineage and shares a common ancestor with 'Candidatus Phytoplasma phoenicium'. Molecular comparison revealed that the 16S rRNA gene sequences of the four CWB strains (IM-1, IM-2, IM-3 and IM-4) identified in symptomatic C. italica samples were nearly identical (99.6-100 % similarity). The closest relatives were members of the pigeon pea witches'-broom phytoplasma ribosomal group (16SrIX; 95-97 % sequence similarity). On the basis of unique 16S rRNA gene sequences and biological properties, the phytoplasma associated with witches'-broom of C. italica in Oman represents a coherent but discrete novel phytoplasma, 'Candidatus Phytoplasma omanense', with GenBank/DDBJ/EMBL accession number EF666051 representing the reference strain.

Classification of aster yellows-group phytoplasmas based on combined analyses of rRNA and tuf gene sequences.

Seventy phytoplasma isolates, including 10 previously characterized reference strains, of the aster yellows group were examined by RFLP analysis of PCR-amplified rDNA and RFLP and sequence analysis of the tuf gene. On the basis of rDNA restriction profiles, seven previously proposed 16S rDNA subgroups (16SrI-A, -B, -C, -D, -E, -F and -K) were recognized in the material examined. In addition, three new subgroups that differ in the RFLP profiles were identified and designated 16SrI-L, 16SrI-M and 16SrI-N. Of the two types of rDNA sequences used, an 1800 bp fragment comprising the entire 16S rRNA gene and the 16S-23S rDNA spacer region proved more suitable for AY-group phytoplasma differentiation than a 1240 bp fragment of the 16S rRNA gene. Many differences in the rDNA profiles between the subgroups could be explained by sequence heterogeneity of the two phytoplasmal rRNA operons. The subgroups delineated by RFLP analysis of a 940 bp tuf gene fragment are consistent with subgroups defined on the basis of rDNA sequences. However, subgroups 16SrI-D, -L and -M showed the same tuf gene restriction profiles as subgroup 16SrI-B. This result was confirmed by sequence analysis in which these subgroups differed slightly in their tuf gene sequence, when compared with members of subgroup 16SrI-B. On the basis of combined analyses of rDNA and tuf gene sequences and in view of pathological aspects, the taxonomic distinction of AY-subgroups 16SrI-A, -B, -C, -D, -E, -F, -K and -N appears to be substantial.

Classification of phytoplasma strains in the elm yellows group (16SrV) and proposal of 'Candidatus Phytoplasma ulmi' for the phytoplasma associated with elm yellows.

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 EY1(T) represents a novel taxon, 'Candidatus Phytoplasma ulmi'.

'Candidatus phytoplasma ziziphi', a novel phytoplasma taxon associated with jujube witches'-broom disease.

Phylogenetic relationships of five jujube witches'-broom (JWB) phytoplasma isolates from four different districts, and other phytoplasmas, were investigated by 16S rDNA PCR amplification and sequence analysis. The 16S rDNA sequences of any pair of the five isolates of JWB phytoplasmas were > 99.5% similar. The JWB phytoplasma 16S rDNA sequences were most closely related to that of the elm yellows (EY) phytoplasma in 16S-group VIII. Phylogenetic analysis of the 16S rDNA sequences from the JWB phytoplasma isolates, together with sequences from most of the phytoplasmas archived in GenBank, produced a tree in which the JWB isolates clustered as a discrete subgroup. The uniqueness of the JWB phytoplasma appears to be correlated with a specific insect vector (Hishimonus sellatus) and the host plant (Zizyphus jujuba), or with a specific geographical distribution. The unique properties of the JWB phytoplasma sequences clearly indicate that it represents a novel taxon, 'Candidatus Phytoplasma ziziphi'.

Multiple phytoplasmas associated with potato diseases in Mexico.

In recent years, the potato crop in Mexico has been notably affected by diseases recognized as potato purple top (PPT) in foliage and potato hair sprouts (PHS) in germinating tubers. In both cases, these syndromes reduce production by affecting viability of the tubers used as seeds. There is evidence indicating that phytoplasmas are associated with these syndromes. This study presents data on the molecular detection, characterization, and ecology of the pathogens related to PPT and PHS. Restriction fragment length polymorphism (RFLP) and sequence analysis indicated that PPT phytoplasma belongs to the 16SrI group and PHS phytoplasma fits in the 16SrII group. In this paper, we report that the two different phytoplasmas have been found coexisting in the same potato plant, which demonstrates the presence of mixed infection in the field. These phytoplasmas were also detected in weeds surrounding potato fields; therefore they should be considered as alternative hosts or natural reservoirs of PPT and PHS phytoplasmas.

'Candidatus phytoplasma phoenicium' sp. nov., a novel phytoplasma associated with an emerging lethal disease of almond trees in Lebanon and Iran.

Almonds (Prunus amygdalus) represent an important crop in most Mediterranean countries. A new and devastating disease of almond trees in Lebanon was recently reported, characterized by the development of severe witches'-brooms on which no flowers or fruits developed, and leading to tree death within a few years. A phytoplasma was detected in diseased trees by PCR amplification of rRNA operon sequences, and RFLP patterns of amplified DNA indicated that the phytoplasma belonged to the pigeon pea witches'-broom (PPWB) group. In the present work, the presence of a phytoplasma in symptomatic plants was confirmed by electron microscopy; this phytoplasma was graft-transmissible to almond, plum and peach seedlings. The phytoplasma was characterized by sequence analysis of rRNA genes and was shown to be different from the phytoplasmas previously described in the PPWB group. A 16S rDNA phylogenetic tree identified the almond tree phytoplasma as a member of a distinct subclade of the class Mollicutes. Oligonucleotides have been defined for specific detection of the new phytoplasma. The almond phytoplasma from Lebanon was shown to be identical to a phytoplasma that induces a disease called 'almond brooming' in Iran, but different from another PPWB-group phytoplasma that infects herbaceous annual plants in Lebanon. Based on its unique properties, the name 'Candidatus Phytoplasma phoenicium' is proposed for the phytoplasma associated with almond witches'-broom in Lebanon and Iran.

The phytoplasma associated with ash yellows and lilac witches'-broom: 'Candidatus phytoplasma fraxini'.

Phytoplasmas associated with the plant diseases ash yellows (AshY, occurring in Fraxinus) and lilac witches'-broom (LWB, occurring in Syringa) represent a putative species-level taxon. Phytoplasmal DNA from 19 ash or lilac sources across the known geographic range of AshY (71-113 degrees W) was examined to determine if AshY and LWB phytoplasmas are a coherent group, if variability exists in both conserved and anonymous DNA, and if variability in 16S rDNA is related to host or geographic origin. The 16S rRNA gene and the 16S-23S spacer were amplified using primer pair P1/P7 and analysed using 15 restriction enzymes. RFLPs were detected in digests obtained with Alul, Hhal or Taql, for a total of four RFLP profile types. Sequencing of the amplimers from strains AshY1T, AshY3, AshY5 and LWB3 (which represent the four 16S rDNA RFLP profile types) revealed only three positions in the 16S rRNA gene and one position in the 16S-23S spacer at which differences occurred; these were single nucleotide substitutions. Sequence homology between any two strains was > 99.8%. A portion of a ribosomal protein operon, amplified with primer pair rpF1/R1 from each of the four strains noted above, was analysed with six restriction enzymes, resulting in the detection of two RFLP profiles with Msel. Southern analysis, utilizing two non-specific probes from other phytoplasma groups, revealed three RFLP profile types in anonymous chromosomal DNA of strains representing the four 16S rDNA genotypes. Two strains, AshY3 and LWB3, had unique combinations of characters in the various assays. On the basis of RFLP profiles, the strains from the other plants sampled comprised two groups. The grouping was not clearly related to host or geographic origin. The genome size of strain AshY3 was estimated from PFGE data to be 645 kbp. Phylogenetic analysis of a 1423 bp 16S rDNA sequence from strains AshY1T, AshY3, AshY5 and LWB3, together with sequences from 14 other mollicutes archived in GenBank, produced a tree on which the AshY and LWB strains clustered as a discrete group, consistent with previous analyses utilizing only type strain AshY1T. Thus, the AshY phytoplasma group is coherent but heterogeneous. The name 'Candidatus Phytoplasma fraxini' is proposed for this group.