Zhao, Y.

Sweet cherry (Prunus avium L.) is a deciduous tree originating in the Black Sea/Caspian Sea region where Asia and Europe converge. Being highly valued for its timber and fruit, sweet cherry has been cultivated and naturalized on all continents. Over the past decade, the area of sweet cherry cultivation increased rapidly in China and has reached 140,000 ha. In April 2013, sweet cherry trees (cv. Summit) exhibiting floral virescence symptoms were observed in two orchards located in suburban Taian, Shandong Province, China. The diseased trees developed flowers having white petals with green veins or abnormal floral structures having cupped, green petals. The affected flowers failed to set fruit. A month following the first appearance of the virescence symptoms, the diseased trees became wilted and eventually died. Leaf and stem samples were collected from nine symptomatic and two nearby symptomless trees. Total DNA was extracted from each sample using the Plant Quick DNA Extract Kit (TianGen, Beijing, China). Nested-PCR was carried out using phytoplasma-universal primer pairs P1/P7 and R16F2n/R16R2 (1). All PCR assays with DNA templates from symptomatic samples yielded an amplicon of 1.25 kb, corresponding to the full-length F2nR2 region of phytoplasmal 16S rDNA. No amplicon was generated in PCRs containing DNA templates from symptomless plants. The amplicons were cloned into plasmid vector pMD18-T (TaKaRa, Dalian, China) and sequenced. The obtained sequences were nearly identical, and a representative sequence was deposited into GenBank (Accession No. KF268424). An analysis of the sequence through the iPhyClassifier (4) revealed that the sweet cherry virescence (SCV) disease was associated with infection by a phytoplasma closely related to the reference strain of ‘Candidatus Phytoplasma ziziphi.’ The 16S rDNA F2nR2 region of the SCV phytoplasma shared 99.8% nucleotide sequence identity with that of ‘Candidatus Phytoplasma ziziphi’ reference strain (Accession No. AB052876). A computer-simulated restriction fragment length polymorphism (RFLP) analysis of the SCV phytoplasma 16S rDNA F2nR2 sequence with a set 17 restriction enzymes (3) resulted in a collective RFLP profile identical to the reference pattern of the elm yellows phytoplasma group, subgroup B (16SrV-B). Phytoplasmal diseases of sweet cherry were reported previously in Europe and the etiological agents were phytoplasmas of other groups, including the aster yellows group (16SrI), the X-disease group (16SrIII), and the apple proliferation group (16SrX) (2). To our knowledge, this is the first report of a phytoplasmal disease of sweet cherry in China, and the SCV phytoplasma is a new member of the subgroup 16SrV-B. Presence of 16SrV-B phytoplasmas and their etiological association with various plant diseases in China have been reported previously; affected host plants included jujube, hemp fiber, paper mulberry, Chinese cherry, plum, apricot, red barberry, clover, dianthus, elm, and sunshine tree. Our identification of the SCV phytoplasma expands the known plant host range of the 16SrV-B phytoplasma lineage. The impact of the SCV phytoplasma in the regional ecosystem and in sweet cherry production is being assessed. References: (1) I. M. Lee et al. Int. J. Syst. Bacteriol. 48:1153, 1998. (2) S. Paltrinieri et al. Acta Hort. 550:365, 2001. (3) W. Wei et al. Int. J. Syst. Evol. Microbiol. 57:1855, 2007. (4) Y. Zhao et al. Int. J. Syst. Evol. Microbiol. 59:2582, 2009.

Phytoplasmas classified in group 16SrXII infect a wide range of plants and are transmitted by polyphagous planthoppers of the family Cixiidae. Based on 16S rRNA gene sequence identity and biological properties, group 16SrXII encompasses several species, including ‘Candidatus Phytoplasma australiense ’, ‘Candidatus Phytoplasma japonicum ’ and ‘Candidatus Phytoplasma fragariae ’. Other group 16SrXII phytoplasma strains are associated with stolbur disease in wild and cultivated herbaceous and woody plants and with bois noir disease in grapevines (Vitis vinifera L.). Such latter strains have been informally proposed to represent a separate species, ‘Candidatus Phytoplasma solani’, but a formal description of this taxon has not previously been published. In the present work, stolbur disease strain STOL11 (STOL) was distinguished from reference strains of previously described species of the ‘Candidatus Phytoplasma ’ genus based on 16S rRNA gene sequence similarity and a unique signature sequence in the 16S rRNA gene. Other stolbur- and bois noir-associated (‘Ca. Phytoplasma solani’) strains shared >99 % 16S rRNA gene sequence similarity with strain STOL11 and contained the signature sequence. ‘Ca. Phytoplasma solani’ is the only phytoplasma known to be transmitted by Hyalesthes obsoletus. Insect vectorship and molecular characteristics are consistent with the concept that diverse ‘Ca. Phytoplasma solani’ strains share common properties and represent an ecologically distinct gene pool. Phylogenetic analyses of 16S rRNA, tuf, secY and rplV–rpsC gene sequences supported this view and yielded congruent trees in which ‘Ca. Phytoplasma solani’ strains formed, within the group 16SrXII clade, a monophyletic subclade that was most closely related to, but distinct from, that of ‘Ca. Phytoplasma australiense ’-related strains. Based on distinct molecular and biological properties, stolbur- and bois noir-associated strains are proposed to represent a novel species level taxon, ‘Ca. Phytoplasma solani’; STOL11 is designated the reference strain.

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.

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.

Symptoms of abnormal proliferation of shoots resulting in formation of witches’-broom growths were observed on diseased plants of passion fruit (Passiflora edulis f. flavicarpa Deg.) in Brazil. RFLP analysis of 16S rRNA gene sequences amplified in PCRs containing template DNAs extracted from diseased plants collected in Bonito (Pernambuco) and Viçosa (Minas Gerais) Brazil, indicated that such symptoms were associated with infections by two mutually distinct phytoplasmas. One phytoplasma, PassWB-Br4 from Bonito, represents a new subgroup, 16SrIII-V, in the X-disease phytoplasma group (‘Candidatus Phytoplasma pruni’-related strains). The second phytoplasma, PassWB-Br3 from Viçosa, represents a previously undescribed subgroup in group 16SrVI. Phylogenetic analyses of 16S rRNA gene sequences were consistent with the hypothesis that strain PassWB-Br3 is distinct from previously described ‘Ca. Phytoplasma ’ species. Nucleotide sequence alignments revealed that strain PassWB-Br3 shared less than 97.5 % 16S rRNA gene sequence similarity with previously described ‘Ca. Phytoplasma ’ species. The unique properties of its DNA, in addition to natural host and geographical occurrence, support the recognition of strain PassWB-Br3 as a representative of a novel taxon, ‘Candidatus Phytoplasma sudamericanum’.

A novel phytoplasma, designated strain SoyST1c1, associated with a newly emerging disease in soybean (Glycine max), known as soybean stunt (SoyST), was found in 2002 in a soybean plantation in Alajuela Province, Costa Rica. The same phytoplasma, or a very closely related strain, also infected sweet pepper (Capsicum annuum) with purple vein syndrome (SwPPV) and passion fruit vine (Passiflora edulis) with bud proliferation disease (PasFBP) in the same region. Sequence analysis of cloned 16S rRNA gene sequences (GenBank accession nos FJ226068–FJ226073 and HQ225624–HQ225635) indicated that all three affected plants were infected by phytoplasmas that shared <97.5 % sequence similarity with previously described phytoplasmas. The SoyST-causing phytoplasma represents a new taxon, most closely related to phytoplasma group 16SrI and 16SrXII strains. Virtual RFLP analysis indicated that the SoyST-causing phytoplasma and its closely related strains represent a novel 16Sr group, designated 16SrXXXI. Phylogenetic analysis of 16S rRNA gene sequences from the new phytoplasma strains, those previously described as ‘Candidatus Phytoplasma spp.’ and other distinct, as yet unnamed, phytoplasmas indicated that the SoyST-causing phytoplasma represents a distinct lineage within the aster yellows/stolbur branch on the phylogenetic tree. On the basis of its unique 16S rRNA gene sequence and biological properties, strain SoyST1c1 represents a novel taxon, for which the name ‘Candidatus Phytoplasma costaricanum’ is proposed with SoyST1c1 as the reference strain.

Cacti (Opuntia spp.) are perennial, evergreen, succulent plants native to arid areas of the Americas. Because of their aesthetic appearance, many cacti have been cultivated and introduced to other parts of the world as ornamentals. Cacti are susceptible to phytoplasma infections and develop witches'-broom (WB) disease. Currently, all reported cactus WB cases are associated with infections by phytoplasmas in the peanut witches'-broom group (16SrII) (1,2,4). During a phytoplasma diversity survey carried out during 2004 in Yunnan, China, we collected 29 malformed and 14 healthy-looking naturally occurring cactus plants from 14 locations representing five geographical regions. Each of the 29 malformed plants exhibited stunted growth and possessed clusters of highly proliferating cladodia, typical symptoms of cactus WB disease. Nested-PCR was carried out on the DNA samples extracted from young cladodia of these plants using phytoplasma-universal 16S rDNA primers P1A/P7A and R16F2n/R16R2 (3). Results revealed that all 29 diseased plants that were examined were infected by phytoplasmas, whereas all 14 healthy-looking plants were negative for phytoplasmas. Subsequent restriction fragment length polymorphism (RFLP) analysis of the PCR-amplified 1.25-kb 16S rDNA fragments indicated that 28 diseased plants were infected by a phytoplasma of group 16SrII, whereas one plant (from Suan Village) was infected by a ‘Candidatus Phytoplasma asteris’-related (group 16SrI) phytoplasma designated as strain YN26. Nucleotide sequence analysis of the strain YN26 partial rRNA operon (GenBank Accession No. EF190970), covering a near full-length 16S rRNA gene, a 16S-23S rRNA intergenic spacer, a tRNA-Ile gene, and a partial 23S rRNA gene, suggested that this phytoplasma is most closely related to an ash witches'-broom phytoplasma (GenBank Accession No. AY566302, 99.7% identity) and an epilobium phyllody phytoplasma (GenBank Accession No. AY101386, 99.7% identity), both members of subgroup16SrI-B. This YN26-infected cactus plant was transferred to a greenhouse and maintained for more than 2 years, during which time DNA samples were extracted and tested two additional times. The same 16S rDNA RFLP pattern type was consistently obtained in these tests, confirming that the plant remained infected by the 16SrI phytoplasma. To our knowledge, this is the first report of a natural infection of a cactus species by a group 16SrI phytoplasma. Since this 16SrI-cactus WB phytoplasma was found in the same geographical location where 16SrII-cactus WB phytoplasma was detected both in this and a previous study (1), the findings raised the question whether 16SrI- and 16SrII-cactus WB phytoplasmas have overlapping geo- and bioecological niches. References: (1) H. Cai et al. Plant Pathol. 51:394, 2002. (2) E. Choueiri et al. Plant Dis. 89:1129, 2005. (3) I. M. Lee et al. Int. J. Syst. Evol. Microbiol 54:337, 2004. (4) N. Leyva-Lopez et al. Phytopathology (Abstr.) 89(suppl):S45, 1999.

Phytopathogenic mollicutes, which include spiroplasmas and phytoplasmas, are cell wall-less bacteria that parasitize plant hosts and insect vectors. Knowledge of the evolution of these agents is important in understanding their biology. The availability of the first complete phytoplasma and several partial spiroplasma and phytoplasma genome sequences made possible an investigation of evolutionary relationships between phytopathogenic mollicutes and other micro-organisms, especially Gram-positive bacteria, using a comparative genomics approach. Genome data from a total of 41 bacterial species were used in the analysis. Sixty-one conserved proteins were selected from each species for the construction of a hypothetical phylogenetic tree. The genes encoding these selected proteins are among a core of genetic elements that constitute a hypothetical minimal genome. The proteins were concatenated into five superproteins according to their functional categories, and phylogenetic trees were reconstructed using distance, parsimony and likelihood methods. Phylogenetic trees based on the five sets of concatenated proteins were congruent in both clade topology and relative branching length. Spiroplasma kunkelii and phytoplasmas clustered together with other mollicutes, forming a monophyletic group. Phytoplasmas diverged from spiroplasmas and mycoplasmas at early stages in the evolution of mollicutes. Branch lengths on the phylogenetic trees were noticeably longer in the Mollicutes clade, suggesting that the genes encoding the five sets of proteins evolved at a greater rate in this clade than in other clades. This observation reinforces the concept that mollicutes have rapidly evolving genomes.