X-disease Phytoplasma Research Articles

Colladonus spp. (Hemiptera: Cicadellidae) vectors of X-disease: biology and management in Western United States

Abstract The US cherry and stone fruit industries have periodically experienced devastating outbreaks of X-disease phytoplasma (XDP) since the 1930s, with an ongoing epidemic occurring since 2010. This disease is critically impacting Prunus spp. and the stone fruit industry, causing underdeveloped, bitter, and misshapen fruit on economically important crops, primarily cherries, peaches, and nectarines, making the fruit unmarketable. Trees cannot recover from this disease and eventually die if they are not removed. It takes at least 1 yr for symptoms to appear, postinfection, which hinders disease management efforts. The phytoplasma, Candidatus Phytoplasma pruni, is a phloem-limited intracellular bacterium that is spread by grafting and leafhopper (Hemiptera: Cicadellidae) feeding. Several leafhopper species are known to transmit XDP, of which Colladonus spp. are considered the most important vectors in the Western United States and are native to this region. Colladonus spp. feed and oviposit on a wide variety of broadleaf perennial plants, including mallow, alfalfa, and clover. These plants are also hosts of the phytoplasma. In Western United States, these leafhoppers have 3 periods of adult activity: May–June, late July–August, and October. The highest rates of pathogen transmission likely occur during the last 2 activity periods due to the high abundance of leafhoppers and a postharvest increase of XDP titers in trees. Using known activity periods and host plants of the leafhoppers, growers can monitor and strategically manage the vectors, which, when combined with the removal of infected trees and ground-cover hosts, should help limit the spread of X-disease.

Recovery Plan for X-Disease in Stone Fruit Caused by ‘Candidatus Phytoplasma pruni’

Stone fruits are a multibillion-dollar industry for the United States and Canada, one that has repeatedly suffered significant economic losses due to outbreaks of the X-disease phytoplasma (‘ Candidatus Phytoplasma pruni’) over the last century. Orchards and entire production areas have been abandoned, with corresponding losses to growers, fruit packers, and consumers. The most recent outbreak, in the U.S. Pacific Northwest, resulted in an estimated $65 million (USD) loss in revenue between 2015 and 2020 and is only increasing in incidence. Already present across much of the continental United States and Canada, the phytoplasma has a broad host range beyond stone fruit and is transmitted by at least eight leafhopper species, therefore stone fruit production in every state is at significant risk. This recovery plan was produced as part of the National Plant Disease Recovery System and is intended to provide a review of pathogen biology, assess the status of critical recovery components, and identify disease management research, extension, and education needs.

Estimated Impact of X-disease and Little Cherry Disease in Washington and Oregon from 2015 to 2020

X-disease phytoplasma and little cherry virus 1 and 2 have resulted in substantial loss of stone fruit acreage in Washington and Oregon. The extent and financial impact on the industry was not previously known. A Washington and Oregon State University survey documented 238,856 trees equivalent to 974 acres of sweet cherries removed due to X-disease and little cherry disease between 2015 and 2020. Removed trees reduced revenue to the industry by an estimated $30 million in 2020 and $65 million between 2015 and 2020. Over the seven-year re-establishment period estimated lost revenue and establishment costs to growers is an estimated $115 million.

Unraveling the Etiology of North American Grapevine Yellows (NAGY): Novel NAGY Phytoplasma Sequevars Related to 'Candidatus Phytoplasma pruni'.

North American grapevine yellows (NAGY) disease has sometimes been attributed to infection of Vitis vinifera L. by Prunus X-disease phytoplasma ('Candidatus Phytoplasma pruni') but this attribution may not be fully adequate. In this study, phytoplasma strains related to 'Ca. Phytoplasma pruni' were found in NAGY-diseased grapevines in Maryland, Pennsylvania, Virginia, Ohio, Missouri, and New York State. Based on restriction fragment length polymorphism analysis of 16S ribosomal RNA gene (16S rDNA) sequences, the strains (termed NAGYIII strains) were classified in group 16SrIII (X-disease group) but they contained a recognition site for the restriction endonuclease MseI that is not present in the 16S rDNA of 'Ca. Phytoplasma pruni'. The 16S rDNA of the strains differed by three or four nucleotides from that of 'Ca. Phytoplasma pruni', indicating that they belonged to two novel 16S rDNA sequevars, designated NAGYIIIα and NAGYIIIβ. Both sequevars differed from 'Ca. Phytoplasma pruni' by a single base in each of three regions corresponding to species-unique (signature) sequences described for 'Ca. Phytoplasma pruni'. Phylogenetic analyses of 16S rRNA genes and SecY proteins, and single-nucleotide polymorphism analyses of secY and ribosomal protein genes, further distinguished the two grapevine sequevar lineages from one another and from 'Ca. Phytoplasma pruni'. The NAGYIIIα and NAGYIIIβ sequevars also differed from 'Ca. Phytoplasma pruni' in regions of the folded SecY protein that are predicted to be near or exposed at the outer surface of the phytoplasma membrane. No evidence indicated that diseased grapevines contained any phytoplasma strain conforming to 'Ca. Phytoplasma pruni' sensu stricto. Because the NAGYIII sequevars have not been reported in X-disease, a question is raised as to whether NAGYIII and Prunus X-disease are caused by different phytoplasma genotypes.

CURRENT STATUS OF PHYTOPLASMA DISEASES OF PEACH

Peach (Prunus persica) is in many countries severely affected by yellows and decline diseases of phytoplasmal etiology. These diseases are caused by phylogenetically different phytoplasmas. X-disease is one of the most serious diseases of peach. It is widespread in North America and has not been observed elsewhere. This disease is induced by a distinct phytoplasma, the X-disease agent ‘Candidatus Phytoplasma pruni’, which is a member of the X-disease phytoplasma group or 16SrIII group, subgroup 16SrIII-A. ‘Ca. P. pruni’ is, at the level of 16S rDNA sequences, a homogeneous pathogen. Peach rosette, peach red suture, and peach yellows, also called little peach, are three other diseases which are mainly known from eastern North America. These diseases are currently of minor importance. They are also caused by the X-disease phytoplasma. Peach yellow leaf roll (PYLR) is a major disease of peach that occurs in California. The causative agent, the PYLR phytoplasma, is a member of the apple proliferation (AP) group and is most closely related to the pear decline phytoplasma. In Europe, peach is affected by an economically important disorder known as European stone fruit yellows. This disorder is induced by ‘Ca. P. prunorum’ which is a member of the AP group, subgroup 16SrX-B. ‘Ca. P. prunorum’ is closely related to other important temperate fruit tree phytoplasmas including the PYLR agent. In Lebanon and Iran, peach is affected by almond witches’-broom, a lethal disease that is induced by ‘Ca. P. phoenicium’. This phytoplasma is a member of the 16SrIX group, subgroup 16SrIX-B. Phytoplasmas of other phylogenetic groups, which are known to infect a wide range of plant hosts, have been identified in declining peach trees in several fruit growing areas worldwide. However, the pathological relevance of these non-peach phytoplasmas needs to be investigated in many cases.

First Report of a 16SrIII-L Phytoplasma Associated with Frogskin Disease in Cassava (Manihot esculenta Crantz) in Brazil.

Cassava (Manihot esculenta Crantz) is a major staple crop in developing countries and a large source of raw material for industrial purposes as flour, starch, and ethanol. In July 2012, 24 cassava genotypes (corresponding to 1.85% of the accessions) with typical symptoms of frogskin disease (CFSD) were observed in one of the maintenance areas of the Brazilian Cassava Germplasm (located at Embrapa Cassava & Fruits, Cruz das Almas, Bahia State, Brazil). All diseased plants were asymptomatic on the aboveground parts (leaves and stem). However, for accessions BGM 880, BGM 1094, BGM 1100, BGM 1212, BGM 1218, and BGM 1526, all roots showed a woody appearance, thickened cork-like peel with opaque aspect, and coalescent lip-like slits in a honeycomb pattern. Based on literature description, two pathogens could be associated with CFSD: a dsRNA virus (belonging to family Reoviridae) and a 16SrIII-L phytoplasma (1). To investigate the presence of phytoplasma associated with the CFSD symptoms, total DNA was extracted from 0.5 g of root tissue collected from both symptomatic and asymptomatic roots by scratching the secondary vessel at the center of the cassava root with a CTAB method. The nested PCR was carried out using phytoplasma-specific primer set P1/Tint followed by R16F2n/R16R2, targeting the 16S rRNA gene sequence of 1.2 kb in length, for the final reaction (4). No phytoplasma was detected in asymptomatic cassava roots that were sampled from the same field. A posterior extraction of total RNA was made but no dsRNA was noticed on the agarose gel, and reaction of RT-PCR with specific primers (2) had no amplification. In order to characterize the strains, the 1.2-kb amplicon was digested with BamHI, MseI, MspI, KpnI, and TaqI endonucleases. The resulting patterns indicated that the symptomatic accessions were infected with a phytoplasma belonging to the 16SrIII group, sharing similarities with pseudo gel mapping from the reference strain of Peach X-Disease Phytoplasma (GenBank Accession No. L33733). Nested PCR products from accessions BGM 1526 and BGM 1212 were purified and sequenced by Macrogen, (Seoul, South Korea) in both directions, manually edited, and the consensus sequences were deposited in the NCBI database (GenBank Accession Nos. KF019184 and KF019185). Phylogenetic studies were conducted based on maximum parsimony, neighbor-joining, and maximum likelihood analysis for 16S rRNA. The phytoplasma 16S rRNA gene sequences from both strains had 99% identity (P < 0.0001) with the 16SrIII-L CFSD phytoplasma (EU346761 and AY737647), described by Alvarez et al. (1) infecting cassava in Colombia. To our knowledge, this is the first report of a phytoplasma associated with Cassava Frogskin Disease in Brazil, where only the dsRNA virus was recognized as causing this symptom (3). This is not likely to be an isolated case, and possibly more cassava plants are infected with this phytoplasma in Brazil. Due to the difficulties to observe the symptoms at the field level, this could be an emerging disease in that country.

Quantitative phenotyping of X-disease resistance in chokecherry using real-time PCR

A quantitative real-time SYBR Green PCR (qPCR) assay has been developed to detect and quantify X-disease phytoplasmas in chokecherry. An X-disease phytoplasma-specific and high sensitivity primer pair was designed based on the 16S rRNA gene sequence of X-disease phytoplasmas. This primer pair was specific to the 16SrIII group (X-disease) phytoplasmas. The qPCR method can quantify phytoplasmas from a DNA mix (a mix of both chokecherry and X-disease phytoplasma DNA) at as low as 0.001ng, 10-fold lower than conventional PCR using the same primer pair. A significant correlation between the copy number of phytoplasmas and visual phenotypic rating scores of X-disease resistance in chokecherry plants was observed. Disease resistant chokecherries had a significantly lower titer of X-disease phytoplasmas than susceptible plants. This suggests that the qPCR assay provides a more objective tool to phenotype phytoplasma disease severity, particularly for early evaluation of host resistance; therefore, this method will facilitate quantitative phenotyping of disease resistance and has great potential in enhancing plant breeding.

First report of a 16SrIII-B subgroup phytoplasma associated with leaf reddening, virescence and phyllody of purple coneflower

Purple coneflower plants showing leaf reddening and flower abnormalities were observed in South Bohemia (Czech Republic). Transmission electron microscopy observations showed phytoplasmas in sieve cells of symptomatic plants but not in healthy ones. Polymerase chain reactions with universal and group specific phytoplasma primers followed by restriction fragment length polymorphism analyses of 16S rDNA allowed us to classify the detected phytoplasmas into the X-disease group, ribosomal subgroup 16SrIII-B. Sequence analyses of the 16S-23S ribosomal operon (1684 bp), ribosomal protein L15, and protein translocase genes (1566 bp) confirmed the closest relationship with phytoplasmas belonging to the 16SrIII ribosomal group, specifically the 16SrIII-B subgroup. The current study reports purple coneflower as a new host for the X-disease phytoplasma group in the Czech Republic and worldwide.

‘Candidatus Phytoplasma pruni’, a novel taxon associated with X-disease of stone fruits, Prunus spp.: multilocus characterization based on 16S rRNA, secY, and ribosomal protein genes

X-disease is one of the most serious diseases known in peach (Prunus persica). Based on RFLP analysis of 16S rRNA gene sequences, peach X-disease phytoplasma strains from eastern and western United States and eastern Canada were classified in 16S rRNA gene RFLP group 16SrIII, subgroup A. Phylogenetic analyses of 16S rRNA gene sequences revealed that the X-disease phytoplasma strains formed a distinct subclade within the phytoplasma clade, supporting the hypothesis that they represented a lineage distinct from those of previously described 'Candidatus Phytoplasma' species. Nucleotide sequence alignments revealed that all studied X-disease phytoplasma strains shared less than 97.5 % 16S rRNA gene sequence similarity with previously described 'Candidatus Phytoplasma' species. Based on unique properties of the DNA, we propose recognition of X-disease phytoplasma strain PX11CT1(R) as representative of a novel taxon, 'Candidatus Phytoplasma pruni'. Results from nucleotide and phylogenetic analyses of secY and ribosomal protein (rp) gene sequences provided additional molecular markers of the 'Ca. Phytoplasma pruni' lineage. We propose that the term 'Ca. Phytoplasma pruni' be applied to phytoplasma strains whose 16S rRNA gene sequences contain the oligonucleotide sequences of unique regions that are designated in the formally published description of the taxon. Such strains include X-disease phytoplasma and--within the tolerance of a single base difference in one unique sequence--peach rosette, peach red suture, and little peach phytoplasmas. Although not employed for taxon delineation in this work, we further propose that secY, rp, and other genetic loci from the reference strain of a taxon, and where possible oligonucleotide sequences of unique regions of those genes that distinguish taxa within a given 16Sr group, be incorporated in emended descriptions and as part of future descriptions of 'Candidatus Phytoplasma' taxa.

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

Symptoms of abnormal proliferation of shoots resulting in formation of witches'-broom growths were observed on diseased plants of passion fruit (Passiflora edulis f. flavicarpa Deg.) in Brazil. RFLP analysis of 16S rRNA gene sequences amplified in PCRs containing template DNAs extracted from diseased plants collected in Bonito (Pernambuco) and Viçosa (Minas Gerais) Brazil, indicated that such symptoms were associated with infections by two mutually distinct phytoplasmas. One phytoplasma, PassWB-Br4 from Bonito, represents a new subgroup, 16SrIII-V, in the X-disease phytoplasma group ('Candidatus Phytoplasma pruni'-related strains). The second phytoplasma, PassWB-Br3 from Viçosa, represents a previously undescribed subgroup in group 16SrVI. Phylogenetic analyses of 16S rRNA gene sequences were consistent with the hypothesis that strain PassWB-Br3 is distinct from previously described 'Ca. Phytoplasma' species. Nucleotide sequence alignments revealed that strain PassWB-Br3 shared less than 97.5 % 16S rRNA gene sequence similarity with previously described 'Ca. Phytoplasma' species. The unique properties of its DNA, in addition to natural host and geographical occurrence, support the recognition of strain PassWB-Br3 as a representative of a novel taxon, 'Candidatus Phytoplasma sudamericanum'.

Occurrence of Poinsettia Stem Flat Disease Caused by Phytoplasma in Korea.

In December 2009, commercially grown poinsettia (Euphorbia pulcherrima Willd cv. Ichibang) exhibited typical phytoplasma-like symptoms in 95% of an affected field in Yongin, Korea (Gyeonggi Province). Symptoms consisted of flat stems and fascicles and an abnormal number of apexes resulting in a cockscomb form of stem and flower bud proliferation. Leaf narrowing with curling of bracts was also associated with the disease. Symptomatic poinsettia plants were not marketable. Poinsettias, cv. Ichibang, without symptoms were obtained from a breeding collection in a glasshouse of the National Institute of Horticultural and Herbal Science in Suwon (Gyeonggi Province). The presence of phytoplasmas in symptomatic and healthy-looking flowers and stems of cv. Ichibang was demonstrated by PCR analysis with primer pair R16mF2/R16mR1 (1), which amplifies phytoplasma 16S rDNA regions. PCR products (~1,427 bp), were obtained from both symptomatic and healthy-looking plants, sequenced, and registered under the GenBank Accession Nos. of GU461275 and GU461277, respectively. Symptomatic poinsettia and healthy poinsettia sequences had 99.6 and 100% identity with U.S. PoiBI isolate FJ376625, indicating poinsettia stem flat disease is caused by PoiBI. A branch-inducing factor in poinsettia has been known for several decades, but only since 1997 was this graft transmissible factor identified as a PoiBI phytoplasma (1), belonging to the peach X-disease phytoplasma group (16S rRNA III) subgroup 16SrIII-H (2). Normally, phytoplasmas are associated with host quality loss, but abnormally, infection in poinsettia generates a desirable, free-branching growth habit. In this study we found that PoiBI could be detrimental to the quality of poinsettia depending on the cultivar and agronomic practice. Poinsettia stem flat disease presumably occurred because of increased levels of phytoplasma caused by successive stem cutting for commercial use. To our knowledge, this is the first report of PoiBI phytoplasma that affected marketability of poinsettia in Korea.

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).

Construction of an interactive online phytoplasma classification tool, iPhyClassifier, and its application in analysis of the peach X-disease phytoplasma group (16SrIII).

Phytoplasmas, the causal agents of numerous plant diseases, are insect-vector-transmitted, cell-wall-less bacteria descended from ancestral low-G+C-content Gram-positive bacteria in the Bacillus–Clostridium group. Despite their monophyletic origin, widely divergent phytoplasma lineages have evolved in adaptation to specific ecological niches. Classification and taxonomic assignment of phytoplasmas have been based primarily on molecular analysis of 16S rRNA gene sequences because of the inaccessibility of measurable phenotypic characters suitable for conventional microbial characterization. In the present study, an interactive online tool, iPhyClassifier, was developed to expand the efficacy and capacity of the current 16S rRNA gene sequence-based phytoplasma classification system. iPhyClassifier performs sequence similarity analysis, simulates laboratory restriction enzyme digestions and subsequent gel electrophoresis and generates virtual restriction fragment length polymorphism (RFLP) profiles. Based on calculated RFLP pattern similarity coefficients and overall sequence similarity scores, iPhyClassifier makes instant suggestions on tentative phytoplasma 16Sr group/subgroup classification status and ‘Candidatus Phytoplasma’ species assignment. Using iPhyClassifier, we revised and updated the classification of strains affiliated with the peach X-disease phytoplasma group. The online tool can be accessed at http://www.ba.ars.usda.gov/data/mppl/iPhyClassifier.html.

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 a New Phytoplasma Subgroup, 16SrIII-T, Associated with Decline Disease Affecting Sweet and Sour Cherry Trees in Lithuania

During July 2007, sweet (Prunus avium) and sour cherry (P. cerasus) trees exhibiting disease symptoms suggestive of possible phytoplasma infection were observed in a large orchard in the Kaunas Region of Lithuania. Samples of leaf tissue were collected from 13 sweet cherry trees that were affected by a decline disease (designated cherry decline, ChD) characterized by symptoms that included leaf reddening and premature leaf drop and two sour cherry trees exhibiting proliferation of branches and nonseasonal flowering. To assess the diseased trees for phytoplasma infection, DNA was extracted with a Genomic DNA Purification Kit (Fermentas, Vilnius, Lithuania) and used as template in nested PCRs, primed by phytoplasma universal primer pairs P1/P7 and R16F2n/R16R2 for amplification of 16S ribosomal (r) DNA sequences (1,2). The 1.2-kbp DNA sequences amplified from all 15 trees were subjected to restriction fragment length polymorphism (RFLP) analyses with AluI, MseI, KpnI, HhaI, HaeIII, HpaII, RsaI, HinfI, TaqI, Sau3AI, and BfaI. The collective profiles indicated that DNAs were derived from two different phytoplasmas. One of them, designated ChD phytoplasma, was found in 11 sweet cherry trees and two sour cherry trees and tentatively classified as a member of new subgroup designated 16SrIII-T in 16S rDNA RFLP group 16SrIII (X-disease phytoplasma group). It was observed that the ChD phytoplasma caused different symptoms in sweet and sour cherry. The amplified ChD phytoplasma 16S rDNA was cloned in Escherichia coli, sequenced, and the sequence deposited in the GenBank database (Accession No. FJ231728). The ChD phytoplasma 16S rDNA shared 98.4 and 98.6% sequence identity with the 16S rDNAs from stone fruit-infecting phytoplasmas associated with western X-disease (GenBank Accession No. L04682) and Canada X-disease (GenBank Accession No. L33733), respectively, indicating that the three strains are closely related. Interestingly, the ChD phytoplasma 16S rDNA shared 99.8% sequence identity with 16S rDNA from one operon (rrnB, GenBank Accession No. AF370120) from a phytoplasma previously found to be associated with dandelion virescence (DanVir) disease in Lithuania. The operon rrnA (GenBank Accession No. AF370119) shared 99.3% sequence identity (2). The high similarity of the ChD 16S rRNA gene sequence to that of DanVir rrnB suggests the possibility that ChD and DanVir may belong to a single phytoplasma species and that dandelion is possibly an alternate host of ChD phytoplasma. The other phytoplasma, found in two sweet cherry trees, was classified in subgroup 16SrI-B of 16S rDNA RFLP group 16SrI ('Candidatus Phytoplasma asteris' and related strains) and was designated cherry proliferation phytoplasma (GenBank Accession No. FJ231729). Thus, in Europe, cherry may be affected by diseases associated with phytoplasmas belonging to groups 16SrI, 16SrIII, 16SrX, and 16SrXII (3,4). The infections by diverse phytoplasma strains and species underscore the need for production of phytoplasma-free planting stock and for intensified research to reduce ecological and economic impacts of these phytoplasmas.

First report of a phytoplasma associated with Spondias purpurea (Jocote de Corona) in El Salvador

A new disease of the fruit tree Jocote de Corona (Spondias purpurea L.) was observed in El Salvador, Central America. The symptoms included small chlorotic leaves, highly proliferating shoots, and shortened internodes. Absence of sweet pulp in fruits made them inedible, causing considerable yield losses for farmers. The disease etiology was investigated using polymerase chain reaction with phytoplasma-specific primers, sequencing, and phylogenetic analysis. We obtained no amplification products from symptomless plants, whereas all tests were positive from plants with symptoms. Phylogenetic analysis indicated that this phytoplasma clustered in the 16S rIII group, the type member of which is X-disease phytoplasma. This is the first report of a phytoplasma associated with Jocote de Corona disease in El Salvador and Central America.

Effect of Benzothiadiazole on Transmission of X-Disease Phytoplasma by the Vector Colladonus montanus to Arabidopsis thaliana, a New Experimental Host Plant.

Colladonus montanus (Van Duzee), a leafhopper vector of X-disease phytoplasma (Xp), efficiently transmitted the pathogen to Arabidopsis thaliana Columbia wild type. During transmission trials, the phytoplasma was inoculated into 22-, 34-, and 40-day-old plants. Phytoplasma infections were confirmed by polymerase chain reaction (PCR) using primers specific for Xp Symptoms in Xp-positive A. thaliana were overall stunting and reduced or no fruit (silques). All symptom-free plants were PCR negative. Leafhopper nymphs free of Xp that fed on diseased A. thaliana acquired and transmitted Xp to celery plants, a diagnostic host, causing typical X-disease symptoms. Foliar spray applications of the plant resistance elicitor benzothiadiazole (BTH) to A. thaliana 1 week before phytoplasma inoculation significantly reduced phytoplasma infection, ranging from an infection rate of 73.7% for untreated plants to 50 and 35% for plants treated with 1.2 and 4.8 mM BTH, respectively. Vector leafhoppers survival was significantly reduced on BTH-treated A. thaliana compared with leafhoppers on nontreated plants, suggesting that systemic acquired resistance in this plant may have some detrimental effect on the leafhopper C. montanus.

POSSIBLE CAUSE OF EUROPEAN BLUEBERRY DISEASE IS RELATED TO NORTH AMERICAN MILKWEED YELLOWS PHYTOPLASMA

SUMMARY A phytoplasma lineage previously found in Germany and North America was identified in diseased plants of wild European blueberry (Vaccinium myrtillus L.) exhibiting symptoms of shoot proliferation in Lithuania. On the basis of RFLP and nucleotide sequence analyses of 16S rDNA, the phytoplasma (strain VAC-L) was classified in the group 16SrIII (X-disease phytoplasma group), subgroup F. Phylogenetic analysis based on 16SrRNA gene sequences confirmed that the VAC-L phytoplasma is closely related to phytoplasma strain VAC from diseased, cultivated blueberry in Germany and to North American milkweed yellows phytoplasma strain MW1, both of which are classified in subgroup III-F. The results extend the known geographic range of phytoplasmal diseases in blueberry, and raise questions concerning intercontinental movement of subgroup III-F strains and their potential to infect cultivated Vaccinium spp. in North America.

Short communication. First detection of a phytoplasma infecting faba bean (Vicia faba L.) in Spain

Faba bean is one of the most widely grown protein-producing food legumes. Faba bean plants showing symptoms of shoe stringed leaves, phyllody and flower abortion were observed in fields of Antequera (Málaga, Southern Spain). The etiology of this disease was investigated using graft transmission to periwinkle plants, polymerase chain reaction (PCR) with phytoplasma-specific primers, sequencing and phylogenetic analysis. We could not obtain amplification products from asymptomatic plants, whereas all tests were positive for phytoplasma infection from plants showing symptoms. Phylogenetic analysis indicated that this phytoplasma clustered in the 16SrIII group, the type member of which is X-disease phytoplasma. This is the first report of a phytoplasma infecting faba bean in Spain, and the first report of a phytoplasma in this group infecting faba bean.

Cosmid cloning and sample sequencing of the genome of the uncultivable mollicute, Western X-disease phytoplasma, using DNA purified by pulsed-field gel electrophoresis

Western X-disease (WX) phytoplasma is an uncultivable, intracellular pathogen of plants and insects with an AT-rich genome of 670 kb. As part of the genome sequencing project of WX phytoplasma, we have cloned approximately 50% of its genome into the pcosRW2 cosmid vector using DNA purified from pulsed-field gels. One of the cosmid clones with an insert of 24.6 kb was sequenced, which along with the cosmid end sequences, represents 60 kb of unique sequence from the WX phytoplasma genome. The putative genes identified in this sequence represent a wide variety of functions and many had not been previously identified in a phytoplasma.