Diversity of the 'Candidatus Phytoplasma asteris' and 'Candidatus Phytoplasma fraxini' isolates that infect urban trees in Bogotá, Colombia.

Phytoplasmas have been associated with a disease that affects trees of at least 11 species from different botanic families in Bogotá, Colombia. 'Candidatus Phytoplasma asteris' and 'Candidatus Phytoplasma fraxini' are the major groups of phytoplasma in the area of Bogotá. In this study, the genetic diversity within 'Ca. P. asteris' and 'Ca. P. fraxini' was studied in five urban tree species: Croton species (Euphorbiaceae), Fraxinus uhdei (Oleaceae), Magnolia grandiflora (Magnoliaceae), Populus nigra (Salicaceae) and Quercus humboldtii (Fagaceae). Analyses of the 16S rRNA gene using nested PCR, RFLP and sequencing showed that phytoplasmas of 'Ca. P. asteris' could be assigned to: subgroup 16SrI-B; a new subgroup named 16SrI-AF, with a restriction pattern similar to that of 16SrI-B; and a new subgroup named 16SrI-AG, with a restriction pattern similar to that of 16SrI-K and 16SrI-AH with a restriction pattern similar to that of 16SrI-AC. 'Ca. P. fraxini' isolates belonged to a new subgroup named 16SrVII-G, with a restriction pattern similar to that of 16SrVII-A. To complement the identification of the phytoplasma strains, we amplified nonribosomal genes such as leuS and secA. Unexpectedly, it was observed that in 16 trees in which 16S rRNA gene analysis showed the presence of 'Ca. P. fraxini' only, the leuS or secA primers amplified sequences exclusively affiliated to 'Ca. P. asteris. In those plants, sequences belonging to 'Ca. P. fraxini' leuS or secA genes were not amplified. The present work contributes to the identification of novel strains of both species in Colombia, and supports previous suggestions that phytoplasmas in South America are highly variable.

Phytoplasmas diversity and identification of new aster yellows subgroup (16SrI) associated with weed species in Argentina.

Symptoms of phytoplasma infection were observed in different weed species, Bidens subalternans, Conyza bonariensis, Heterosperma ovatifolium and Conium maculatum, collected from diverse geographical regions in Argentina. To confirm the association of phytoplasma infection with symptomatic plants, PCR, RFLP and phylogenetic analyses based on 16S rRNA-encoding sequences were performed. In this work, we report the presence of phytoplasmas from group 16SrVII (subgroup 16VII-B) infecting C. bonariensis and B. subalternans and from group 16SrIII (subgroup 16SrIII-X) B. subalternans, H. ovatifolium, and C. maculatum. Phytoplasmas from the aster yellows group were detected infecting C. bonariensis and B. subalternans. Analysis of 16S rRNA-encoding genes revealed the presence of two distinct operons, rrnB (16SrI-B) and newly described rrnA, which is different from the reference RFLP patterns of all previously established 16SrI-subgroups. A single rp operon sequence analysis reveals the presence of simple infection and confirms a description of a novel subgroup. On the basis of these results we propose a designation of new subgroup 16SrI-(B/AJ) AJ (rp-AJ). To our knowledge, this is the first report of phytoplasmas infecting Bidens subalternans¸ Heterosperma ovatifolium and Conium maculatum.

Identification of a 'Candidatus Phytoplasma hispanicum'-related strain, associated with yellows-type diseases, in smoke-tree sharpshooter (Homalodisca liturata Ball).

The 16SrXIII group from phytoplasma bacteria were identified in salivary glands from Homalodisca liturata, which were collected in El Comitán on the Baja California peninsula in Mexico. We were able to positively identify 15 16S rRNA gene sequences with the corresponding signature sequence of 'CandidatusPhytoplasma' (CAAGAYBATKATGTKTAGCYGGDCT) and in silico restriction fragment length polymorphism (RFLP) profiles (F value estimations) coupled with a phylogenetic analysis to confirm their relatedness to 'CandidatusPhytoplasma hispanicum', which in turn belongs to the 16SrXIII group. A restriction analysis was carried out with AluI and EcoRI to confirm that the five sequences belongs to subgroup D. The rest of the sequences did not exhibit any known RFLP profile related to a subgroup reported in the 16SrXIII group.

Delineation of a novel subgroup 16SrXIII-J phytoplasma, a 'Candidatus Phytoplasma hispanicum'-related strain, based on computer-simulated RFLP and phylogenetic analysis.

Symptoms of fruit phyllody and slow growth, which are suggestive of phytoplasma infection, were observed in strawberry plants cultivated in commercial fields. In order to provide evidence of association of phytoplasma with affected plants, assays for detecting and identifying were performed through computer-simulated restriction fragment length polymorphism (RFLP) and phylogenetic analysis. Total DNA was extracted from symptomatic and asymptomatic samples and used as template in nested PCR primed by the primers P1/Tint followed by R16F2n/16R2. Amplified DNA fragments of 1.2 kb from the 16S rRNA gene revealed the presence of phytoplasma in all symptomatic samples. Molecular detection was confirmed by electron transmission microscopy, which evidenced pleomorphic bodies in the phloem vessels. Nucleotide sequence representative of the strawberry phytoplasma shared 97.2 to 99 % similarity with phytoplasmas currently classified as members of the distinct subgroups within the 16SrXIII group. Similarity coefficient (F) values ranged from 0.70 to 0.92, indicating that strawberry phytoplasma delineates a new strain in addition to 'Candidatus Phytoplasma hispanicum'-related strains. The evolutionary tree displayed that this strain emerges as a new branch in relation to those previously described. The novel strain, designated SFP (strawberry fruit phyllody) phytoplasma represents the new 16SrXIII-J subgroup and its sequence, denominated SFP-Br02, was deposited in the GenBank database (EU719108). These findings contribute for the knowledge of the genetic diversity existing among members of the group 16SrXIII and establishes strawberry as an additional host of representatives of this group in Brazil.

Molecular diagnostic assays based on cpn60 UT sequences reveal the geographic distribution of subgroup 16SrXIII-(A/I)I phytoplasma in Mexico.

Geographically diverse samples from strawberry exhibiting symptoms of Strawberry Green Petal (SbGP), periwinkle plants with virescence, and blackberry, blueberry, and raspberry plants displaying yellowing and inedible fruits, were assayed for the presence of phytoplasma DNA. PCR targeting the 16S rRNA-encoding gene and chaperonin-60 (cpn60) showed that the plants were infected with phytoplasma subgroup16SrXIII-(A/I)I (SbGP/MPV). To examine the geographic distribution of this pathogen in Mexico, we designed an array of cpn60-targeted molecular diagnostic assays for SbGP/MPV phytoplasma. A fluorescent microsphere hybridization assay was designed that was capable of detecting SbGP/MPV phytoplasma in infected plant tissues, successfully differentiating it from other known phytoplasma cpn60 UT sequences, while identifying a double infection with SbGP/MPV and aster yellows (16SrI) phytoplasma. Two quantitative assays, quantitative real-time PCR (qRT-PCR) and droplet digital PCR (ddPCR), gave similar results in infected samples. Finally, a loop-mediated isothermal amplification (LAMP) assay provided rapid detection of SbGP/MPV phytoplasma DNA. Application of these assays revealed that SbGP/MPV phytoplasma is widely distributed in Central Mexico, with positive samples identified from eleven localities within three states separated by hundreds of kilometres. These results also provide tools for determining the presence and geographic distribution of this pathogen in plant and insect samples in other localities.

Molecular characterization of a phytoplasma affiliated with the 16SrVII group representative of the novel 16SrVII-F subgroup.

Vernonia brasiliana is a wild perennial shrub frequently found in pasture areas. Plants of this species have been observed displaying typical symptoms induced by phytoplasmas, which were characterized by shoot proliferation, deformed leaves and leaf chlorosis. The present study confirmed the presence of phytoplasmas in association with affected plants. Sequencing of the 16S rRNA gene, computer-simulated RFLP analysis and phylogenetic analysis revealed that one of the phytoplasmas identified was representative of novel subgroup. The sequence identity scores between the novel strain and those of previously described 'CandidatusPhytoplasma fraxini'-related strains was 99 %, while similarity coefficient values were lower than 0.97. These findings provide support to delineate the phytoplasma found in vernonia plants as a reference phytoplasma for a novel subgroup designated 16SrVII-F. This representative of the novel subgroup was denominated VbSP phytoplasma (Vernonia brasiliana Shoot Proliferation; GenBank KX342018). The results of the present study revealed V. brasiliana to be a host of phytoplasmas, evidenced a novel phytoplasma associated with phytoplasmal disease in Brazil and extended the knowledge of the genetic diversity existing within the 16SrVII group.

Identification of three novel subgroups within the X-disease group phytoplasma associated with strawberry redness disease.

Strawberry plants showing symptoms of lethal redness disease were found in production fields located in Tucumán province, Argentina. The presence of phytoplasmas was confirmed by PCR of 16S rDNA gene using phytoplasma universal primers. According to the 16S rDNA gene sequence identity, the four isolates analysed are related to the X-disease group (16SrIII) (identity ~99 %). These results were confirmed by in silico RFLP, actual RFLP and also by phylogenetic analyses of the 16S rDNA gene. This new phytoplasma was named as Strawberry X-Redness (StrawXR). The results from virtual and actual RFLP analyses of 16S rDNA gene revealed the presence of subgroup 16SrIII-J and three new 16SrIII subgroups. This is the first record of phytoplasmas from X-disease group associated strawberry in Argentina. These results confirm the prevalence of X-disease group and also contribute to the knowledge of diversity of phytoplasmas in this region.

Description of 'Candidatus Phytoplasma meliae', a phytoplasma associated with Chinaberry (Melia azedarach L.) yellowing in South America.

China tree yellows (ChTY) phytoplasma is associated with the yellowing disease of the China tree (Melia azedarach) in Argentina. According to partial 16S rRNA gene analysis, ChTY phytoplasma belongs to the 16Sr XIII group, subgroup G. Strains of species of ChTY have 98-99 % 16S rDNA gene sequence similarity with 16SrXIII-group phytoplasmas, and less than 97.5 % when compared to all 'CandidatusPhytoplasma' described so far, except for the novel 'CandidatusPhytoplasma hispanicum'. However, strains of species of ChTY are differentiated from the latter due to having additional molecular and biological attributes. The presence of unique features in the 16S rDNA sequence distinguishes ChTY from all species of 'CandidatusPhytoplasma' currently described. The in silico RFLP profile of 16S rDNA (1.2 kb) and rpLV-rpsC (1.3 kb) genes distinguished ChTY, as in the 16SrXIII-G subgroup within the 16SrXIIII group. The phylogenetic analyses, based on 16S rDNA, rpLV-rpsC and secA gene sequences, in addition to the restricted host range, characteristic symptoms and geographical distribution, confirm that the collective strains of the species ChTY represent a distinct lineage within the phytoplasma clade and support the description of a novel species of 'CandidatusPhytoplasma meliae' with the reference strain being ChTY-Mo3 (Montecarlo, Argentina).

Detection and identification of the heterogeneous novel subgroup 16SrXIII-(A/I)I phytoplasma associated with strawberry green petal disease and Mexican periwinkle virescence.

Phytoplasmas (species of the genus 'CandidatusPhytoplasma') are insect-vectored phytopathogenic bacteria associated with economically and ecologically important crop diseases. Strawberry production represents an important part of agricultural activity in Mexico and elsewhere, and infection of plants with phytoplasma renders the fruit inedible by altering plant development, resulting in virescence and phyllody. In this study we examined samples taken from four strawberry plants showing symptoms associated with strawberry green petal disease and from two periwinkle plants showing virescence, sampled in different areas of Mexico. Analysis of the 16S rRNA-encoding sequences showed that the plants were infected with a phytoplasma previously identified as Mexican periwinkle virescence (MPV; 16SrXIII). Examination of bacterial sequences from these samples revealed that two distinct 16S rRNA gene sequences were present in each sample along with a single chaperonin-60 (cpn60) sequence and a single rpoB sequence, suggesting that this strain displays 16S rRNA gene sequence heterogeneity. Two distinct rrn operons, identified with subgroup 16SrXIII-A and the newly described subgroup 16SrXIII-I, were identified from the six samples analyzed, delineating the novel subgroup 16SrXIII-(A/I)I, following the nomenclature proposed for heterogeneous subgroups.

'Candidatus Phytoplasma hispanicum', a novel taxon associated with Mexican periwinkle virescence disease of Catharanthus roseus.

Mexican periwinkle virescence (MPV) phytoplasma was originally discovered in diseased plants of Madagascar periwinkle (Catharanthus roseus) in Yucatán, Mexico. On the basis of results from RFLP analysis of PCR-amplified 16S rRNA gene sequences, strain MPV was previously classified as the first known member of phytoplasma group 16SrXIII, and a new subgroup (16SrXIII-A) was established to accommodate MPV phytoplasma. Phylogenetic analysis of 16S rRNA gene sequences indicated that strain MPV represents a lineage distinct from previously described 'CandidatusPhytoplasma' species. Nucleotide sequence alignments revealed that strain MPV shared less than 97.5 % 16S rRNA gene sequence similarity with all previously described 'Ca.Phytoplasma' species. Based on unique properties of the DNA, we propose recognition of Mexican periwinkle virescence phytoplasma strain MPV as representative of a novel taxon, 'CandidatusPhytoplasma hispanicum'.

The underestimated diversity of phytoplasmas in Latin America.

Phytoplasmas ('Candidatus Phytoplasma') are insect-transmitted, cell-wall-less, plant-pathogenic bacteria that cause economically important crop diseases. Because phytoplasmas are difficult or impossible to culture in vitro, they are classified taxonomically according to the convention used for unculturable micro-organisms. The first coherent scheme of classification of phytoplasmas, based on the RFLP pattern of the 16S rRNA-encoding gene generated with 17 restriction endonucleases, was updated several times until the development of the iPhyClassifier. iPhyClassifier is an interactive online tool capable of determining the species, group and subgroup of 'Candidatus Phytoplasma' of unknown samples using the 16S F2nR2 sequence. Latin America, an important geographical area in relation to food production, has a high incidence of plant diseases caused by phytoplasmas. However, many phytoplasmas associated with these diseases have not been properly classified. An extensive literature review and the use of iPhyClassifier allowed us to identify two new tentative groups (16SrXXXIII-A and 16SrXXXIV-A) and the following tentative new subgroups among Latin American strains that were either previously unclassified or misclassified: six in 16SrI, six in 16SrII, one in 16SrIII, one in 16SrVII, one in 16SrIX, one in 16SrXII and two in 16SrXIII.

Detection and identification of a novel 16SrXIII subgroup phytoplasma associated with strawberry red leaf disease in Argentina.

Strawberry red leaf phytoplasma was found in strawberry plants from production fields in Lules (Tucumán province) and Bella Vista (Corrientes province), Argentina. Characteristic strawberry red leaf symptoms were stunting, young leaves with yellowing at the edges, mature leaves which curled and were reddish at the abaxial face, flower and fruit deformation and death. The pathogen was detected with phytoplasma-universal primer pairs P1/P7 followed by R16F2n/R16R2 as nested primers in 13 diseased plants. Based on RFLP and sequence analysis of the amplified 16S rRNA gene, the phytoplasma was related to the 16SrXIII group (Mexican periwinkle virescence). In silico the RFLP profile of all the samples analysed revealed the presence of a unique pattern, showing that the novel phytoplasma is different from all the phytoplasmas currently composing the 16SrXIII group. The phylogenetic analysis was consistent with RFLP analysis as the strawberry red leaf phytoplasma was grouped within the 16SrXIII group, but formed a particular cluster. On this basis, the Strawberry red leaf phytoplasma associated with strawberry red leaf disease was assigned to a new subgroup, 16SrXIII-F.

A novel subgroup 16SrVII-D phytoplasma identified in association with Erigeron witches' broom.

Erigeron sp. plants showing symptoms of witches' broom and stunting were found near orchards of passion fruit in São Paulo state, Brazil. These symptoms were indicative of infection by phytoplasmas. Thus, the aim of this study was to detect and identify possible phytoplasmas associated with diseased plants. Total DNA was extracted from symptomatic and asymptomatic plants and used in nested PCR conducted with the primer pairs P1/Tint and R16F2n/16R2. Amplification of genomic fragments of 1.2 kb from the 16S rRNA gene confirmed the presence of phytoplasma in all symptomatic samples. The sequence identity scores between the 16S rRNA gene of the phytoplasma strain identified in the current study and those of previously reported 'Candidatus Phytoplasma fraxini'-related strains ranged from 98% to 99% indicating the phytoplasma to be a strain affiliated with 'Candidatus Phytoplasma fraxini'. The results from a phylogenetic analysis and virtual RFLP analysis of the 16S rRNA gene sequence with 17 restriction enzymes revealed that the phytoplasma strain belongs to the ash yellows phytoplasma group (16SrVII); the similarity coefficient of RFLP patterns further suggested that the phytoplasma represents a novel subgroup, designated 16SrVII-D. The representative of this new subgroup was named EboWB phytoplasma (Erigeron bonariensis Witches' Broom).

Weedy hosts and prevalence of potential leafhopper vectors (Hemiptera: Cicadellidae) of a phytoplasma (16SrIX group) associated with Huanglongbing symptoms in citrus groves.

Huanglongbing (HLB) is a severe citrus (Citrus spp.) disease associated with the bacteria genus Candidatus Liberibacter, detected in Brazil in 2004. Another bacterium was found in association with HLB symptoms and characterized as a phytoplasma belonging to the 16SrIX group. The objectives of this study were to identify potential leafhopper vectors of the HLB-associated phytoplasma and their host plants. Leafhoppers were sampled every other week for 12 mo with sticky yellow cards placed at two heights (0.3 and 1.5 m) in the citrus tree canopy and by using a sweep net in the ground vegetation of two sweet orange, Citrus sinensis (L.) Osbeck, groves infected by the HLB-phytoplasma in São Paulo state. Faunistic analyses indicated one Agalliinae (Agallia albidula Uhler) and three Deltocephalinae [Balclutha hebe (Kirkaldy), Planicephalus flavicosta (Stål), and Scaphytopius (Convelinus) marginelineatus (Stål)] species, as the most abundant and frequent leafhoppers (Hemiptera: Cicadellidae). Visual observations indicated an association of leafhopper species with some weeds and the influence of weed species composition on leafhopper abundance in low-lying vegetation. S. marginelineatus and P. flavicosta were more frequent on Sida rhombifolia L. and Althernantera tenella Colla, respectively, whereas A. albidula was observed more often on Conyza bonariensis (L.) Cronq. and B. hebe only occurred on grasses. DNA samples of field-collected S. marginelineatus were positive by polymerase chain reaction and sequencing tests for the presence of the HLB-phytoplasma group, indicating it as a potential vector. The association of leafhoppers with their hosts may be used in deciding which management strategies to adopt against weeds and diseases in citrus orchards.

'Candidatus Phytoplasma sudamericanum', a novel taxon, and strain PassWB-Br4, a new subgroup 16SrIII-V phytoplasma, from diseased passion fruit (Passiflora edulis f. flavicarpa Deg.).

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'.

Identification by computer-simulated RFLP of phytoplasmas associated with eggplant giant calyx representative of two subgroups, a lineage of 16SrIII-J and the new subgroup 16SrIII-U.

Symptoms resembling giant calyx, a graft-transmissible disease, were observed on 1-5 % of eggplant (aubergine; Solanum melongena L.) plants in production fields in Sao Paulo state, Brazil. Phytoplasmas were detected in 12 of 12 samples from symptomatic plants that were analysed by a nested PCR assay employing 16S rRNA gene primers R16mF2/R16mR1 followed by R16F2n/R16R2. RFLP analysis of the resulting rRNA gene products (1.2 kb) indicated that all plants contained similar phytoplasmas, each closely resembling strains previously classified as members of RFLP group 16SrIII (X-disease group). Virtual RFLP and phylogenetic analyses of sequences derived from PCR products identified phytoplasmas infecting eggplant crops grown in Piracicaba as a lineage of the subgroup 16SrIII-J, whereas phytoplasmas detected in plants grown in Bragança Paulista were tentatively classified as members of a novel subgroup 16SrIII-U. These findings confirm eggplant as a new host of group 16SrIII-J phytoplasmas and extend the known diversity of strains belonging to this group in Brazil.

A phytoplasma closely related to the pigeon pea witches'-broom phytoplasma (16Sr IX) is associated with citrus huanglongbing symptoms in the state of São Paulo, Brazil.

In February 2007, sweet orange trees with characteristic symptoms of huanglongbing (HLB) were encountered in a region of São Paulo state (SPs) hitherto free of HLB. These trees tested negative for the three liberibacter species associated with HLB. A polymerase chain reaction (PCR) product from symptomatic fruit columella DNA amplifications with universal primers fD1/rP1 was cloned and sequenced. The corresponding agent was found to have highest 16S rDNA sequence identity (99%) with the pigeon pea witches'-broom phytoplasma of group 16Sr IX. Sequences of PCR products obtained with phytoplasma 16S rDNA primer pairs fU5/rU3, fU5/P7 confirm these results. With two primers D7f2/D7r2 designed based on the 16S rDNA sequence of the cloned DNA fragment, positive amplifications were obtained from more than one hundred samples including symptomatic fruits and blotchy mottle leaves. Samples positive for phytoplasmas were negative for liberibacters, except for four samples, which were positive for both the phytoplasma and 'Candidatus Liberibacter asiaticus'. The phytoplasma was detected by electron microscopy in the sieve tubes of midribs from symptomatic leaves. These results show that a phytoplasma of group IX is associated with citrus HLB symptoms in northern, central, and southern SPs. This phytoplasma has very probably been transmitted to citrus from an external source of inoculum, but the putative insect vector is not yet known.

'Candidatus Phytoplasma lycopersici', a phytoplasma associated with 'hoja de perejil' disease in Bolivia.

New diseases known locally as 'hoja de perejil' of tomato (Lycopersicon esculentum Mill) and 'brotes grandes' of potato (Solanum tuberosum L.) were first recognized in surveys of production fields in Bolivia during 2000-2003. Alfalfa (Medicago sativa) witches' broom and little leaf diseases of native weeds Morrenia variegata and mora-mora (Serjania perulacea) were also identified near to production fields. Phytoplasma aetiology was attributed to each of these diseases following detection and initial identification of aster yellows group (16SrI) phytoplasmas in all five diseased plant species. While potato, alfalfa and mora-mora plants contained indistinguishable 16SrI-B strains, 'hoja de perejil' (THP) and morrenia little leaf (MVLL)-associated phytoplasma strains shared 97.5 % 16S rRNA gene sequence similarity with 'Candidatus Phytoplasma asteris' and related strains and <95 % similarity with all other 'Candidatus Phytoplasma' species. Phylogenetic analysis of 16S rRNA gene sequences indicated that the THP and MVLL phytoplasmas represent a novel lineage within the aster yellows (16SrI) group and, on the basis of unique 16S rRNA gene sequences, we propose that THP and MVLL phytoplasmas represent 'Candidatus Phytoplasma lycopersici', with THP as the reference strain.

"Candidatus Phytoplasma americanum", a phytoplasma associated with a potato purple top wilt disease complex.

Potato purple top wilt (PPT) is a devastating disease that occurs in various regions of North America and Mexico. At least three distinct phytoplasma strains belonging to three different phytoplasma groups (16SrI, 16SrII and 16SrVI) have been associated with this disease. A new disease with symptoms similar to PPT was recently observed in Texas and Nebraska, USA. Two distinct phytoplasma strain clusters were identified. One belongs to the 16SrI phytoplasma group, subgroup A, and the other is a novel phytoplasma that is most closely related to, and shares 96.6 % 16S rRNA gene sequence similarity with, a member of group 16SrXII. Phylogenetic analysis of 16S rRNA gene sequences of the novel PPT-associated phytoplasma strains, previously described 'Candidatus Phytoplasma' organisms and other distinct unnamed phytoplasmas indicated that the novel phytoplasma, termed American potato purple top wilt (APPTW) phytoplasma, represents a distinct lineage and shares a common ancestor with stolbur phytoplasma, "Candidatus Phytoplasma australiense", "Candidatus Phytoplasma japonicum", "Candidatus Phytoplasma fragariae", bindweed yellows phytoplasma (IBS), "Candidatus Phytoplasma caricae" and "Candidatus Phytoplasma graminis". On the basis of unique 16S rRNA gene sequences and biological properties, it is proposed that the APPTW phytoplasma represents "Candidatus Phytoplasma americanum", with APPTW12-NE as the reference strain.

'Candidatus Phytoplasma graminis' and 'Candidatus Phytoplasma caricae', two novel phytoplasmas associated with diseases of sugarcane, weeds and papaya in Cuba.

During 2003, surveys of sugarcane yellow leaf disease and papaya bunchy top-like disease were carried out on plantations in Havana province, Cuba, to determine the roles of weeds and Auchenorrhyncha insects in the epidemiology of these diseases. More than 250 plant and insect samples were collected and indexed by using a nested PCR for phytoplasma 16S rDNA with the generic primer pairs P1/P7 and R16F2n/R16R2. The PCR products were further characterized by restriction fragment length polymorphism using HaeIII, AluI, Sau3AI, Tru9I, HhaI, HpaII and TaqI endonucleases, giving patterns that distinguished them from those of the other reference phytoplasmas analysed. Phylogenetic analysis of 16S rRNA gene sequences identified the phytoplasmas present in sugarcane (Saccharum officinarum L.), Cynodon dactylon L., Conyza canadensis L. Cronq., Sorghum halepense L. Pers., Macroptilium lathyroides L. Urb., Saccharosydne saccharivora (Westwood) and Cedusa spp., and those in papaya (Carica papaya L.) and Empoasca papayae, as two novel provisional phytoplasma species. We propose that these phytoplasmas should be given Candidatus status, as 'Candidatus Phytoplasma graminis' and 'Candidatus Phytoplasma caricae', respectively.