Aster Yellows Group Research Articles

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.

First Report of a Phytoplasma Associated with Witches'-Broom of the Giant Coral Tree (Erythrina poeppigiana, Fabaceae) in Costa Rica.

The giant coral tree (Erythrina poeppigiana, Fabaceae) is a common shade tree in coffee plantations in Costa Rica. Coral trees are pruned to decrease fungal infections and increase nitrogen fixation. Recently, severe shoot proliferation, internodes shortening, and leaf reduction were observed in pruned shade trees in the south of San José Province, Costa Rica. Leaf samples from 10 symptomatic E. poeppigiana trees were collected. Also, two samples from symptomless coral trees were collected from areas free of witches'-broom. Total DNA was extracted from 0.5 g of petiole tissue from all samples with the plant extraction mini kit (Qiagen GmbH, Hilden, Germany) with a modified protocol (2) and assayed by nested PCR with phytoplasma universal rDNA primers (P1/P7) (1) and R16F2n/R16R2 (3). All symptomatic trees tested positive for phytoplasmas by PCR, yielding the expected 1.2-kb band. DNA from the symptomless trees was not amplified by PCR. The restriction fragment length polymorphism analyses (HaeIII, AluI, RsaI, BfaI, HpaII, KpnI, HhaI, and MseI) and the sequence of the 1.2-kb PCR fragment (GenBank Accession No. DQ485305) revealed that the phytoplasma associated with coral tree witches'-broom belongs to the aster yellows phytoplasma group (16SrI) (4). To our knowledge, this is the first report of a phytoplasma belonging to the aster yellow group causing witches'-broom in the Erythrina genus.

Phytoplasma-associated Shoot Proliferation and Leaf Yellowing in Lettuce

Phytoplasma was identified from leaf lettuce (Lactuca sativa) cultivated in commercial green-house in Korea. Diseased leaf lettuce revealed proliferation of shoots, and yellowing and shrinking of leaves (lettuce proliferation-K). Polymerase chain reaction (PCR) with universal primer pair P1/P6, and aster yellows (AY) specific primer pair R16F1/R1 amplified 1.5kb and 1.1kb length of DNA fragments, respectively. Nucleotide sequences of 16S rRNA gene were determined (Gen Bank accession no EF489024). Phylogenetic analysis of 16S rDNA showed the closest relationship with AY phytoplasma (GenBank accession no. AY389822 and AY389826), indicating that lettuce proliferation-K is a member of AY. Phytoplasma bodies were detected in phloem sieve tubes of diseased lettuce by transmission electron microscopy. The structures had round or pleomorphic shapes with a diameter of 130-300nm. Phylogenetic analysis of 16S rRNA gene, microscopic observation of phytoplasma bodies and symptomatology indicated that lettuce proliferation-K is caused by phytoplasma in the AY group. This is the first report of phytoplasma disease in lettuce in Korea.

‘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.

First report of Candidatus‘Phytoplasma asteris’ infecting tomatillo (Physalis ixocarpa) in Sinaloa, México

Tomatillo, also called husk or green tomato, is an important vegetable in the Mexican diet since it is widely used to prepare many types of salsas and other traditional dishes. As a result it is produced commercially in 29 of the 32 Mexican states, with a national cultivated area of 53 407 hectares in 2004. In 2005, a plant disorder was observed in tomatillo fields from Sinaloa state. Symptoms were similar to those associated with phytoplasma diseases, such as little leaf, yellowing and leaf deformation (Lee et al., 2000). Leaf samples of 26 plants with symptoms were collected. Total DNA was extracted (Zhang et al., 1998) and used as template in a nested PCR assay to amplify the phytoplasma 16S rDNA gene using primer pairs R16mF2/R16mR1 and R16F2n/R16R2 (Gundersen & Lee, 1996). Almost two thirds of the samples produced the expected PCR fragment (1·25 kb) and all samples tested gave the same profile following restriction digestion with AluI, KpnI, MseI, HaeIII and RsaI. The PCR product was cloned into the pGEM-T easy vector (Promega) and sequenced. The amplified 16S rDNA sequence of the phytoplasmas (1246 bp; GenBank Acc. no. DQ987871) was compared with DNA sequences of other phytoplasmas taken from the GenBank database, using the Clustal V alignment method (MegAlign). The highest sequence similarity (99%) was obtained with tomato and pepper little leaf and ash witches’-broom phytoplasmas (GenBank Acc. nos. DQ375238, DQ168882 and AY566302 respectively), which are isolates of Candidatus‘Phytoplasma asteris’ (16SrI Aster yellows group). This is the first report of a phytoplasma associated with a disease of the tomatillo crop in Mexico. This work was supported by Instituto Politécnico Nacional and Fundación Produce Sinaloa.

First Report of Phytoplasmas Associated with the Degeneration Syndrome of Artichoke in Catalonia (Spain).

Spain is the second largest producer of artichoke (Cynara scolymus L.) in the world with 230,000 tons produced annually. The region of Catalonia, located in northeast Spain, has 3,500 ha dedicated to this crop. Low yield and degeneration anomalies are widespread problems in the Mediterranean area. The degeneration syndrome results in curled leaves and late development of capitulum. The association of this syndrome with different viruses such as Artichoke degeneration virus (ADV), Artichoke latent virus (ALV), Broad bean wilt virus (BBWV), Cucumber mosaic virus (CMV), and many others was not conclusive. Other studies indicated that this phenomenon could be related to a regression of the cultivar toward the species of origin, the wild cardoon (C. cardunculus L. var. sylvestris). The distribution of degenerated plants in the principal artichoke-growing areas of Catalonia together with the presence of plants with normal and degenerated tissues in the same plant suggested the presence of phytoplasmas. Samples from 30 symptomatic and 30 asymptomatic plants of cv. Blanca de Tudela were collected in February of 2006 from different areas of Catalonia and analyzed by PCR amplification of phytoplasma DNA. DNA for PCR analyses was prepared from leaf petioles and midribs according to the Ahrens and Seemüller procedure (1). Nested-PCR was carried out with rRNA primer pairs P1/P7 and fU5/rU3 (2,3). Alternatively, nested-PCR, with primers Tuf 1 f/r in the first step and Tuf AY f/r in the second, amplifying a DNA fragment of the elongation gene Tu of the phytoplasmas belonging to Aster yellows and stolbur groups was conducted (4). Results showed a high correlation between presence of symptoms and phytoplasma detection. Phytoplasmas were detected in 100% of the symptomatic plants (30 of 30) and only in one of the asymptomatic plants. The restriction fragment length profiles of Tuf AY amplicons with HpaII showed two different patterns, the most important belonging to the aster yellows (16SrI) group phytoplasma and the other to the stolbur (16SrXII) group phytoplasma. To our knowledge, this is the first time that phytoplasmas have been detected in artichoke and associated with degeneration of this crop.

First Report of Spirea Witches'-Broom Disease in China.

Bumald spirea (Spiarea bumalda Burv.) is an important ornamental tree widely grown in northern China. In August of 2006, spirea plants exhibiting symptoms of witches'-broom, stunting, yellowing, and shoot dieback were found at an incidence of 5 to 15% in Qingzhou City, Shandong Province, China. Total DNA was extracted separately from 0.1 g of phloem tissue from leaf midribs and stems of six symptomatic and six asymptomatic plants with a modified cetyltriethylammonium bromide (CTAB) method (3). Resulting DNA samples were analyzed for phytoplasma DNA by a nested PCR assay using phytoplasma universal 16S rDNA gene primer pairs R16mF2/R16mR1 and R16F2n/R16R2 (2). These primers amplified 1.5- and 1.2-kb products, respectively, from DNA of all symptomatic plants only. Restriction fragment length polymorphism (RFLP) analysis of the 1.2-kb 16S rDNA product using enzymes AluI, MseI, and HhaI indicated that all symptomatic plants contained a group 16SrI (aster yellows group) subgroup B (16SrI-B) phytoplasma strain (4). A 16S rDNA sequence derived from this strain (GenBank Accession No. EF176608) was most similar (99.8 and 99.6%) to those of severe aster yellows (GenBank Accession No. M86340) and Maryland aster yellows (GenBank Accession No. AF322644) phytoplasmas, respectively, thereby confirming strain identity based on RFLP analysis. A phytoplasma (Spiarea stunt phytoplasma, GenBank Accession No. AF190228), which belongs to X-disease group (16SrIII), was reported to infect spirea and probably be lethal to S. tomentosa in New York (1,4). The phytoplasma reported here shared low identity (90.8%) with Spiarea stunt phytoplasma, but also caused dieback of spirea shoots. The epidemiology and economic impact of this disease need further intensive investigation. To our knowledge, this is the first report of spirea witches'-broom disease and of its association with a subgroup 16SrI-B phytoplasma in China.

Detection and Characterization of Phytoplasmas Infecting Ornamental and Weed Plants in Iran

AbstractDuring field surveys in 2004, ornamental and weed plants showing symptoms resembling those caused by phytoplasmas were observed in Mahallat (central Iran). These plants were examined for phytoplasma infections by polymerase chain reaction (PCR) assays using universal phytoplasma primers directed to ribosomal DNA (rDNA). All affected plants gave positive results. The detected phytoplasmas were characterized and differentiated through restriction fragment length polymorphism (RFLP) and sequence analysis of PCR‐amplified rDNA. The phytoplasmas detected in diseased Asclepias curassavica and Celosia argentea were identified as members of clover proliferation phytoplasma group (16SrVI group) whereas those from the remaining plants examined proved to be members of aster yellow phytoplasma group (16SrI group) (‘Candidatus Phytoplasma asteris’). In particular, following digestion with AluI, HaeIII and HhaI endonucleases, the phytoplasma detected in Limonium sinuatum showed restriction profiles identical to subgroup 16SrI‐C; phytoplasmas from Gomphocarpus physocarpus, Tanatacetum partenium, Lactuca serriola, Tagetes patula and Coreopsis lanceolata had the same restriction profiles as subgroup 16SrI‐B whereas Catharanthus roseus‐ and Rudbeckia hirta‐infecting phytoplasmas showed restriction patterns of subgroup 16SrI‐A. This is the first report on the occurrence of phytoplasma diseases of ornamental plants in Iran.

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.

First Report of a 16SrI-B Group Phytoplasma Associated with a Yellows-Type Disease Affecting Tomato Plants in the Baja California Peninsula of Mexico.

Since 2000, a phytoplasma-like disease (locally known as "permanent yellowing") was observed on tomatoes (Lycopersicon esculentum Mill.) grown in the Valle de San Quintín in northern Baja California Peninsula. Affected plants showed general chlorosis, severe stunting, upwardly rolled leaves, bronzing of mature leaves, purple discoloration of veins, "little leaf", abnormal floral structures, and excessive branching of axillary shoots. Total DNA extracted from symptomatic and asymptomatic plants was used in nested (n)-PCR assays driven by phytoplasma-universal primer pair P1/P7 (3), followed by primer pair R16F2n/R16R2 (1) targeting the 16S ribosomal RNA gene of the putative phytoplasma. PCR conditions (direct and nested) were conducted as previously described (l,3). Restriction fragment length polymorphism (RFLP) patterns of nPCR-amplified products (≈ 1.25-kbp 16S rDNA fragments) digested with enzymes AluI, MseI, HhaI, and HpaII showed that 85% (17 of 20) of PCR-positive tomato samples had restriction patterns typical of phytoplasmas belonging to the aster yellows group, subgroup B (16SrI-B) "Candidatus Phytoplasma asteris" (2). Only 10% (2 of 20) of the samples were associated with a phytoplasma related to the 16SrXIII-A Mexican periwinkle virescence group (formerly group 16SrI, subgroup I). None of the symptomless plants tested positive. Subsequently, these results were confirmed by nPCR using 16SrI specific primer pair P1/Aint (4) and specific primers rp(I-B)F1/rp(I-B)R1 that amplify the ribosomal protein (rp) gene operon of aster yellows phytoplasma subgroup B (16SrI-B[rp-B]) (1). The presence of the phytoplasmas in symptomatic plants was confirmed by scanning electron microscopy. Characteristic yellow symptoms could be experimentally reproduced by graft inoculation of tomato seedlings (cv. Maya) with tissue of field-infected plants. Symptoms similar to those of field-grown diseased plants were observed consistently in most of the plants, and when graft transmitted from tomato to periwinkle (Catharantus roseus (L.) G. Don), symptoms of virescencent, small flowers were observed. In contrast, no symptoms were observed on plants grafted with tissues from healthy plants. In Baja California, it appears that at least two distinct phytoplasmas are involved in the disease complex. To our knowledge, this is the first molecular evidence of the presence of a phytoplasma associated with yellows-type diseases in the major tomato cultivation areas of the peninsula.

Detection, characterisation and transmission by Macrosteles leafhoppers of watercress yellows phytoplasma in Hawaii

AbstractA new yellows disease of watercress (Nasturtium officinale) in Hawaii has symptoms of reduced leaf size, leaf yellowing and crinkling, and occasionally witches’ brooms. This disease is found on all watercress farms on Oahu but has not yet been found on other Hawaiian islands. Watercress plants were tested for phytoplasma infection by polymerase chain reaction assays using phytoplasma‐specific primers. Amplicons of the expected sizes were produced from all symptomatic plants but not from healthy plants raised from seed. Phylogenetic analysis of the 16S rRNA gene indicated that watercress yellows was caused by a phytoplasma in the aster yellows group, with sequence similarity to onion yellows from Japan. Six weed species collected from the vicinity of affected watercress farms, Amaranth sp., Eclipta prostrata, Emilia sonchifolia, Plantago major, Myriophyllum aquaticum and Sonchus oleraceus, were also determined to be hosts of this phytoplasma. Leafhoppers, identified as Macrosteles sp. (Hemiptera, Cicadellidae), collected from symptomatic watercress transmitted this phytoplasma to watercress, plantain and lettuce (Lactuca sativa) in greenhouse experiments.

Basil little leaf: a new disease associated with a phytoplasma of the 16SrI (Aster Yellows) group in Cuba

In Cuba, the development of urban and peri-urban agriculture, using a combined organic and hydroponic (‘organoponic’) system of intensive vegetable production, currently plays a very important role for the sustainability of food security. Basil ( Ocimum basilicum ) has become a very popular barrier crop for organoponic gardens, where it is commonly used to repel undesirable pests. During a survey in 2005 of weeds from the ‘Alamar’ organoponic in Havana province, typical phytoplasma-symptoms of little leaf and witches’ broom (Jones, 2002) were observed in all basil plants used as barrier crops. Leaf samples from 27 plants with symptoms and 16 apparently healthy (symptomless) plants were collected and the DNA extracted. This was used as the template in a nested PCR with universal phytoplasma rRNA primer pairs P1/P7 and R16F2n/R16R2. All 27 extracts from plants with symptoms produced a 1250 bp product, which gave typical phytoplasma profiles when digested with the restriction endonuclease Hae III. No amplicons were produced when DNA of symptomless plants was used as the template. RFLP using Rsa I and Alu I gave identical profiles for all 27 samples. The P1-P7 PCR products were directly sequenced and compared by BLAST analysis with those of other phytoplasmas in GenBank. The highest similarity (99%) was to that of a 16SrI group phytoplasma (Aster Yellows; ‘ Candidatus Phytoplasma asteris’) affecting watercress (GenBank accession no. AY665676). The 16S rRNA sequence from basil was deposited in GenBank (Accession no. DQ286577). This report from Cuba is the first record of a new disease for which the name basil little leaf is proposed.

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 "Candidatus Phytoplasma asteris"-Related Strains in Brassica rapa in Saskatchewan, Canada.

"Candidatus phytoplasma asteris" and related strains (i.e., aster yellows group 16SrI) have been associated with diseases of numerous plant species worldwide. Symptoms of aster yellows (AY) have been reported on rapeseed/canola (Brassica napus and B. rapa) crops in Saskatchewan (SK) and Manitoba, Canada since 1953 (2). Symptoms generally include stunting, virescence, leaf yellowing or purpling, phyllody, and formation of bladder-like siliques. A total of 120 mature B. rapa cv. AC Sunbeam plants exhibiting AY symptoms were collected in commercial fields near Medstead, SK during 2003 and 2004 (one field per year). As described previously (4), total genomic DNA was extracted from leaf, stem, roots, and seeds collected from the 120 plants, from seeds from the seed lots sown in 2003 and 2004, and from leaf and stem tissue of 20 greenhouse-grown plants from each seed lot. The latter DNA samples were assayed for phytoplasma DNA by a nested polymerase chain reaction (PCR) assay incorporating phytoplasma universal 16S rRNA primer pairs P1/P6 (1) followed by R16R2/R16F2 (4). Seed samples analyzed from the 2003 and 2004 seed lots and tissues of the 40 greenhouse-grown plants all tested negative for phytoplasma DNA using this assay. Leaf, stem, and/or root tissues of all plants collected in the field in 2003 (60 plants) and 2004 (60 plants) and 71.1% (315 of 443) of seed samples (five seeds per sample) tested positive for the presence of phytoplasma DNA, as evidenced by the presence of an expected band of 1.2 kb on the gels after the second amplification with primers R16R2/R16F2. Nested PCR products from plant samples collected in 2003 were cloned, sequenced, and compared with phytoplasma sequences archived in the GenBank nucleotide database. On this basis, phytoplasmas detected in plants or their seeds collected in 2003 were found to be most similar (98.8%) to CHRY (Accession No. AY180956), a 16SrI-A subgroup strain, or were most similar (98.9%) to isolate 99UW89 (Accession no. AF268407), a known 16SrI-B subgroup strain. Sequences of phytoplasmas detected in plants or their seeds in 2004 were obtained by direct sequencing of rRNA products amplified from samples using PCR incorporating primer pairs P1/P6 and P4/P7 (3). Analysis of sequence data revealed that phytoplasmas in these plants were all most similar (99.5%) to AY-WB (Accession no. AY389828), a 16SrI-A subgroup member. The nucleotide sequences have been deposited with GenBank under Accession nos. DQ404346, DQ404347, and DQ411470. To our knowledge, this is the first report of 16SrI-A and 16SrI-B subgroup phytoplasmas infecting plants and seed of B. rapa in Saskatchewan.

Development of a Real-Time, Quantitative PCR for Detection of the Columbia Basin Potato Purple Top Phytoplasma in Plants and Beet Leafhoppers.

A quantitative, real-time "TaqMan" polymerase chain reaction assay (real-time PCR) was developed which was capable of detecting and quantifying a group 16SrVI phytoplasma in DNA extracts prepared from infected tomatoes, potatoes, and beet leafhoppers (Circulifer tenellus). Primers and probe were designed from the 16S rRNA gene of the Columbia Basin potato purple top phytoplasma, which is closely related to the beet leafhopper transmitted virescence agent. The detection limit in phytoplasma-infected tomato DNA was approximately 50 pg. The concentration of phytoplasma varied considerably among potato plants showing symptoms of purple top. The pathogen was readily detected in extracts from single or groups of five beet leafhoppers. As with infected potatoes, the concentration of phytoplasma in individual leafhoppers was variable. The assay also detected aster yellows (group 16SrI) and pigeon pea witches'-broom (group 16SrIX) phytoplasmas in infected periwinkle plants. The real-time PCR was at least as sensitive as the commonly used and more labor-intensive nested PCR for detection of the pathogen.

A New Phytoplasma Infecting Lettuce in Iran.

Lettuce phyllody (LP) is an economically important disease of romaine lettuce (Lactuca sativa) in vegetative crop and seed crop fields in the Fars Province of Iran. Wild lettuce phyllody (WLP) occurs also in epidemic proportions in wild lettuce (L. serriola) during the fall in Iranian central and southern provinces, especially in Fars. Among various leafhopper species tested, Neoaliturus fenestratus successfully transmitted agents of LP and WLP to lettuce, wild lettuce, periwinkle, and sowthistle directly after field collection or after 5 to 7 days of feeding on diseased source plants. With primer pair P1/P7 in polymerase chain reaction (PCR), target DNA fragments (1.8 kbp) were amplified from total nucleic acid samples extracted from diseased lettuce and wild lettuce plants, but not from healthy counterparts. On the basis of disease symptoms, leafhopper transmission, and positive reaction in phytoplasma-specific PCR, Iranian lettuce and wild lettuce phyllodies agents have phytoplasmal etiology. Phylogenetic and putative restriction site analysis of 16S/23S rDNA spacer region (SR) indicated that LP and WLP phytoplasmas are members of pigeon pea witches'-broom (16SrIX) group and are closely related. Reciprocal transmission of LP phytoplasma to wild lettuce and WLP phytoplasma to lettuce by the same leafhopper species and induction of similar symptoms in common hosts are other evidences that agents of LP and WLP may be related or identical phytoplasmas. This is the first report of lettuce as a new host in pigeon pea witches'-broom group and N. fenestratus as a vector of a pigeon pea witches'-broom group phytoplasma. In other countries, phytoplasmas of aster yellows group (16SrI) are commonly associated with phytoplasmal lettuce diseases (2) and Macrosteles quadrilineatus leafhopper is reported as the vector (1). Relatedness of WLP phytoplasma to 16SrIX group was previously reported (3). On the basis of the results of this study, wild lettuce and sowthistle are two reservoirs of LP phytoplasma.

SecY gene sequence analysis for finer differentiation of diversestrains 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.

Distinctions between phytoplasmas at the subgroup level detected by heteroduplex mobility assay

A total of 62 phytoplasma isolates were collected from North America, Europe and Asia and analysed by heteroduplex mobility assay (HMA) of the 16/23S spacer region amplified by the polymerase chain reaction. The results revealed wide genetic diversity among the phytoplasmas studied and a number of new phytoplasma strains were identified from known or new plant hosts in Alberta, Canada. Two distinctive subgroups were revealed by HMA in phytoplasmas associated with canola yellows, Chinese aster yellows, dandelion yellows and monarda yellows. In Alberta, two subgroups of the aster yellows group of phytoplasmas, I‐A and I‐B, were prevalent in naturally infected field crops and ornamentals in open gardens. The results indicated that HMA is a simple, but rapid and accurate, alternative method for the detection and estimation of genetic divergence of phytoplasmas when finer molecular characterization of phytoplasmas is required at the subgroup level.

Detection and characterization of the phytoplasma associated with a phyllody disease of black pepper ( Piper nigrum L.) in India

Using polymerase chain reaction (PCR), the phytoplasma was detected in black pepper ( Piper nigrum) with phyllody symptoms in India. A 1.20 kb DNA fragment encoding the portion of phytoplasma 16S rDNA consistently amplified by nested PCR was cloned and sequenced. The sequenced region contained 1230 nucleotides. Sequence analyses showed that the gene was most closely related to members of aster yellows group (16Sr I) of phytoplasma. The sequence identity with members of aster yellows group (16Sr I) was >98% while that with members of other groups (16Sr II to 16Sr XV and other undesignated groups) ranged from 87 to 96%. On the basis of sequence identity and phylogenetic relationship studies, it is concluded that phytoplasma infecting black pepper in India belongs to aster yellows group. This is the first report of identification of phytoplasma in black pepper.

The Occurrence of Phytoplasmas in Apple Trees Showing Branch Twisting

AbstractApple trees showing malformation of branches were found at different locations in the Czech Republic. Ultrathin sections of tissues from diseased trees showed the presence of pleomorphic bodies resembling phytoplasma. Nested‐polymerase chain reaction (PCR) assays with primers amplifying16S–23S ribosomal RNA (rRNA) sequences specific for phytoplasma and the subsequent restriction fragment length polymorphism (RFLP) analyses allowed to classify the detected phytoplasmas in the aster yellows group, subgroup 16SrI‐C in 12 trees, single (11 plants) or mixed with phytoplasma belonging to subgroup 16SrI‐B (1 plant). Phytoplasma of apple proliferation group (16SrX‐A subgroup) were identified in cv. Matčino. Apple tree cv. Přeštické růžové was infected by phytoplasma of the 16SrX‐A and 16SrI‐B subgroups. No phytoplasmas were detected in asymptomatic apple trees.