Davis, R. E.

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.

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.

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.

Catharanthus roseus (L.) G. Don (periwinkle) is well known as an experimental host for diverse phytoplasmas that are artificially transmitted to it through the use of dodder (Cuscuta sp.), laboratory vector insects, or grafting. However, few phytoplasma taxa have been reported in natural infections of C. roseus, and the role of C. roseus in phytoplasma dissemination and natural disease spread is not clear. In this study, naturally diseased plants of C. roseus exhibiting yellowing and witches' broom symptoms indicative of phytoplasma infection were observed throughout the year in the state of Rio de Janeiro, Brazil. Shoots and leaves of four diseased plants were assayed for the presence of phytoplasma DNA sequences by nested polymerase chain reactions (PCR) as previously described (2,3). Phytoplasma rDNA was amplified from diseased periwinkle plants in PCR primed by primer pair P1/P7 and was reamplified in nested PCR primed by primer pair R16F2n/R16R2 (F2n/R2). The results indicated the presence of phytoplasma in all four diseased plants. Phytoplasma identification was accomplished by restriction fragment length polymorphism (RFLP) analysis, using 11 restriction enzymes, of 16S rDNA amplified in PCR primed by F2n/R2. Phytoplasmas were classified according to the system of Lee et al. (1). On the basis of collective RFLP patterns of 16S rDNA, the phytoplasma infections in the four periwinkle plants could not be distinguished from one another. Furthermore, the collective RFLP patterns were indistinguishable from those reported previously for hibiscus witches' broom phytoplasma, “Candidatus Phytoplasma brasiliense” (2). The phytoplasma found in C. roseus, designated strain HibWB-Cr, was classified in group 16SrXV (hibiscus witches' broom phytoplasma group). HibWB-Cr is tentatively considered a new strain of “Ca. P. brasiliense”. C. roseus is the first known, naturally diseased alternate plant host of “Ca. P. brasiliense”. The present study identified strain HibWB-Cr in Rio de Janeiro State, where hibiscus witches' broom disease is prevalent (2). How this economically important disease of hibiscus spreads is not known. Our findings raise the possibility that a polyphagous insect vector is involved in the natural transmission of “Ca. P. brasiliense” and that C. roseus or other plant species serve as reservoirs for the spread of this phytoplasma taxon. References: (1) I.-M. Lee et al. Int. J. Syst. Bacteriol. 48:1153, 1998. (2) H. G. Montano et al. Int. J. Syst. Evol. Microbiol. 51:1109, 2001. (3) H. G. Montano et al. Plant Dis. 84:429, 1999.