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.

Genetic diversity among phytoplasmas infecting Opuntia species: virtual RFLP analysis identifies new subgroups in the peanut witches'-broom phytoplasma group.

Phytoplasmas were detected in cactus (Opuntia species) plants exhibiting witches'-broom disease symptoms in Yunnan Province, south-western China. Comparative and phylogenetic analyses of 16S rRNA gene sequences indicated that an overwhelming majority of the cactus-infecting phytoplasmas under study belonged to the peanut witches'-broom phytoplasma group (16SrII). Genotyping through use of computer-simulated restriction fragment length polymorphism (RFLP) analysis of 16S rRNA genes revealed a remarkable genetic diversity among these cactus-infecting phytoplasma strains. Based on calculated coefficients of RFLP pattern similarities, seven new 16SrII subgroups were recognized, bringing the total of described group 16SrII subgroups to 12 worldwide. Geographical areas differed from one another in the extent of genetic diversity among cactus-infecting phytoplasma strains. The findings have implications for relationships between ecosystem distribution and the emergence of group 16SrII subgroup diversity.

'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 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'.

Differential amplification of sequence heterogeneous ribosomal RNA genes and classification of the 'Fragaria multicipita' phytoplasma.

Ribosomal (r) RNA interoperon sequence heterogeneity in the 'Fragaria multicipita' phytoplasma, a member of group 16SrVI, was initially observed in RFLP patterns of rDNA amplified in the polymerase chain reaction (PCR), and was confirmed through sequence analysis of cloned rDNA. Sequences from operons rrnA and rrnB were amplified in PCR primed by primer pair P1/P7 but from only rrnA in PCR primed by primer pair R16mF2/R16mR1. Preferential amplification of DNA from operon rrnA was explained by base mismatches between the R16mF2/R16mR1 primers and primer annealing sites in rrnB. The results revealed potential for classification of a phytoplasma into two different subgroups within a 16S rRNA group, if the phytoplasma's 16S rRNA gene sequences are independently characterized. It is suggested that the rRNA operon containing species-specific signature sequence(s) should be specified, and where possible sequences from both 16S rRNA genes should be included, in descriptions of new 'Candidatus Phytoplasma species'.

'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'.

'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.

'Candidatus Phytoplasma castaneae', a novel phytoplasma taxon associated with chestnut witches' broom disease.

In Korea, Japanese chestnut trees (Castanea crenata Sieb. and Zucc.) showing symptoms indicative of witches' broom disease, including abnormally small leaves and yellowing of young leaves, were examined. Since the symptoms were suggestive of a phytoplasma infection, tissues were assayed for phytoplasmas by PCR analysis using a pair of universal primers that amplify a 1.4-kbp phytoplasma 16S rDNA fragment. The phytoplasma-specific fragment was amplified from diseased plants, but not from healthy plants, indicating that a phytoplasma was the causal agent of the chestnut witches' broom (CnWB) disease. The phylogenetic relationship of the CnWB phytoplasma to other phytoplasmas was examined by sequence analysis of the 16S rDNA. A phylogenetic analysis of 16S rDNA sequences of the phytoplasmas placed the CnWB phytoplasma within a distinct subgroup in the phytoplasma clade of the class Mollicutes. The phylogenetic tree indicated that the CnWB phytoplasma is related most closely to coconut phytoplasmas and suggested that they share a common ancestor. The unique properties of the CnWB phytoplasma sequences clearly establish that it represents a novel taxon, 'Candidatus Phytoplasma castaneae'.

Complete nucleotide sequence of the S10-spc operon of phytoplasma: gene organization and genetic code resemble those of Bacillus subtilis.

An 11.4-kbp region of genomic DNA containing the complete S10-spc operon was constructed by an integrative mapping technique with eight plasmid vectors carrying ribosomal protein sequences from onion yellows phytoplasma. Southern hybridization analysis indicated that phytoplasmal S10-spc is a single-copy operon. This is the first complete S10-spc operon of a phytoplasma to be reported, although only a part of six serial genes of the S10 operon is reported previously. The operon has a context of 5'-rps10, rpl3, rpl4, rpl23, rpl2, rps19, rpl22, rps3, rpl16, rpl29, rps17, rpl14, rpl24, rpl5, rps14, rps8, rpl6, rpl18, rps5, rpl30, rpl15, SecY-3', and is composed of 21 ribosomal protein subunit genes and a SecY protein translocase subunit gene. Resembling Bacillus, this operon contains an rpl30 gene that other mollicutes (Mycoplasma genitalium, M. pneumoniae, and M. pulmonis) lack. A phylogenetic tree based on the rps3 sequence showed that phytoplasmas are phylogenetically closer to acholeplasmas and bacillus than to mycoplasmas. In the S10-spc operon, translation may start from either a GTG codon or an ATG codon, and stop at a TGA codon, as has been reported for acholeplasmas and bacillus. However, in mycoplasmas, GTG was found as a start codon, and TGA was found not as a stop codon, but instead as a tryptophan codon. These data derived from the gene organization, and the genetic code deviation support the hypothesis that phytoplasmal genes resemble those of acholeplasmas and Bacillus more than those of other mollicutes.

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 brasiliense', a new phytoplasma taxon associated with hibiscus witches' broom disease.

Hibiscus rosa-sinensis is a valuable ornamental species widely planted in Brazil. Many plants are affected by witches' broom disease, which is characterized by excessive axillary branching, abnormally small leaves, and deformed flowers, symptoms that are characteristic of diseases attributed to phytoplasmas. A phytoplasma was detected in diseased Hibiscus by amplification of rRNA operon sequences by PCRs, and was characterized by RFLP and nucleotide sequence analyses of 16S rDNA. The collective RFLP patterns of amplified 16S rDNA differed from the patterns described previously for other phytoplasmas. On the basis of the RFLP patterns, the hibiscus witches' broom phytoplasma was classified in a new 16S rRNA RFLP group, designated group 16SrXV. A phylogenetic analysis of 16S rDNA sequences from this and other phytoplasmas identified the hibiscus witches' broom phytoplasma as a member of a distinct subclade (designated subclade xiv) of the class Mollicutes. A phylogenetic tree constructed on the basis of 16S rRNA gene sequences was consistent with the hypothesis that there was divergent evolution of hibiscus witches' broom phytoplasma and its closest relatives (members of 16S rRNA RFLP group 16SrII) from a common ancestor. On the basis of unique properties of the DNA from hibiscus witches' broom phytoplasma, it is proposed that it represents a new taxon, namely 'Candidatus Phytoplasma brasiliense'.

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.

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.

A test for measuring growth responses of mollicutes to serum and polyoxyethylene sorbitan.

A test is described that is useful for characterizing mollicutes in terms of the ability to maintain growth in medium containing 15 to 20% fetal bovine serum or in serum-free media with or without 0.04% Tween 80 (polyoxyethylene sorbitan). Representative Acholeplasma species maintained growth in serum-free medium, and about half of the strains tested grew well in Tween 80-supplemented medium. Representative Mycoplasma and Entomoplasma species did not maintain growth in either serum-free medium alone or when Tween 80 was added. Spiroplasma species and group representatives generally failed to sustain growth in serum-free medium with or without Tween 80, but at least four of the spiroplasmas tested maintained growth in serum-free medium. The representative Mesoplasma species grew in serum-free media only when Tween 80 was added, as did Mycoplasma lactucae. Although the test has obvious determinative uses for members of the class Mollicutes, it does not supplant the conventional methodology for assaying the cholesterol requirements of these organisms.

Membrane properties of a plant-pathogenic mycoplasmalike organism.

In terms of biosystematics, the plant-pathogenic mycoplasmalike organisms (MLOs) have been tentatively placed into the class Mollicutes. Certain physiological tests have been used to distinguish families within this class: the sterol-nonrequiring Acholeplasmataceae differ from the sterol-requiring Mycoplasmataceae in that the former are more resistant to lysis by digitonin and more sensitive to lysis in hypotonic salt solutions. To test MLOs for these membrane properties and thus assist in their definitive classification, a dot-blot microassay procedure was used to detect nucleic acids released from lysed cells. The results show that MLOs resemble acholeplasmas grown in the absence of sterols in that they are resistant to digitonin and sensitive to hypotonic salt solutions. The MLOs can be differentiated from acholeplasmas grown without sterols by their greater resistance to lysis in hypotonic sucrose solutions.

Biochemical and serological study of two mycoplasma strains isolated from geese.

2 mycoplasma strains were isolated, one from the phallic lymph of a gander and the other from a cloacal swab of a laying goose. The strains proved to be different from mycoplasma species isolated from geese before. Strain No. 1223 is a glucose-negative and arginine-negative species belonging to the genus Mycoplasma. In the growth inhibition test, it fails to react with hyperimmune sera raised in rabbits against the presently known mycoplasma species of avian origin nor with sera produced against mammalian mycoplasma species sharing its biochemical properties. Strain No. 1225 belongs to the digitonin resistant Acholeplasmataceae family. It is glucose-positive and aesculin-positive. It is negative by all the other tests and fails to react with sera produced against the presently known acholeplasma species.