Publications

3879

Cauliflower stunt, caused by a phytoplasma of the group 16SrIII-J, was reported in the beginning of 2012 and has occurred with high incidences of infected plants (up to 90%) in crops located in the state of São Paulo in the southeast region of Brazil (3). Diseased plants exhibit general stunting, malformation of inflorescence, reddening leaves, and vessel necrosis (3). Further investigations with plants displaying identical symptoms collected in Nova Bassano, state of Rio Grande do Sul, Brazilian south region, have revealed the presence of a phytoplasma distinct from 16SrIII-J subgroup. Four symptomatic plus four asymptomatic samples were assayed from a field, and the presence of phytoplasma was evidenced by nested PCR assays performed with primers P1/Tint followed by R16F2n/16R2 in three affected plants, which amplified genomic fragments of 1.2 kb from the 16S rRNA gene. No amplification occurred in non-affected samples. Nested PCR products analyzed by conventional RFLP (2) using the enzymes AluI, RsaI, KpnI, HpaII, MseI, HhaI, MboI, and BstUI pointed to the presence of a phytoplasma belonging to group 16SrXV-A in all three phytoplasma-positive samples. Virtual RFLP analysis based on restriction patterns, derived from in silico digestion with 17 endonucleases (4), confirmed the previous results obtained from those samples by conventional RFLP. The 16S rDNA sequences of this phytoplasma identified in cauliflower (GenBank Accession No. JN818845) shared 99% sequence similarity with the reference phytoplasma for subgroup 16SrXV-A (Hibiscus witches'-broom phytoplasma, AF147708), designated ‘Candidatus Phytoplasma brasiliense.’ Analysis of putative restriction sites showed excellent identity between the phytoplasma studied here and the reference phytoplasma. In addition, the arrangement of branches of a phylogenetic tree constructed with phytoplasmas representing diverse 16Sr groups and subgroups supported that the phytoplasma found in cauliflower is closed related to the representative of the subgroup 16SrXV-A. Association of distinct phytoplasmas with the same kind of disease is not rare and the present pathosystem constitutes a new example. Members of this subgroup have been described almost exclusively in Brazil and previously reported in Sida sp., periwinkle, and hibiscus (1). In some European countries, as well as in the United States and Canada, phytoplasmas belonging to group 16SrI has been associated with this type of disease, which has been reported for various species of the genus Brassica, as published in previous works (3). However, a representative of the group 16SrVI was described in infected plants in Iran (3). Although the 16SrIII-J phytoplasma is currently the most important agent of cauliflower stunt in Brazil, and members of 16SrI are prevalent in other countries, this study revealed that a 16Sr XV-A phytoplasma may be also associated with this important disease of brassicas. Besides, the findings here reported expand the natural host range, including cauliflower as new host for phytoplasmas affiliated with 16SrXV-A. References: (1) B. Eckstein et al. Plant Dis. 95:363, 2009. (2) I. M. Lee et al. Int. J. Syst. Bacteriol. 48:1153, 1998. (3) M. C. C. Rappussi et al. Eur. J. Plant. Pathol. 133:829, 2012. (4) Wei et al. Int. J. Syst. Evol. Microbiol. 57:1855, 2007.

ABSTRACT Three cohorts of farmed yellowtail kingfish ( Seriola lalandi ) from South Australia were examined for Chlamydia -like organisms associated with epitheliocystis. To characterize the bacteria, 38 gill samples were processed for histopathology, electron microscopy, and 16S rRNA amplification, sequencing, and phylogenetic analysis. Microscopically, the presence of membrane-enclosed cysts was observed within the gill lamellae. Also observed was hyperplasia of the epithelial cells with cytoplasmic vacuolization and fusion of the gill lamellae. Transmission electron microscopy revealed morphological features of the reticulate and intermediate bodies typical of members of the order Chlamydiales . A novel 1,393-bp 16S chlamydial rRNA sequence was amplified from gill DNA extracted from fish in all cohorts over a 3-year period that corresponded to the 16S rRNA sequence amplified directly from laser-dissected cysts. This sequence was only 87% similar to the reported “ Candidatus Piscichlamydia salmonis” (AY462244) from Atlantic salmon and Arctic charr. Phylogenetic analysis of this sequence against 35 Chlamydia and Chlamydia -like bacteria revealed that this novel bacterium belongs to an undescribed family lineage in the order Chlamydiales . Based on these observations, we propose this bacterium of yellowtail kingfish be known as “ Candidatus Parilichlamydia carangidicola” and that the new family be known as “ Candidatus Parilichlamydiaceae.”

X-disease is one of the most serious diseases known in peach (Prunus persica). Based on RFLP analysis of 16S rRNA gene sequences, peach X-disease phytoplasma strains from eastern and western United States and eastern Canada were classified in 16S rRNA gene RFLP group 16SrIII, subgroup A. Phylogenetic analyses of 16S rRNA gene sequences revealed that the X-disease phytoplasma strains formed a distinct subclade within the phytoplasma clade, supporting the hypothesis that they represented a lineage distinct from those of previously described ‘Candidatus Phytoplasma ’ species. Nucleotide sequence alignments revealed that all studied X-disease phytoplasma strains shared less than 97.5 % 16S rRNA gene sequence similarity with previously described ‘Candidatus Phytoplasma ’ species. Based on unique properties of the DNA, we propose recognition of X-disease phytoplasma strain PX11CT1R as representative of a novel taxon, ‘Candidatus Phytoplasma pruni’. Results from nucleotide and phylogenetic analyses of secY and ribosomal protein (rp) gene sequences provided additional molecular markers of the ‘Ca. Phytoplasma pruni’ lineage. We propose that the term ‘Ca. Phytoplasma pruni’ be applied to phytoplasma strains whose 16S rRNA gene sequences contain the oligonucleotide sequences of unique regions that are designated in the formally published description of the taxon. Such strains include X-disease phytoplasma and - within the tolerance of a single base difference in one unique sequence - peach rosette, peach red suture, and little peach phytoplasmas. Although not employed for taxon delineation in this work, we further propose that secY, rp, and other genetic loci from the reference strain of a taxon, and where possible oligonucleotide sequences of unique regions of those genes that distinguish taxa within a given 16Sr group, be incorporated in emended descriptions and as part of future descriptions of ‘Candidatus Phytoplasma ’ taxa.

This study addressed the taxonomic position and group classification of a phytoplasma responsible for virescence and phyllody symptoms in naturally diseased Madagascar periwinkle plants in western Malaysia. Unique regions in the 16S rRNA gene from the Malaysian periwinkle virescence (MaPV) phytoplasma distinguished the phytoplasma from all previously described ‘ Candidatus Phytoplasma ’ species. Pairwise sequence similarity scores, calculated through alignment of full-length 16S rRNA gene sequences, revealed that the MaPV phytoplasma 16S rRNA gene shared 96.5 % or less sequence similarity with that of previously described ‘ Ca. Phytoplasma ’ species, justifying the recognition of the MaPV phytoplasma as a reference strain of a novel taxon, ‘Candidatus Phytoplasma malaysianum’. The 16S rRNA gene F2nR2 fragment from the MaPV phytoplasma exhibited a distinct restriction fragment length polymorphism (RFLP) profile and the pattern similarity coefficient values were lower than 0.85 with representative phytoplasmas classified in any of the 31 previously delineated 16Sr groups; therefore, the MaPV phytoplasma was designated a member of a new 16Sr group, 16SrXXXII. Phytoplasmas affiliated with this novel taxon and the new group included diverse strains infecting periwinkle, coconut palm and oil palm in Malaysia. Three phytoplasmas were characterized as representatives of three distinct subgroups, 16SrXXXII-A, 16SrXXXII-B and 16SrXXXII-C, respectively.