An Emerging Potato Purple Top Disease Associated with a New 16SrIII Group Phytoplasma in Montana.

Potato purple top (PPT) is a devastating disease that occurs in the United States, Canada, Mexico, Russia, and elsewhere causing great economic loss to the potato industry through substantially reduced tuber yield and quality. Chips and fries processed from infected tubers often develop brown discoloration, greatly reducing their marketability. At least seven distinct phytoplasma strains belonging to five different phytoplasma groups (16SrI, 16SrII, 16SrVI, 16SrXII, and 16SrXVIII) have been reported to cause purple top and related symptoms in potato (3). During an unusual drought in 2007, a newly emerging potato disease with extensive yellowish or reddish purple discoloration of terminal shoots and leaves, similar to PPT symptoms, was observed in isolated potato fields in Montana where over 50% of plants exhibited symptoms. Shoot tissues were collected from three symptomatic plants and 17 tubers randomly collected from 17 other symptomatic plants. The tubers were cold treated to induce sprouting and then planted in the greenhouse. All tubers produced plants of which seven exhibited PPT symptoms including severe stunting. Total nucleic acid was extracted from leaf veinal tissue, stolons, or tubers of 10 symptomatic and 10 asymptomatic plants (both field-collected and greenhouse samples) as previously described (3). A nested-PCR assay, using universal primer pair P1/16S-SR followed by R16F2n/R16R2n, was performed as previously described (2,3) to detect phytoplasmas in these samples. Phytoplasma strains were detected in all symptomatic plants. Restriction fragment length polymorphism (RFLP) analyses of nested-PCR products (approximately 1.2 kb) with seven key restriction enzymes (AluI, MseI, HhaI, Tsp509I, HpaII, RsaI, and BfaI) indicated that all samples contained a very similar or identical phytoplasma most closely related to reference strain MW1 (belonging to subgroup 16SrIII-F) (1). Analysis of cloned 16S rDNA sequences confirmed the identity of this new phytoplasma and sequences of three representative PPT-MT strains were deposited in GenBank with Accession Nos. FJ226074-FJ226076. Computer-simulated RFLP analyses of 1.2-kb 16S rDNA sequences of this new phytoplasma and representative members in the peach X-disease phytoplasma group (16SrIII) available in GenBank indicated the strain is distinct and represents a new subgroup, 16SrIII-M (4). This study also indicated that the phytoplasma is tuber transmissible since approximately 35% of plants produced from infected tubers collected in this study developed symptoms. Transmission via infected tubers may pose a potential threat for disease spread by planting uncertified seed potatoes. To our knowledge, this is the first report of 16SrIII group phytoplasmas-associated diseases in potato. A phytoplasma closely related to the PPT-MT strains has recently been detected in potato seedlings exhibiting purple top, rosette, and stunting in Alaska (GenBank Accession No. FJ376628). References: (1) I.-M. Lee et al. Int. J. Syst. Bacteriol. 48:1153, 1998. (2) I.-M. Lee et al. Int. J. Syst. Evol. Microbiol. 54:337, 2004. (3) I.-M. Lee et al. Int. J. Syst. Evol. Microbiol. 56:1593, 2006. (4) W. Wei et al. Int. J. Syst. Evol. Microbiol. 58:2368, 2008.

Identification of phytoplasma species associated with potato diseases in Russia.

Four out of six known potato diseases attributed to phytoplasma infection were previously reported to occur in Russia based on a combination of biological properties such as symptomatology and/or vector relationships and electron microscopy of infected phloem tissue. In 2007, the first molecular identification of potato diseases causing symptoms including purple top, round leaves, stunting, bud proliferation and formation of aerial tubers was carried out using PCR methods. A nested PCR using primer pair P1/P7 in the first amplification followed by R16F2n/R16R2n in the second amplification was performed to detect phytoplasma in infected potato samples. PCR products were digested singly with several restriction enzymes. Comparison of RFLP profiles with published profiles was used for identification of the putative phytoplasma detected. The majority of 49 PCR positive potato samples showed RFLP profiles of 16S rDNA sequences very similar or identical to stolbur phytoplasma, a strain belonging to stolbur phytoplasma group (16Sr XII), subgroup 16SrXII-A, and only two showed RFLP profiles similar to those of aster yellow phytoplasma strains ('Candidatus Phytoplasma asteris') belonging to aster yellows phytoplasma group (16SrI), subgroup 16SrI-A and 16SrI-B. The results demonstrated that stolbur phytoplasma is prevalent in several potato growing regions of Russia.

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. References: (1) I.-M. Lee et al. Int. J. Syst. Bacteriol. 48:1153, 1998. (2) G. R. Stanosz et al. Plant Dis. 81:424, 1997.

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. References: (1) D. Z. Bogoutdinov. Potato Phytoplasmas and Methods of Their Study. Samara State Agricultural University, Samara, 2000. (2) M. J. Green et al. Plant Dis. 83:482, 1999. (3) I.-M. Lee et al. Int. J. Syst. Bacteriol. 48:1153, 1998. (4) I.-M. Lee et al. Plant Dis. 90:989, 2006.

Ribosomal protein gene-based phylogeny for finer differentiation and classification of phytoplasmas.

Extensive phylogenetic analyses were performed based on sequences of the 16S rRNA gene and two ribosomal protein (rp) genes, rplV (rpl22) and rpsC (rps3), from 46 phytoplasma strains representing 12 phytoplasma 16Sr groups, 16 other mollicutes and 28 Gram-positive walled bacteria. The phylogenetic tree inferred from rp genes had a similar overall topology to that inferred from the 16S rRNA gene. However, the rp gene-based tree gave a more defined phylogenetic interrelationship among mollicutes and Gram-positive walled bacteria. Both phylogenies indicated that mollicutes formed a monophyletic group. Phytoplasmas clustered with Acholeplasma species and formed one clade paraphyletic with a clade consisting of the remaining mollicutes. The closest relatives of mollicutes were low-G+C-content Gram-positive bacteria. Comparative phylogenetic analyses using the 16S rRNA gene and rp genes were performed to evaluate their efficacy in resolving distinct phytoplasma strains. A phylogenetic tree was constructed based on analysis of rp gene sequences from 87 phytoplasma strains belonging to 12 16Sr phytoplasma groups. The phylogenetic relationships among phytoplasmas were generally in agreement with those obtained on the basis of the 16S rRNA gene in the present and previous works. However, the rp gene-based phylogeny allowed for finer resolution of distinct lineages within the phytoplasma 16Sr groups. RFLP analysis of rp gene sequences permitted finer differentiation of phytoplasma strains in a given 16Sr group. In this study, we also designed several semi-universal and 16Sr group-specific rp gene-based primers that allow for the amplification of 11 16Sr group phytoplasmas.

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. References: (1) I.-M. Lee et al. Int. J. Syst. Bacteriol. 48:1153, 1998. (2) I.-M. Lee et al. Int. J. Syst. Evol. Microbiol. 54:337, 2004.

SecY gene sequence analysis for finer differentiation of diverse strains in the aster yellows phytoplasma group.

Aster yellows (AY) group (16SrI) phytoplasmas are associated with more than 100 economically important diseases worldwide and represent the most diverse and widespread phytoplasma group. Phylogenetic analysis of secY gene sequences resolved 10 genetically distinct lineages. The 10 lineages coincide with those delineated by phylogenetic analysis based on ribosomal protein (rp) gene sequences. However, greater genetic variability among the 10 lineages was revealed based on secY gene sequences. The distinct phylogenetic lineages can be readily identified through restriction fragment length polymorphism (RFLP) analysis of secY gene sequences. Ten subgroups were differentiated among the AY group phytoplasmas based on RFLP analysis of secY gene sequences. Phylogenetic analysis based on secY gene sequences in this study, and previous studies on the 16S rRNA gene, tuf gene, and rp gene sequences reinforced the notion that most subgroups identified by RFLP analysis of secY and rp gene sequences represent distinct phylogenetic lineages.

First Report of a Defect of Processing Potatoes in Texas and Nebraska Associated with a New Phytoplasma.

An outbreak of a new potato disease occurred in Texas and Nebraska causing a serious defect in potato chips produced from commercial processing potatoes. The defect consists of patchy brown discoloration of chips and can be a cause for rejection of contracted potatoes by the processor. Infected potato plants exhibit symptoms of the purple top wilt syndrome similar to those of the purple top disease in processing potatoes caused by clover proliferation phytoplasma recently found in Washington and Oregon (3). Foliar symptoms include stunting, chlorosis, slight purple coloration of new growth, swollen nodes, proliferated axillary buds, and aerial tubers. Tuber symptoms include mild vascular discoloration and brown flecking of medullary rays. Seed potatoes from affected plants produce hair sprouts. Total nucleic acid was extracted from leaf and stolon tissue of symptomatic plants in the field and from tuber samples exhibiting the defect from commercial storages. Nested polymerase chain reactions (PCR) were performed using phytoplasma-universal 16SrDNA-based primers (P1/P7 and R16F2n/R16R2) (2) to detect the presence of phytoplasmas in these samples. A negative control, devoid of DNA templates in the reaction mix, was included in all PCR assays. In 2004, 13 foliar samples tested positive for phytoplasmas using PCR. None of the apparently symptomless plants or tubers tested positive. Restriction fragment length polymorphism (RFLP) analysis of the PCR-amplified 16S rDNA using enzymes AluI, MseI, HhaI, BfaI, and Tsp509I indicated that four samples are associated with a phytoplasma belonging to subgroup A (16SrI-A) of the "Candidatus Phytoplasma asteris" (aster yellows phytoplasma) group (16SrI), and nine plant samples were associated with a new phytoplasma related to, but distinct from, the stolbur phytoplasma group (16SrXII). Nucleotide sequence analysis of cloned 16S rDNAs (GenBank Accession Nos. DQ174114-DQ174123) confirmed the results on the basis of RFLP analyses. Sequences of cloned 16S rDNAs were analyzed with previously described phytoplasma strains available in GenBank using DNAStar's (Madison, WI) Lasergene software MegAlign program. The new phytoplasma is only distantly related to the stolbur phytoplasma, sharing 96.6% sequence homology. In 2005, 14 defective tuber samples from storage and 16 symptomatic plants from the field tested positive for the new phytoplasma. In Texas and Nebraska, it appears that at least two distinct phytoplasmas seem to be involved in the disease complex contributing to the defects of processed products produced from infected potatoes. Previous reports have suggested a similar defect of chipping potatoes, but the phytoplasma associated with the disease was not identified (1). To our knowledgek, this the first report of this new phytoplasma associated with disease and defects of potato and the first report of this phytoplasma in the United States. References: (1) E. E. Bantarri et al. Trans. ASAE 33:221, 1990. (2) I.-M. Lee et al. Int. J. Sys. Bacteriol. 48:1153, 1998. (3) I.-M. Lee et al. Plant Dis. 88:429, 2004.

Novel insertion sequence-like elements in phytoplasma strains of the aster yellows group are putative new members of the IS3 family.

Novel insertion sequence (IS)-like elements were isolated and characterized from phytoplasma strains in the aster yellows (AY) group (16SrI). The IS-like elements were cloned from phytoplasma strains AY1 and NJAY or PCR-amplified from 15 additional strains representing nine subgroups in the AY group using primers based on sequences of the putative transposases (Tpases). All IS-like elements contained sequences encoding similar Tpases of 321 amino acids (320 for strain CPh). Substantial amino acid sequence variability suggested multiple species of Tpases or IS-like elements exist in the AY phytoplasma group. These Tpases have an identical DDE motif that is most similar to the DDE consensus of Tpases in the IS3 family.

'Candidatus Phytoplasma asteris', a novel phytoplasma taxon associated with aster yellows and related diseases.

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.

Clover Proliferation Group (16SrVI) Subgroup A (16SrVI-A) Phytoplasma is a Probable Causal Agent of Dry Bean Phyllody Disease in Washington.

During 2003, a new disease, dry bean phyllody (DBPh), was observed in the Columbia Basin of Washington in dry bean (Phaseolus vulgaris L.) cultivars of Andean origin grown in Mattawa and Paterson, WA that caused great reduction in dry bean production. Symptoms of DBPh became apparent during mid-to-late pod development and were characterized by leafy petals (phyllody) and aborted seed pods resembling thin, twisted, and corrugated leaf-like structures. Deformed sterile pods that were small, sickle-shaped, upright, and leathery were also observed. The infected plants generally exhibited chlorosis, stunting, or bud proliferation from leaf axils. Symptoms of DBPh were indicative of possible infection by phytoplasmas. Restriction fragment length polymorphism (RFLP) and phylogenetic analyses of amplified 16S rDNA sequences were used for phytoplasma identification. Four symptomatic bean plants were analyzed and tested positive for phytoplasma infection on the basis of results of initial polymerase chain reaction (PCR) and subsequent nested-PCR amplifications (2). RFLP analyses of 16S rDNA sequences with restriction enzymes, MseI, AluI, HhaI, RsaI, and HpaII indicated that the phytoplasma strains associated with DBPh belonged to the clover proliferation group (16SrVI) subgroup A (16SrVI-A) (2). This subgroup currently consists of three members, clover proliferation (CP; GenBank Accession No. AY500130), potato witches'-broom (PWB; GenBank Accession No. AY500818), and vinca virescence (VR; GenBank Accession No. AY500817), a strain of beet leafhopper-transmitted virescence agent (BLTVA) phytoplasmas (1,2). The taxonomic affiliations of the DBPh phytoplasma strains were confirmed by phylogenetic analysis of cloned 16S rRNA gene sequences (GenBank Accession Nos. DBPh2, AY496002; DBPh3, AY496003). Among the existing members of subgroup 16SrVI-A, the four DBPh strains were closely related to the VR strain with 99.7% 16S rDNA sequence homology and to the CP strain with 99.2% sequence homology. To gain further evidence on the role of 16SrVI-A phytoplasma strains in DBPh disease, a modified test of Koch's postulates was conducted. Infected tissue from one phytoplasma-positive dry bean sample was grafted onto three Pinto UI-114 bean seedlings in the greenhouse. Within 60 days, the bean seedlings exhibited corrugated leaf-like structures from aborted seedpods, a lack of flower formation, general chlorosis, and stunting similar to the original diseased plants. The lower leaves of the inoculated bean plants became epinastic and leathery. The transmitted phytoplasma was detected in each of the grafted symptomatic seedlings, and the RFLP patterns of its 16S rRNA gene sequences were identical to those of the phytoplasmas in the scions. A high correlation between the presence of disease symptoms and the presence of subgroup 16SrVI-A phytoplasmas in the bean plants suggests that these phytoplasmas play an etiological role in DBPh disease. To our knowledge, these findings provide the first confirmed case of phytoplasma-associated DBPh in the United States. References: (1) D. A. Golino et al. Plant Dis. 73:850, 1989. (2) I.-M. Lee et al. Int. J. Syst. Bacteriol. 48:1153, 1998.

Clover Proliferation Group (16SrVI) Subgroup A (16SrVI-A) Phytoplasma is a Probable Causal Agent of Potato Purple Top Disease in Washington and Oregon.

An epidemic of purple top disease of potato (Solanum tuberosum) occurred in the Columbia Basin Region of Washington and Oregon in 2002 and 2003, causing great economic loss in the potato industry (1). Symptoms of potato purple top (PPT) were characterized by upright terminal shoots, upward leaf rolling, chlorosis, red or purplish discoloration of new leaves, proliferation of axillary shoots with basal swelling, and the formation of aerial tubers. Preliminary studies on PPT disease suggested phytoplasma as a possible cause (1). In this study, 78 potato samples (including five asymptomatic) were collected from five fields throughout the region. A nested polymerase chain reaction (PCR) with primer pair P1/P7 in the first amplification followed with primer pair R16F2n/R16R2 was performed to detect the presence of phytoplasmas in infected plants (2). Restriction fragment length polymorphism (RFLP) and phylogenetic analyses of amplified 16S rDNA sequences were used for phytoplasma identification. Eighty-four percent (63% in the first amplification) of the symptomatic samples and 60% (0% in the first amplification) of the asymptomatic samples tested positive. Low phytoplasma titers and the presence of PCR inhibitors accounts for the low detection rate in the first PCR amplifications. RFLP analyses of 16S rDNA with enzymes MseI, AluI, HhaI, RsaI, and HpaII indicated that the phytoplasma associated with PPT belonged to the clover proliferation (CP) group (16SrVI) subgroup A (16SrVI-A) (2). 16SrVI-A currently consists of three members, CP (GenBank Accession No. AY500130), potato witches'-broom (GenBank Accession No. AY500818), and vinca virescence (VR) (GenBank Accession No. AY500817), a strain of beet leafhopper-transmitted virescence agent (BLTVA) phytoplasma (2). The taxonomic affiliation of PPT phytoplasma was confirmed by phylogenetic analysis of cloned 16S rDNA (GenBank Accession Nos. PPT4, AY496004; PPT8, AY496005). The 16S rDNA sequences of the PPT strains were closely related to VR with 99.7% sequence homology compared with 99.2% with CP. A high correlation between the symptoms and the presence of 16SrVI-A phytoplasmas in the potato plants suggests that these phytoplasmas play an etiological role in PPT disease. To gain further evidence, a modified test of Koch's postulates was conducted. Infected tissues from four phytoplasma-positive potato samples (including PPT4 and PPT8) were grafted onto healthy potato seedlings. Within 60 days after grafting, the potato seedlings developed symptoms similar to those in the original diseased samples. The newly infected plants were maintained through cuttings. RFLP analysis of 16S rDNA indicated that the phytoplasmas detected in each of the seedlings and cuttings were identical to those in the scions. These results confirmed the probable etiological role of CP group, subgroup 16SrVI-A phytoplasma strains in PPT disease in Washington and Oregon. There are two other confirmed cases of phytoplasmas (BLTVA and aster yellows phytoplasma) associated with PPT disease in Utah (4) and Mexico (3). References: (1) P. B. Hamm et al. Potato Prog. Vol. 3, No. 1, 2003. (2) I.-M. Lee et al. Int. J. Syst. Bacteriol. 48:1153, 1998. (3) N. E. Leyva-Lopez et al. Can. J. Microbiol. 48:1062, 2002. (4) C. D. Smart et al. Phytopathology 83:1399, 1993.

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.

A New Member of the Clover Proliferation Phytoplasma Group (16SrVI) Associated with Elm Yellows in Illinois.

A disease with symptoms similar to elm yellows (EY) was noticed in the early 1990s in suburban Chicago, IL. More than 1,000 mature American elms (Ulmus americana) have since died. Infected trees varied in the incidence and severity of canopy yellowing, leaf epinasty, butterscotch discoloration, and wintergreen odor of the phloem, but all developed a sparse and clumpy crown, uniformly necrotic phloem, and died within 2 years of showing canopy symptoms. Because symptoms were expressed irregularly and phytoplasma detection results by a commercial diagnostic company were inconsistent, a study was initiated to determine if EY phytoplasma was the causal agent. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) methods using universal or EY phytoplasma specific primers were employed to detect putative phytoplasma(s) associated with 10 trees of varied disease severity within the outbreak region and 10 asymptomatic trees from an uninfected area (controls). Nested PCR using universal primers revealed that 90% of trees from the outbreak region were positive for phytoplasma while asymptomatic elms from another location (controls) tested negative. Phytoplasma-positive trees ranged in disease severity from 1 (asymptomatic) to 5 (near death). Inner bark samples chiseled from the lower trunk had higher phytoplasma detection rates than foliage or drill shavings. RFLP analyses and DNA sequencing of 16S rDNA indicated that the phytoplasma recovered from dying elms in Arlington Heights is not related to the reference EY phytoplasma (group16SrV). It is most closely related to clover proliferation (CP) phy-toplasma (group 16SrVI), and we have designated it Illinois Elm Yellows (ILEY) phytoplasma, and assigned it to a new taxonomic subgroup (16SrVI-C). EY phytoplasma was not detected in any samples, but two ILEY phytoplasma positive trees also were positive for aster yellows (AY) phytoplasma. ILEY phytoplasma was not detected in local leafhopper populations trapped in elm trees between May and September 2000. This is the first report of a phytoplasma related to CP phytoplasma causing elm yellows disease symptoms.

First Report of an Aster Yellows Subgroup 16SrI-B Phytoplasma Infecting Chayote in Costa Rica.

An outbreak of a witches' broom disease affected approximately 20% of plants in several chayote (Sechium edule (Jacq.) Schwartz) fields in the commercial production area of the Ujarrás Valley, Cartago Province, Costa Rica during 2000 and 2001. Affected chayote plants exhibited symptoms, including basal proliferation with severe foliage reduction, aborted flowers, and deformed fruits, suggestive of phytoplasmal infection. Two other symptomatic cucurbit species growing near the chayote fields were also identified. These species were tacaco plants (S. tacaco (Pitt.) C. Jeffrey), an edible cucurbit for domestic marketing in Costa Rica, showing severe size reduction of leaves and fruits, and Rytidostylis carthaginensis (Jacq.) Kuntze, a weed in chayote and tacaco fields, exhibiting abnormal tendril proliferation. Plants were analyzed for phytoplasma infection by a nested polymerase chain reaction (PCR) assay, using the universal rRNA primer pair P1/P7 followed by R16F2n/R16R2 (2). Phytoplasmas were detected in all symptomatic samples (18 chayote, 6 tacaco, and 3 weed) tested but were undetectable in all asymptomatic samples (10 chayote, 6 tacaco, and 2 weed). Restriction fragment length polymorphism (RFLP) analysis of PCR products (16S rDNA sequences) by separate digestion with eight restriction enzymes (RsaI, HhaI, KpnI, BfaI, HaeIII, HpaII, AluI, MseI) revealed that a phytoplasma belonging to subgroup 16SrI-B in the aster yellows phytoplasma group (16SrI) was associated with chayote witches' broom (CWB). The same or very similar phytoplasmas were found in both symptomatic tacaco and R. carthaginensis plants. Phylogenetic analysis of 16SrDNA sequences also confirmed the CWB phytoplasma to be most similar to members of subgroup 16SrI-B. Similar diseases in chayote and other cucurbits have been reported in Brazil (3), Taiwan (1), and Mexico (4). The CWB phytoplasma differs from the phytoplasma (16SrIII-J subgroup) associated with chayote in Brazil. The identities of phytoplasmas associated with cucurbits in Taiwan and Mexico are unknown. The occurrence of an aster yellows group phytoplasma in chayote may pose a potential threat to continued production and exportation of this cash crop. To our knowledge, this is the first report of 16SrI-B subgroup phytoplasmas in naturally infected cucurbits in Costa Rica. References: (1) T. G. Chou et al. Plant Dis. Rep. 60:378, 1976. (2) I.-M. Lee et al. Int. J. Syst. Bacteriol. 48:1153, 1998. (3) H. G. Montano et al. Plant Dis. 84:429, 2000. (4) E. Olivas. Rev. Fitopatol. (Lima) 13:14, 1978.