Subgroup 16SrI-A Research Articles

Genome wide sequence analysis grants unbiased definition of species boundaries in “Candidatus Phytoplasma”

The phytoplasmas are currently named using the Candidatus category, as the inability to grow them in vitro prevented (i) the performance of tests, such as DNA-DNA hybridization, that are regarded as necessary to establish species boundaries, and (ii) the deposition of type strains in culture collections. The recent accession to complete or nearly complete genome sequence information disclosed the opportunity to apply to the uncultivable phytoplasmas the same taxonomic approaches used for other bacteria. In this work, the genomes of 14 strains, belonging to the 16SrI, 16SrIII, 16SrV and 16SrX groups, including the species “Ca. P. asteris”, “Ca. P. mali”, “Ca. P. pyri”, “Ca. P. pruni”, and “Ca. P. australiense” were analyzed along with Acholeplasma laidlawi, to determine their taxonomic relatedness. Average nucleotide index (ANIm), tetranucleotide signature frequency correlation index (Tetra), and multilocus sequence analysis of 107 shared genes using both phylogenetic inference of concatenated (DNA and amino acid) sequences and consensus networks, were carried out. The results were in large agreement with the previously established 16S rDNA based classification schemes. Moreover, the taxonomic relationships within the 16SrI, 16SrIII and 16SrX groups, that represent clusters of strains whose relatedness could not be determined by 16SrDNA analysis, could be comparatively evaluated with non-subjective criteria. “Ca. P. mali” and “Ca. P. pyri” were found to meet the genome characteristics for the retention into two different, yet strictly related species; representatives of subgroups 16SrI-A and 16SrI-B were also found to meet the standards used in other bacteria to distinguish separate species; the genomes of the strains belonging to 16SrIII were found more closely related, suggesting that their subdivision into Candidatus species should be approached with caution.

Multilocus sequences confirm the close genetic relationship of four phytoplasmas of peanut witches'‐broom group 16SrII‐A

Four witches'-broom diseases associated with Arachis hypogaea (peanut), Crotalaria pallida, Tephrosia purpurea, and Cleome viscosa were observed in Hainan Province, China during field surveys in 2004, 2005, and 2007. In previously reported studies, we identified these four phytoplasmas as members of subgroup 16SrII-A, and discovered that their 16S rRNA gene sequences were 99.9-100% identical to one another. In this study, we performed extensive phylogenetic analyses to elucidate relationships among them. We analyzed sequences of the 16S rRNA gene and rplV-rpsC, rpoB, gyrB, dnaK, dnaJ, recA, and secY combined sequence data from two strains each of the four phytoplasmas from Hainan province, as well as strains of peanut witches'-broom from Taiwan (PnWB-TW), "Candidatus Phytoplasma australiense", "Ca. Phytoplasma mali AT", aster yellows witches'-broom phytoplasma AYWB, and onion yellows phytoplasma OY-M. In the 16S rRNA phylogenetic tree, the eight Hainan strains form a clade with PnWB-TW. Analysis of the seven concatenated gene regions indicated that the four phytoplasmas collected from Hainan province cluster most closely with one another, but are closely related to PnWB-TW. The results of field survey and phylogenetic analysis indicated that Cr. pallida, T. purpurea, and Cl. viscosa may be natural plant hosts of peanut witches'-broom phytoplasma.

First Report of a 16SrII-A Subgroup Phytoplasma Associated with Purple Coneflower (Echinacea purpurea) Witches'-Broom Disease in Taiwan.

Purple coneflower (Echinacea purpurea), widely grown as an ornamental and medicinal plant, is a perennial flowering plant that is native to eastern North America. In July 2011, symptoms indicative of phytoplasma disease, including floral virescence, phyllody, and witches'-broom (WB), were observed to be affecting plants in coneflower fields in Wufeng, Taichung City, Taiwan. Incidence of infected plants was estimated to be greater than 90% within a single field. Phytoplasmas previously associated with purple coneflower WB disease have all been classified as aster yellows group (16SrI) strains (GenBank Accession Nos. EU333395, AY394856, EU416172, and EF546778) except for pale purple coneflower (Echinacea pallida) WB in Australia, which was identified as a subgroup 16SrII-D member (2). Three diseased plants were uprooted and transplanted in a greenhouse for further study. Transmission electron microscopy revealed clusters of phytoplasma cells ranging from 170 to 490 nm in diameter in phloem sieve elements of virescent and phylloid flowers and stems from diseased plants. Comparable tissues from symptomless plants were devoid of phytoplasma. Total DNA was extracted from plant tissue samples (50 to 100 mg each) including stems, leaves, and flowers by a modified CTAB method (1) from three symptomatic plants as well as from three asymptomatic coneflower plants seedlings. Analyses by a nested PCR using universal primer pairs P1/P7 followed by R16F2n/R16R2 were performed to detect putative phytoplasma (2). Each primer pair amplified a single PCR product of either 1.8 or 1.2 kb, respectively, from diseased plant tissues only. The nested PCR products (1.2 kb) amplified from phylloid flowers of the three diseased plants were cloned separately and sequenced (GenBank Accession Nos. JN885460, JN885461, and JN885462). Blast analysis of the sequences revealed a 99.7 to 99.8% sequence identity with those of Echinacea WB phytoplasma strain EWB5 and EWB6 (GenBank Accession Nos. JF340076 and JF340080), which reportedly belonged to the 16SrII-D subgroup (2). Moreover, iPhyClassifier software (3) was used to perform sequence comparison and generate the virtual restriction fragment length polymorphism (RFLP) profile. The 16S rDNA sequences share a 99.4 to 99.5% similarity with that of the 'Candidatus Phytoplasma australasiae' reference strain (Y10097) and the RFLP patterns are identical to that of the 16SrII-A subgroup. Taken together, these results indicated that the phytoplasma infecting purple coneflower in Taiwan is a 'Ca. Phytoplasma australasiae'-related strain and belongs to the 16SrII-A subgroup. To our knowledge, this is the first report of a 16SrII-A subgroup phytoplasma causing WB disease on purple coneflower in Taiwan. The occurrence of phytoplasma on purple coneflower could have direct implication for the economically important ornamental, medicinal plant, and floral industry in Taiwan, especially to the growers and breeders that eagerly promote the purple coneflower industry.

Detection of 16SrII Group Phytoplasma in China Aster (Callistephus chinensis)

China aster, Callistephus chinensis, is widely grown in Myanmar as an ornamental plant. Symptoms of flower virescence were observed in diseased China aster plants in Yezin, Myanmar. The presence of a phytoplasma was detected and identified by applying Polymerase Chain Reaction (PCR)/ Restriction Fragment Length Polymorphism (RFLP) techniques and sequencing the 16S ribosomal DNA. The phytoplasma was identified as belonging to ribosomal subgroup 16SrII-A, never reported before in China aster. It showed almost 100 % similarity with a 16S rDNA sequence of sunn hemp witches' broom phytoplasma (AB558143), which belongs to the peanut witches' broom phytoplasma group. This is the first record of China aster as a new host for a subgroup 16SrII-A phytoplasma.

A disease associated with phytoplasma in Parthenium hysterophorus

A disease on parthenium weed (Parthenium hysterophorus L.) was observed in June 2008 in Danzhou of Hainan Province. Infected weeds showed phytoplasma-like associated symptoms such as severe stunting, excessive proliferation of shoots, inflorescence-clustering, green petal, small leaves and witches’-broom. The original cause of phytoplasma was further confirmed by polymerase chain reaction (PCR). PCR products of 1.8 kb were obtained using the universal primers pair (P1/P7) designed to amplify the entire 16S rDNA and the 16/23S intergenic spacer region in a direct PCR assay. The primers pair R16F2n/R2 was used to amplify a PCR product of 1.2 kb. Restriction fragment length polymorphism (RFLP) was used to analyze the partial 16S rDNA sequences (1.2 kb) of all phytoplasma DNA digested with five endonucleases (Kpn I, Hpa II, Taq I, Rsa I, EcoR I). The RFLP patterns of the strain were found to be identical with that of the reference peanut witches’-broom phytoplasma. Based on the RFLP data, it is suggested that the phytoplasma strain belongs to subgroup 16SrII-A. This is the first demonstration of a 16SrII-A group phytoplasma associated with parthenium weed.

First Report of Spiroplasma citri in Carrot in Europe.

In 2008 and 2009, symptoms of curling, yellow and purple discoloration of leaves, stunting of shoots and tap roots, and formation of bunchy, fibrous secondary roots were observed in commercial carrot (Daucus carota L.) fields located in several production areas of Spain (Alicante, Albacete, Segovia, and Valladolid). Incidence of this disease was almost 100% in individual affected fields. Similar symptoms were reported from 1997 to 1998 in various carrot production areas of Spain (the Canary Islands, Segovia, and Madrid) and were associated with infection of stolbur and aster yellows phytoplasmas (2). Moreover, the observed symptoms resembled those caused by Spiroplasma citri in carrots affected by the carrot purple leaf disease recently reported in the United States (4). Studies were conducted to investigate whether S. citri and phytoplasmas were associated with the observed carrot symptoms. Total DNA was extracted from 0.5 g of phloem tissue of 13 symptomatic and 3 asymptomatic plants with DNeasy Plant Mini Kit (Qiagen, Valencia, CA). DNA samples were analyzed by nested-PCR assays using primers pair P1/P7 (1) and R16F2n/R16R2n (3) for phytoplasmas and ScR16F1/ScR16R1 followed by ScR16F1A/ScR16R2 (4) for S. citri detection. DNA of a known strain of S. citri (Sediag, Longvic, France) was used as a positive control of the assay. Analyses revealed that 8 of the 13 symptomatic plants tested positive for S. citri; the plants were collected from three different provinces of Spain, namely, Alicante, Valladolid, and Segovia. Two symptomatic plants were double infected by S. citri and a phytoplasma strain belonging to the Aster yellows group (16SrI), subgroup 16SrI-A. However, none of the symptomatic plants presented single infection with phytoplasmas. S. citri identity was determined by sequencing two nested PCR products (1.1 kb) that yielded identical sequences deposited in the GenBank database (Accession Nos. HM124555 and HM124556). BLAST analysis showed 100% nt identity with a sequence of S. citri from carrot (Accession No. DQ112019) associated with the new carrot disease referred to as 'carrot purple leaf reported in Washington State (4). To our knowledge, this is the first report of S. citri associated with carrot in Europe.

Detection of aster yellows phytoplasma DNA in seed and seedlings of canola (Brassica napus and B. rapa) and AY strain identification

A study was conducted to identify the strains of aster yellows (AY) disease present in crops of Brassica napus and B. rapa grown near Medstead, Saskatchewan. AY phytoplasma DNA was detected in midrib, stem and root tissues of several symptomless plants as well as plants exhibiting typical AY disease symptoms. Most symptomatic and symptomless, AY-infected plants produced normal-looking and misshapen seeds. However, for both Brassica species, symptomatic plants produced significantly more seeds containing phytoplasma DNA than symptomless, AY-infected plants. Also, significantly more misshapen seeds contained phytoplasma DNA than normal seeds. Phytoplasma DNA belonging to subgroups 16SrI-A and 16SrI-B was detected in symptomatic and symptomless, AY-infected plants and in seed of these plants. The new AY strain sequences were registered in Genbank. The study reports for the first time the detection of AY strains in seedling tissues of both Brassica species. The research also showed that spiral cleaning has the potential to remove seeds that contain phytoplasma DNA in B. napus.

ONION IS HOST FOR TWO PHYTOPLASMA LINEAGES, SUBGROUPS 16SrI-A AND 16SrI-(B/L)L, IN LITHUANIA: A HinfI SITE REVEALED A SNP MARKING DIVERGENT BRANCHES OF EVOLUTION

Onions (Allium cepa L.) grown for seed production in the Kaunas region of Lithuania exhibited mild yellowing of leaves and stems, stunting, phyllody, and proliferation of flowers. RFLP and sequence analysis of PCR-amplified 16S rRNA, ribosomal protein (rp), and secY genes revealed the presence of phytoplasmas belonging to subgroups 16SrI-A (rpI-A) and 16SrI-L (rpIB, secYI-B). The results indicated that phytoplasma strains in subgroup16SrI-A (rpI-A) have potential to damage onions in Europe, as well as in North America, and for the first time demonstrated onion as a host for subgroup 16SrI-L. Subgroup 16SrI-L was distinguished based on a composite HinfI RFLP pattern of 16S rDNA that revealed the presence of two sequence heterogeneous rRNA operons in this subgroup, thus showing the significance of composite RFLP patterns for phytoplasma identification and classification. A single nucleotide polymorphism (SNP) in the first base of one HinfI recognition site (5’-GANTC-3’) marked the divergence of major phylogenetic branches, supporting the concept that SNPs provide powerful molecular markers of phytoplasma evolution.

Leafhoppers and cixiids in phytoplasma-infected carrot fields: Species composition and potential phytoplasma vectors

The first molecular analysis of samples collected in southern Backa (Serbia) confirmed the presence of aster yellows (16SrI) and stolbur phytoplasmas (16SrXII) in insects belonging to the family Cicadellidae, as well as in carrot plants where the insects were collected. A correct identification of the phytoplasmas and their vectors is essential to arrange effective control strategies to prevent diseases associated with phytoplasmas from spreading to carrots and other vegetable crops. In order to enhance knowledge about insect vectors of aster yellows and stolbur phytoplasmas in Serbia, Cicadellidae and Cixiidae (Homoptera Auchenorrhyncha), the most common vectors of these phytoplasmas, were monitored in southern Backa during 2008. Adults leaf- and planthoppers were collected and identified at species level using standard entomological methods, and tested for phytoplasma presence by means of PCR/RFLP. A total of 13 insect species of Cicadellidae were identified, as follows: a) three species of the subfamily Agallinae: Anaceratagallia ribauti (Ossiannilsson), Anaceratagallia venosa (Fourcroy), and Anaceratagallia laevis (Ribaut); b) seven species of the subfamily Deltocephalinae: Psammotettix confinis (Dahlbom), Psammotettix striatus (Linnaues) Psammottettix alienus (Dahlbom), Macrosteles sexnotatus (Fall?n), Ophiola decumana (Kontkanen), Errastunus ocellaris Fall?n, and Scaphoideus titanus Ball; c) three species of the subfamily Typhlocibinae: Eupteryx atropunctata (Goeze), Eupteryx mellissae Curtis, Zyginidia pullula (Boheman). Female specimens of the genus Euscelis (Deltocephalinae) were also collected, as well as one species of Reptalus quinquecostatus (Dufour) of the family Cixiidae. Stolbur phytoplasmas were detected in A. laevis, A. ribauti, A. venosa, P. striatus, P. confinis and P. alienus. The species: A. laevis, O. decumana, and P. confinis were AY-infected (subgroup 16SrI-A), while subgroup 16SrI-C was found only in one specimen of P. confinis. Since some aster yellows- and stolbur-infected species of the genera Psammotettix and Anaceratagallia (especially P. confinis and A. laevis) were regularly and commonly found in the infected carrot fields during the whole vegetative period, they could play a significant role in transmitting and spreading these pathogens in natural environment.

First Report of Aster Yellow Phytoplasmas (‘Candidatus Phytoplasma asteris’) in Canadian Grapevines

In North America, elm yellows, aster yellows (AY), and X-disease phytoplasmas have been detected in American grapevines (1), and recently, Bois noir was detected in Canadian vineyards from British Columbia (BC) and Ontario (ON) (2). Typical symptoms of grapevine yellows (GY) include leaf rolling and chlorosis, uneven or total lack of lignification of canes, flower abortion or berry withering, and stunting. In 2006 and 2007, independent surveys were conducted by the Canadian Food Inspection Agency (CFIA) and Agriculture and Agri-Food Canada (AAFC) to detect phytoplasmas in Canadian vineyards containing different cultivars in BC, ON, Québec (QC), Nova Scotia, New Brunswick, and Prince Edward Island. The CFIA collected and tested 651 fresh leaf samples from recently imported grapevines and older grapevines in the same or neighboring blocks displaying symptoms typical of those associated with disease caused by phytoplasmas. Many vineyards were surveyed only once. AAFC collected and tested 3,485 samples from symptomatic and asymptomatic grapevines from established vineyards in ON, BC, and QC. The same vineyards were sampled in ON and BC both years; QC vineyards were only sampled in 2007. AAFC-collected leaf samples were freeze dried and stored at -20°C before processing. CFIA samples were tested by a modified real-time PCR assay and TaqMan probe targeting the 16S ribosomal RNA gene that detects a wide range of known phytoplasmas (2). Positive samples were confirmed by conventional PCR using the phytoplasma-specific primers P1/P7 (3) and the resulting ~1,800-bp fragment was cloned and sequenced as previously described (2). DNA extracted by AAFC was amplified by nested PCR technology with universal phytoplasma specific primer pairs P1/P6 and R16R2/R16F2 (3) and the resulting 1,200-bp fragment was cloned and sequenced. Two plants, one located in ON in 2006 and the other in BC in 2007, were found to be infected with an AY-like phytoplasma by the CFIA. The phytoplasmas detected in both infected plants had a 99.9% nt sequence identity with AY phytoplasma sequences from GenBank (Accession Nos. AF222063 and AY665676, respectively), with the BC isolate also showing 100% identity to a strain of AY, ash witches'-broom phytoplasma (GenBank Accession No. AY566302). AAFC detected phytoplasma DNA in both years in a total of 17 symptomatic plants and 21 asymptomatic plants from different vine varieties in ON, BC, and QC. Positive samples were found to have a 99.0% nt sequence identity to AY subgroup 16SrI-A (GenBank Accession No. AY180956). Sequences were exchanged for confirmation of phytoplasma identity and were deposited in Genbank under Accession Nos. FJ659844 and FJ824597. Phytoplasma strains were identified for all plants in which phytoplasmas were detected. Results show that AY is present in vineyards in the provinces of ON, BC, and QC. To our knowledge, this is the first report of AY being detected in grapevines in Canada.

First Report of Purple Coneflower Phyllody Associated with a 16SrI-B Phytoplasma in Maryland.

Purple coneflower (Echinacea purpurea (L.) Moench) is a flowering perennial plant native to North America and is widely grown as an ornamental flower. It is also grown commercially to make herbal teas and extracts purported to help strengthen the immune system. Propagation is by seed or root cuttings. Aster yellows phytoplasmas (strains belonging to group 16SrI) have been reported to be associated with purple coneflower exhibiting virescence and phyllody symptoms in the northern United States and Canada. A subgroup 16SrI-A phytoplasma was identified to be associated with symptomatic purple coneflower in Wisconsin (2). During the summers of 1994 and 2007, purple coneflower plants in Maryland sporadically exhibited symptoms resembling those caused by phytoplasma infection. Symptoms included stunting, virescence, phyllody, and abnormal flower bud proliferation from the cone. Samples from four symptomatic and two asymptomatic purple coneflower plants were collected. Total nucleic acid was extracted from leaf tissue. To assess the etiology of the disease, nested PCR with universal phytoplasma primer pair P1/P7 followed by R16F2n/R16R2 was employed for the detection of phytoplasmas (1). An amplicon of approximately 1.2 kb was amplified from all four symptomatic purple coneflower plants but not from the two asymptomatic plants. Restriction fragment length polymorphism (RFLP) patterns of 16S rDNA digested singly with restriction enzymes AluI, KpnI, HpaI, MseI, HhaI, and RsaI indicated that affected purple coneflower plants were infected by a phytoplasma belonging to aster yellows group 16SrI ('Candidatus Phytoplasma asteris'-related strains), subgroup 16SrI-B (1). Nucleotide sequence analysis of cloned 16S rDNA (GenBank Accession Nos. EU333394 and EU333395) confirmed the results from RFLP analyses. To our knowledge, this is the first report of a 16SrI-B phytoplasma infecting an Echinacea sp. in Maryland.

Molecular Detection and Identification of Group 16SrI and 16SrXII Phytoplasmas Associated with Diseased Potatoes in Russia

Phytoplasmal diseases have long been suspected to occur in several potato-growing regions in Russia on the basis of symptoms and the presence of insect vectors. Symptoms resembling stolbur are most prevalent, but round leaf disease, potato witches'-broom, and potato purple top wilt also occur (1). The phytoplasma etiologies of these diseases have never been verified by molecular means. During the summer of 2006, 33 potato plants exhibiting various symptoms including purple top, round leaves, and stolbur-like symptoms characterized by purple top, stunting, bud proliferation, and formation of aerial tubers were randomly collected from the Volga River Region, Central Region, and Northern Caucasian Region in Russia. DNA extracts were prepared from 1.0 g of petioles and leaf mid veins according to a modified procedure with the Qiagen DNeasy Plant Mini Kit (Qiagen, Valencia, CA) as previously described (2). A nested PCR with primer pair P1/P7 in the first amplification followed by R16F2n/R16R2n in the second amplification was performed to detect phytoplasmas in infected potato samples (4). Potato plants maintained in the greenhouse were used as healthy controls. A negative control devoid of DNA templates was included in all PCR assays. One microliter of diluted (1:30) PCR product from the first amplification was used as the template in the nested PCR. Eight of 33 potato samples tested positive in the first PCR. Twelve of 33 potato samples tested positive in nested PCR. Nine of the 12 potato samples that tested positive for phytoplasma exhibited stolbur-like symptoms; the other three samples exhibited round leaves, stunting, or proliferation. The remaining symptomatic samples that exhibited nonspecific purple or yellow discoloration of terminal leaves, without other specific stolbur-like symptoms, may be infected by other pathogens. Restriction fragment length polymorphism (RFLP) analysis of nested PCR products (R16F2n/R16R2n amplicons, 1.2 kb) was performed. PCR products (6 μl) were digested singly with the restriction enzymes AluI, HaeIII, HhaI, HpaII, KpnI, MseI, RsaI, and Tsp509I. Comparison of RFLP profiles with published profiles (3) was used for identification of the putative phytoplasmas detected. Among the 12 PCR positive potato samples, 10 showed very similar or identical RFLP profiles to stolbur phytoplasma, a strain belonging to stolbur phytoplasma group (16Sr XII), subgroup 16SrXII-A and closely related strains, and two showed RFLP profiles similar to those of aster yellows phytoplasma group (16SrI). Nucleotide sequence analysis of cloned 16S rDNA (GenBank Accession Nos. EU344884-EU344890 and EU333396-EU333400) confirmed the results of the RFLP analyses and also indicated that the two samples showing 16SrI profiles were simultaneously infected with two phytoplasma strains belonging to subgroups 16SrI-A and 16SrI-B. To our knowledge, this is the first confirmation by molecular procedures that stolbur phytoplasma (16SrXII-A) is prevalent in several potato-growing regions and is the first report of 16SrI-A and 16SrI-B phytoplasmas associated with potatoes in Russia.

DNA sequencing reveals false positives during the detection of aster yellows phytoplasmas in leafhoppers

During the summer of 2001 and 2002, 850 and 865 carrot plants and 926 and 2584 leafhoppers associated with aster yellow (AY)-type disease were collected from five fields in Manitoba, Canada. DNA was extracted from 999 individual leafhoppers and 381 leaf tissues from both apparently healthy and AY-like infected carrot plants. All DNA samples were examined by PCR for the presence of phytoplasmas using three universal primer pairs P1/P6, P1/P7 and R16F2n/R2 derived from phytoplasma rDNA sequences. DNA amplification with these three primer pairs generated the expected amplification products of 1.7, 1.5 and 1.2 kb, respectively. Diluted PCR products obtained using universal primer pair P1/P6 were nested with R16F2n/R16R2n. The latter set of primers amplified DNA samples from 92 carrot plants and 83 leafhopper samples. In order to assess the diversity among insect and plant phytoplasmas, nested PCR products from all 92 carrot and 83 leafhopper samples were subjected to RFLP analysis using restriction endonucleases KpnI, MseI, and HhaI. This RFLP analysis showed similar patterns among carrot and leafhopper samples. Phytoplasmas detected in most samples belonged to the subgroup 16Sr-IA. To understand why the R16F2n/R16R2n of primers did not amplify the PCR product obtained from the first PCR in the remaining samples, four PCR products of P1/P6 from two plants (representing 16Sr-IA and 16Sr-IB) and two leafhopper samples that did not amplify with nested PCR, were used for DNA sequencing. 1 1 The GenBank accession numbers for the sequences reported in this paper are: D2 ( DQ679962), E4 ( DQ679963), A4 ( DQ679964), and B2 ( DQ679965). The BLAST analysis of the obtained sequences showed that the PCR amplicons from the two carrot samples precisely matched with the GenBank sequences of known phytoplasmas. Alignments of these two sequences have shown very slight variations (transition/transversion ratio mean of 0.539) that would correspond to the minor differences at the 16S level between the 16Sr-IA and 16Sr-IB phytoplasma subgroups. The sequences of PCR products obtained from the two insect samples had similarity (>98%) with the sequences of phytoplasma in carrot except that their length differed from the carrot samples by 6 bp. They actually matched bacterial sequences from the GenBank, indicating that a single PCR using P1/P6 was not enough to detect phytoplasma in leafhoppers.

First Report of Yucca Phyllody Associated with 16SrI-A Phytoplasmas in Texas.

Buckley's yucca (Yucca constricta Buckl.) is a native flowering perennial plant widely distributed in Texas and northeast Mexico. It is also grown as an ornamental plant in its native range as well as in other dry regions in the United States and Mexico. In 2006, during an extended drought, Buckley's yucca plants sporadically exhibited phyllody and abnormal bud proliferation on the inflorescence in Uvalde County in southwestern Texas. Symptoms resembled those caused by phytoplasmal infection. Samples from four symptomatic and two asymptomatic yucca plants were collected. Total nucleic acid was extracted from abnormal bud tissue. To assess the etiological aspect of the disease nested PCR using phytoplasma specific primer pair P1/16S-SR or P1/P7 followed by R16F2n/R16R2n was employed for the detection of putative phytoplasmas (2). An amplicon of approximately 1.2 kb was amplified from all four symptomatic yucca plants but not from asymptomatic plants. Restriction fragment length polymorphism (RFLP) patterns of 16S rDNA digested singly with AluI, KpnI, HpaII, MseI, HhaI, and RsaI endonucleases indicated that affected yucca plants were infected by a phytoplasma belonging to aster yellows group 16SrI ('Candidatus Phytoplasma asteris'), subgroup 16SrI-A (1). Nucleotide sequence analysis of cloned 16S rDNA (GenBank Accession No. EF190067) confirmed the results on the basis of RFLP analyses. Yucca phyllody has not been reported elsewhere. This disease appears to be newly emerging in Texas with only a few affected plants. To our knowledge, this is the first report of 16SrI-A phytoplasma infecting a Yucca sp.

Carrot Purple Leaf: A New Spiroplasmal Disease Associated with Carrots in Washington State.

During the growing seasons of 2003 and 2004, a disease occurred in several carrot crops in south central Washington with symptoms suggestive of infection by phytopathogenic mollicutes (phytoplasmas and spiroplasmas). In the fall, many affected carrot plants exhibited extensive purple or yellow-purple leaf discoloration, general stunting of shoots and taproots, and formation of bunchy, fibrous secondary roots. For detection of the putative causal agents, polymerase chain reaction (PCR) assays were performed using primers specific to phytoplasmas as well as primers specific to plant-pathogenic spiroplasmas. Restriction fragment length polymorphism (RFLP) analyses of PCR-amplified 16S rDNA sequences revealed that about 81% of affected plants showing dark purple or yellow-purple leaf symptoms tested positive for Spiroplasma citri. Of affected plants showing mild purple discoloration of leaf margins, 18% tested positive for a phytoplasma strain belonging to the clover proliferation group (16SrVI), subgroup 16SrVI-A, and 11% for another phytoplasma strain belonging to the aster yellows group (16SrI), subgroup 16SrI-A. Nucleotide sequence analysis of cloned 16S rDNA confirmed the phytoplasma group affiliations. Some symptomatic plants were co-infected with S. citri and either aster yellows phytoplasma or clover proliferation group phytoplasma. To our knowledge, this is the first documentation of spiroplasma infection of carrot in the United States.

First Report of a 16SrII Group Phytoplasma Associated with Shoot Proliferation of a Cactus (Opuntia monacantha) in Lebanon.

In October 2003, during a survey to evaluate the incidence of phytoplasma diseases in Lebanon, symptoms suggestive of phytoplasma infection in Opuntia monacantha (Haworth) were observed in Saghbine, Bekaa Valley. Symptoms were excessive stem and shoot proliferation. Three symptomatic and as well as symptomless plants were collected and analyzed for the presence of phytoplasmas. Nucleic acids were extracted from 0.5 g of shoot tissue and tested using polymerase chain reaction (PCR) with universal phytoplasma primers (fU5rU3) for partial amplification of the ribosomal 16SrDNA (4). PCR resulted in amplification of an expected 881-bp rDNA fragment from the symptomatic but not from symptomless samples. For characterization, sequence of the amplified DNA was determined (Genbank Accession No. AY939815). The sequence showed a high similarity with several isolates of the 16srII group of phytoplasmas. The highest similarity has been oserved with 16S rDNA of two isolates of cactus witches'-broom phytoplasma found in China (1) and Mexico (3) (Genbank Accession Nos. AJ293216 and AF320575, respectively) (99.8%) as well as faba bean phyllody phytoplasma (Genbank Accession No. X83432) (99.7%) and "Candidatus Phytoplasma aurantifolia" (Genbank Accession No. U15442) (99.3%). The presence of phytoplasmas was confirmed using nested-PCR with primers R16mF2/R1 and R16F2n/R2 (2). The Tru9I digestion pattern of the amplified product R16F2n/F16R2 detected in O. monacantha was identical to the digestion pattern obtained from periwinkle infected by "Ca. P. aurantifolia" (subgroup 16SrII-B) and soybean phyllody phytoplasma (subgroup 16SrII-C), but different from the Tru9I digestion pattern observed for cleome phyllody phytoplasma (subgroup 16SrII-A) and tomato big bud phytoplasma (subgroup 16SrII-E). To our knowledge, this is the first report of an infection with a phytoplasma belonging to16SrII group in Lebanon.

First Report of Barley as Host of a Phytoplasma Belonging to Group 16SrI, Subgroup B, and Ribosomal Protein Subgroup rpI-B in Lithuania.

Numerous diseased plants of barley (Hordeum vulgaris L.) exhibiting twisted, abnormally thin and yellowed awns, reduced spikelets, and general stunting and yellowing were observed in fields in the Vilnius and Kaisiadorys regions of Lithuania. The possible association of a phyto-plasma with the disease, termed barley deformation (BaDef), was assessed using polymerase chain reaction (PCR). Three phytoplasma universal primer pairs (P1/P7, R16F2n/R16R2, and rpF1/rpR1) (1,2,4) were employed to amplify ribosomal (r) RNA gene (rDNA) and ribosomal protein (rp) gene sequences. Template DNA extractions and PCR (direct and nested) were conducted as previously described (4). Although DNA was amplified in PCRs containing template extracted from diseased plants, no amplification was observed in PCRs containing DNA from symptomless plants sampled from the same fields. The BaDef phytoplasma was identified and classified according to Lee et al. (4) through restriction fragment length polymorphism (RFLP) analysis of 1.2-kbp 16S rDNA amplified in the PCR primed by primer pair R16F2n/R16R2 and analysis of the 1.2-kbp rp gene sequences amplified in PCR primed by primer pair rpF1/rpR1. On the basis of collective RFLP patterns of amplified 16S rDNA and rp gene sequences, the BaDef phytoplasma was classified as a member of group 16SrI (group I, aster yellows phytoplasma group), subgroup B (16SrI-B), and rp subgroup rpI-B. Ribosomal protein subgroup B was distinguished from other rp subgroups on the basis of the presence of a recognition site for HpaII. The 1.8-kbp rDNA product of PCR primed by P1/P7 and the 1.2-kbp rpF1/rpR1 PCR product were cloned and sequenced, and the sequences were deposited in GenBank under Accession No. AY734453 for the BaDef 16S rDNA and Accession No. AY735448 for the BaDef rp gene sequence. Previously, only oat proliferation (OatP) phytoplasma, a member of subgroup 16SrI-A, had been characterized in a cereal crop (Avena sativa L.) in Europe (3); BaDef is another phytoplasmal disease threatening cereal crops in the region.

Ecological Implications from a Molecular Analysis of Phytoplasmas Involved in an Aster Yellows Epidemic in Various Crops in Texas

ABSTRACT In the spring of 2000, an aster yellows (AY) epidemic occurred in carrot crops in the Winter Garden region of southwestern Texas. A survey revealed that vegetable crops, including cabbage, onion, parsley, and dill, and some weeds also were infected by AY phytoplasmas. Nested polymerase chain reaction (PCR) and restriction fragment length polymorphism analysis of PCR-amplified phytoplasma 16S rDNA were employed for the detection and identification of phytoplasmas associated with these crops and weeds. Phytoplasmas belonging to two subgroups, 16SrI-A and 16SrI-B, in the AY group (16SrI), were predominantly detected in infected plants. Carrot, parsley, and dill were infected with both subgroups. Onion and three species of weeds (prickly lettuce, lazy daisy, and false ragweed) were predominantly or exclusively infected by subgroup 16SrI-A phytoplasma strains, while cabbage was infected by subgroup 16SrI-B phytoplasmas. Both types of phytoplasmas were detected in three leafhopper species, Macrosteles fascifrons, Scaphytopius irroratus, and Ceratagallia abrupta, commonly present in this region during the period of the epidemic. Mixed infections were very common in individual carrot, parsley, and dill plants and in individual leafhoppers. Sequence and phylogenetic analyses of 16S rDNA and ribosomal protein (rp) gene sequences indicated that phytoplasma strains within subgroup 16SrI-A or subgroup 16SrI-B, detected in various plant species and putative insect vectors, were highly homogeneous. However, based on rp sequences, two rpI subgroups were identified within the subgroup 16SrI-A strain cluster. The majority of subgroup 16SrI-A phytoplasma strains were classified as rp subgroup rpI-A, but phytoplasma strains detected in one onion sample and two leafhoppers (M. fascifrons and C. abrupta) were different and classified as a new rp subgroup, rpI-N. The degree of genetic homogeneity of the phytoplasmas involved in the epidemic suggested that the phytoplasmas came from the same pool and that all three leafhopper species may have been involved in the epidemic. The different phytoplasma population profiles present in various crops may be attributed to the ecological constraints as a result of the vector-phytoplasma-plant three-way interaction.

Identification of aster yellows phytoplasmas in soybean in Wisconsin based on RFLP analysis of PCR-amplified products (16S rDNAs)

Phytoplasmas have been detected for the first time by molecular methods in soybeans in Wisconsin. Nested polymerase chain reaction (PCR) using a combination of two phytoplasma-specific "universal" primer pairs (P1/P7-R16F2n/R16R2 and R16mF2/R16mR1-R16F2n/R16R2) amplified 16S rDNA (with expected fragment size of 1.2 kilobases) in soybean leaf tissue in 48 of 121 plants tested. Restriction fragment length polymorphism analysis of nested PCR products indicated that only 2 of these samples contained phytoplasmas. Gram-positive bacterial 16S rDNA genes were detected in 46 PCR positive samples. This was confirmed by culturing bacteria and direct PCR of soybean seed. In the two soybean samples, phytoplasmas belonging to the aster yellows phytoplasma group (16SrI), subgroup 16SrI-A, and a newly established subgroup, 16SrI-O, were identified. Phylogenetic analysis using partial 16S rDNA sequences (1.2 kilobases) from representative phytoplasma strains clustered soybean phytoplasmas into two distinct phylogenetic lineages that are consistent with the two subgroups defined by restriction fragment length polymorphism analysis. No correlation between the presence of phytoplasmas and the occurrence of "green-stem syndrome" in soybeans in Wisconsin was detected.

Molecular Characterization and Classification of Phytoplasmas Associated with Canola Yellows and a New Phytoplasma Strain Associated with Dandelions.

DNA isolated from symptomatic canola (Brassica napus, Brassica rapa) and dandelion (Taraxacum officinale) was used to amplify 16S ribosomal DNA fragments by polymerase chain reaction using two pairs of universal primers P1/P6 and R16F2n/R2. Restriction fragment length polymorphism (RFLP) analysis of the amplified DNA fragments using endonucleases AluI, HhaI, HpaII, MseI, RsaI, and Sau 3AI revealed two distinct types of phytoplasmas in canola with similar symptoms. One had the same RFLP profiles as the phytoplasmas in subgroup 16SrI-A, whereas the other one had RFLP profiles similar to those of phytoplasmas in subgroup 16SrI-B. Phytoplasmas were detected in symptomatic dandelion plants that were collected from canola and alfalfa fields where severe alfalfa witches'-broom occurred. Comparative studies indicated that two different phytoplasmas were associated with the dandelion plants. One was identified as a member of subgroup 16SrI-A, whereas another one was classified as a member of a distinct subgroup in the aster yellows group on the basis of the unique RFLP patterns.