Elm Yellows Research Articles

Detection and Characterization of an Elm Yellows (16SrV) Group Phytoplasma Infecting Virginia Creeper Plants in Southern Florida.

The polymerase chain reaction (PCR) employing phytoplasma-specific ribosomal RNA primer pair P1/P7 consistently amplified a product of expected size (1.8 kb) from 29 of 36 symptom-less Virginia creeper (Parthenocissus quinquefolia) plants growing in southern Florida. Restriction fragment length polymorphism analysis of P1/P7-primed PCR products indicated that most phytoplasmas detected in Virginia creeper were similar to phytoplasmas composing the elm yellows (16SrV) group. This relationship was verified by reamplification of P1/P7 products using an elm yellows (EY) group-specific rRNA primer pair fB1/rULWS1. rDNA products (1,571 bp) were generated by group-specific PCR from 28 phytoplasma-positive plants and 1 negatively testing plant identified by earlier P1/P7-primed PCR. Analysis of 16S rDNA sequences determined the Virginia creeper (VC) phytoplasma to be phylogenetically closest to the European alder yellows (ALY) agent, an established 16SrV-C subgroup strain. However, presence or absence of restriction sites for endonucleases AluI, BfaI, MspI, RsaI, and TaqI in the 16S rRNA and 16-23S rRNA intergenic spacer region of the VC phytoplasma collectively differentiated this strain from ALY and other 16SrV group phytoplasmas. Failure to detect the VC phytoplasma by PCR employing nonribosomal primer pair FD9f/FD9r suggests that this newly characterized agent varies from known European grapevine yellows (flavescence dorée) phyto-plasmas previously classified as 16SrV subgroup C or D strains.

First Report of Alder Yellows Phytoplasma in the Eastern Baltic Region.

Alnus glutinosa (alder) is widespread in Europe and is an important component of biological diversity in natural forest ecosystems in the Baltic Region. In 2000, diseased trees of A. glutinosa exhibiting characteristically phytoplasmal disease symptoms of shoot proliferation and leaf yellowing were observed in Aukstaitija National Park, Lithuania. In other parts of Europe, alder is affected by a phytoplasmal disease known as alder yellows, which is characterized by symptoms that include yellowing and reduced leaf size, die-back of branches, and decline of trees (2,3). Proliferation of shoots has not been previously reported with this disease. An association between alder yellows and infection by a phytoplasma has been reported in A. glutinosa in Germany and Italy, and a phytoplasma has been found in A. glutinosa in France and Hungary (2,4). We examined symptomatic alder from Lithuania using nested polymerase chain reaction (PCR) (1), primed by P1/P7 and followed by R16F2n/R16R2 (F2n/R2), for amplification of phytoplasmal ribosomal (r) DNA. The results indicated the presence of a phytoplasma, designated ALY-L, in the diseased alder. We classified the ALY-L phytoplasma through restriction fragment length polymorphism (RFLP) analysis of 16S rDNA. A 1.2-kbp fragment (F2n-R2 segment) of rDNA, amplified in PCR primed by F2n/R2, was analyzed using single endonuclease enzyme digestion with AluI, MseI, KpnI, HhaI, HaeIII, HpaI, HpaII, RsaI, HinfI, TaqI, Sau3AI, BfaI, and ThaI. On the basis of collective RFLP patterns, phytoplasma ALY-L was classified as a member of group 16SrV (group V, elm yellows group), subgroup C. The amplified 16S rDNA was cloned in Escherichia coli and sequenced, and the sequence was deposited in the GenBank data library (Accession No. AY028789). Nucleotide sequence alignment revealed that 16S rDNA from phytoplasma ALY-L shared 100% sequence similarity with 16S rDNA (GenBank Accession No. Y16387) from a phytoplasma associated with alder yellows (ALY) disease in Italy. The results support the conclusion that a strain of ALY phytoplasma is present in Lithuania. Phytoplasmas belonging to groups 16SrI (aster yellows phytoplasma group) and III (X-disease phytoplasma group) have been found in herbaceous plant species in Lithuania. This report records the first finding of a group V phytoplasma, and the first finding of a phytoplasma in a tree species in the eastern Baltic Region. These findings contribute knowledge about the diversity of phytoplasmas in the Baltic Region and the distribution of ALY phytoplasma in Europe. Apparently, A. glutinosa may be infected by the phytoplasma but not develop obvious disease symptoms, as has been reported elsewhere (3). Thus, it is possible that ALY-L phytoplasma is widespread, but as yet undetected, throughout the geographic range of alder in the Baltic Region. This possibility is supported by the finding of the monophagous leafhopper vector (Oncopsis alni) of ALY phytoplasma throughout Europe (cited in Maixner and Reinert [3]). Further research is needed to assess the impact of phytoplasmal infections such as those by ALY-related phytoplasma strains on trends in biological diversity in the natural forest ecosystems of the Baltic Region and elsewhere in Europe.

Revised Subgroup Classification of Group 16SrV Phytoplasmas and Placement of Flavescence Dorée-Associated Phytoplasmas in Two Distinct Subgroups.

The subgroup classification of phytoplasmas in 16S rRNA group 16SrV (elm yellows phytoplasma group) was revised and extended on the basis of enzymatic restriction fragment length polymorphism (RFLP) analysis of ribosomal (r) DNA and analysis of putative restriction sites in nucleotide sequences. A 1.85 kbp fragment of the rRNA operon from flavescence dorée (FD) phytoplasma strain FD70 from France was amplified and cloned, and its nucleotide sequence determined (GenBank acc. no. AF176319). Placement of FD70 in subgroup V-C was verified by analysis of amplified DNA and of the cloned sequence. Hemp dogbane phytoplasma HD1 (AF122912), a member of subgroup V-C, was distinguished from other subgroup V-C phytoplasmas by putative restriction site differences in the 16S-23S rRNA spacer region. A previously published FD phytoplasma sequence (GenBank accession no. X76560) differed from FD70 sequence AF176319 by at least eight nucleotide substitutions and differences in putative restriction sites. The X76560 FD phytoplasma was classified in a new subgroup (V-D). Based on analyses of 16S rDNA GenBank sequence Y16395, Rubus stunt phytoplasma was classified in new subgroup V-E. The revised classification was supported by sequence similarities, group 16SrV-characteristic sequences, and a phylogenetic tree constructed on the basis of 16S rDNA sequences.

Elm Yellows Phytoplasma Lethal to Dutch Elm Disease-Resistant Ulmus americana Cultivars.

Ulmus americana (American elm) clonal cultivars Independence, New Harmony, and Valley Forge, together with the triploid putative hybrid cultivar Jefferson, were tested for reaction to the elm yellows (EY) phytoplasma. These cultivars all possess resistance to the fungal pathogen Ophiostoma novo-ulmi (1,4) but had not been screened for EY resistance or tolerance. Procedures and conditions of the test were similar to those used previously for assessing EY tolerance of Eurasian elm cultivars (3). In brief, 9 to 15 saplings of each cultivar and of U. americana raised from seed (susceptible controls), growing in a field plot at Ithaca, NY, were challenged by grafting their mainstems with bark patches from U. americana naturally affected by EY. Six to nine additional trees of each clone and of the seedling group were left untreated as controls. Inoculations were performed in July 1999, and trees were evaluated for symptoms in early September 2000. Multiple individuals in every inoculated group developed the syndrome typical of EY in U. americana: epinasty, foliar yellowing, yellow discoloration and necrosis of root and stem phloem, odor of methyl salicylate from moist discolored phloem on first exposure to air, defoliation or sudden permanent wilting, and death (2). The numbers of trees with these symptoms, of those inoculated, were: 9 of 9 Independence, 7 of 11 New Harmony, 10 of 14 Valley Forge, 3 of 13 Jefferson, and 12 of 15 trees grown from seed. Untreated controls remained asymptomatic, except for one tree of Valley Forge and two trees grown from seed that became infected naturally and had symptoms like those in the grafted trees. Based on these results, the elm cultivars named above are typical of U. americana in susceptibility to, and intolerance of, EY phytoplasmal infection. Effective EY resistance or tolerance in this species, although once thought to occur in rare individuals (2), remains undocumented.

Transmission of 16SrV phytoplasmas by Scaphoi-deus titanus Ball in northern Italy

Flavescence dorée (FD) has been defined as the disease that is caused by a phytoplasmas belonging to group 16SrV (elm yellows) and transmitted to plants by the insect Scaphoideus titanus Ball. We investigated transmission of FD agent by S. titanus in a vineyard located in Veneto region to determine which phytoplasma(s) may be transmitted in this region. Group 16SrV-C phytoplasma was detected and identified in field-collected S. titanus adults, in plant of grapevine (Vitis vinifera L. cv. Gamay) and broadbean (Vicia faba cv. Arlŕ) that had been fed upon by the insects. No evidence of experimental transmission of phytoplasma belonging to 16SrI-B subgroup phytoplasma by S. titanus has been observed.

Detection and Indentification of European Stone Fruit Yellows and Other Phytoplasmas in Wild Plants in the Surroundings of Apricot Chlorotic Leaf Roll-affected Orchards in Southern France

Between 1994 and 1998 a field study was conducted to identify plant hosts of the European stone fruit yellows (ESFY) phytoplasma in two apricot growing regions in southern and southwestern France where the incidence of apricot chlorotic leaf roll was high. A total of 431 samples from 51 different plant species were tested for the presence of phytoplasmas by PCR using universal and ESFY-specific primers. ESFY phytoplasma was detected in six different wild growing Prunus species exhibiting typical ESFY symptoms as well as in symptomless dog rose bushes (Rosa canina), ash trees (Fraxinus excelsior) and a declining hackberry (Celtis australis). The possible role of these plant species in the spread of ESFY phytoplasma is discussed. PCR-RFLP analysis of ribosomal DNA amplified with the universal primers was carried out to characterize the other phytoplasmas found. Thus, elm yellows phytoplasma, alder yellows phytoplasma and rubus stunt phytoplasma were detected in declining European field elm trees (Ulmus carpinifolia Gled), in declining European alder trees (Alnus glutinosa) and in proliferating Rubus spp. respectively. The presence of rubus stunt phytoplasma in great mallow (Malva sylvestris) and dog rose was demonstrated for the first time. Furthermore, the stolbur phytoplasma was detected in proliferating field bindweed (Convolvulus arvensis) and a previously undescribed phytoplasma type was detected in red dogwood (Cornus sanguinea). According to the 16S rDNA-RFLP pattern this new phytoplasma belongs to the stolbur phytoplasmas group.

Responses of Six Eurasian Ulmus Cultivars to a North American Elm Yellows Phytoplasma.

Elms (genus Ulmus) of six clonal cultivars representing Eurasian species and hybrids were grafted when 2 to 3 years old with bark patches from U. rubra infected with an elm yellows phytoplasma or were left untreated as controls. The cultivars were U. glabra × minor 'Pioneer', U. minor × parvifolia 'Frontier', U. parvifolia 'Pathfinder', U. wilsoniana 'Prospector', and the complex hybrids 'Homestead' and 'Patriot'. Trees were evaluated for infection and symptoms 1 or 2 years after inoculation. Infection was detected via the 4',6-diamidino-2-phenylindol e·2HCl (DAPI) fluorescence test in 26 of 86 grafted trees representing five cultivars. Infection of selected trees was confirmed by polymerase chain reaction (PCR) amplification of a fragment of phytoplasmal rDNA, and the phytoplasma was identified by restriction fragment length polymorphism (RFLP) analysis of the amplified DNA using restriction enzymes AluI, RsaI, and TaqI. Elm yellows phytoplasma was also identified by nested PCR and RFLP analysis in two of seven inoculated, healthy-appearing, DAPI-negative trees and one noninoculated control tree. All RFLP profiles were identical to that of reference strain EY1. Phytoplasma-associated symptoms, observed in five cultivars, included suppressed growth, progressive size reduction of apical shoots and leaves, chlorosis, foliar reddening, witches'-brooms, and dieback. Phyto-plasma was not detected in cv. Homestead. Possible resistance of this cultivar to elm yellows phytoplasma was indicated by localized phloem necrosis in stems below inoculum patches.

The occurrence of two phytoplasmas associated with stunted Rubus species in the UK

Polymerase chain reaction using universal primers to sequences in the 16S rRNA gene, and group-specific primers to sequences in the 16S/23S spacer region, revealed two distinct phytoplasmas occurring in Rubus plants showing symptoms of rubus stunt. One phytoplasma appeared similar to phytoplasmas in the elm yellows group; the other appeared to fall into the X disease group. This finding was confirmed by RFLP analysis of PCR products. This is the first identification of phytoplasmas from either of these groups occurring in the UK, and the first report of a phytoplasma belonging to the X disease group in Rubus.

Phytoplasmas Associated with Elm Yellows: Molecular Variability and Differentiation from Related Organisms.

Restriction fragment length polymorphism (RFLP) analyses were performed on polymerase chain reaction (PCR) amplimers of phytoplasmal DNA from eight samples obtained from Ulmus spp. (elms) affected by elm yellows (EY) in Italy and the United States, from Catharanthus roseus infected with strain EY1, and from five other plant species infected with phytoplasmas of the EY group sensu lato (group 16SrV). RFLP profiles obtained with restriction enzyme TaqI from ribosomal DNA amplified with primer pair P1/P7 differentiated elm-associated phytoplasmas from strains originally detected in Apocynum cannabinum, Prunus spp., Rubus fruticosus, Vitis vinifera, and Ziziphus jujuba. RFLP profiles obtained similarly with BfaI differentiated strains from A. cannabinum and V. vinifera from other phytoplasmas of group 16SrV. Elm-associated strains from within the United States had two RFLP patterns in ribosomal DNA based on presence or absence of an RsaI site in the 16S-23S spacer. Elm-associated phytoplasma strains from Italy were distinguished from those of American origin by RFLPs obtained with MseI in the same fragment of non-ribosomal DNA. Strain HD1, which was discovered in A. cannabinum associated with EY-diseased elms in New York State, was unique among the strains studied.

Identification and Epidemic Distribution of Two Flavescence Dorée-Related Phytoplasmas in Veneto (Italy).

Grapevine yellows associated with phytoplasmas of the elm yellows group (16SrV), better known as flavescence dorée (FD), is a serious quarantine problem in some important grapevine growing regions in the European Union. A survey was carried out in 1997 to 1998 in Veneto region (Italy) where a serious outbreak of FD was in progress. Phytoplasma identification by nested polymerase chain reaction (PCR)/restriction fragment length polymorphism (RFLP) analyses on 275 grapevine samples and on batches of Scaphoideus titanus was carried out. RFLP analyses of the 16S rDNA/spacer region with TaqI detected the presence of two distinct elm yellows phytoplasma subgroups designated 16SrV-C and 16SrV-D in 77 FD-infected grapevine samples. Only phytoplasmas of the 16SrV-D subgroup were detected in S. titanus. In 1997, the two phytoplasma subgroups appeared to be located in two diverse geographic areas; but in 1998, the 16SrV-D type also was detected in the provinces where in 1997 only 16SrV-C type was identified. The sequencing of a 400-bp fragment at the 3' end of 16S rDNA plus spacer region allowed a specific primer construction that was successfully employed for detection of both FD types in grapevine by direct PCR.

Chromosome Sizes of Phytoplasmas Composing Major Phylogenetic Groups and Subgroups

ABSTRACT Chromosome sizes of 71 phytoplasmas belonging to 12 major phylogenetic groups including several of the aster yellows subgroups were estimated from electrophoretic mobilities of full-length chromosomes in pulsed-field gels. Considerable variation in genome size, from 660 to 1,130 kilobases (kb), was observed among aster yellows phytoplasmas. Chromosome size heterogeneity was also observed in the stolbur phytoplasma group (range 860 to 1,350 kb); in this group, isolate STOLF contains the largest chromosome found in a phytoplasma to date. A wide range of chromosome sizes, from 670 to 1,075 kb, was also identified in the X-disease group. The other phytoplasmas examined, which included members of the apple proliferation, Italian alfalfa witches' broom, faba bean phyllody, pigeon pea witches' broom, sugarcane white leaf, Bermuda grass white leaf, ash yellows, clover proliferation, and elm yellows groups, all have chromosomes smaller than 1 megabase, and the size ranges within each of these groups is narrower than in the aster yellows, stolbur, and X-disease groups. The smallest chromosome, approximately 530 kb, was found in two Bermuda grass white leaf phytoplasma isolates. This not only is the smallest mollicute chromosome found to date, but also is the smallest chromosome known for any cell. More than one large DNA band was observed in several phytoplasma preparations. Possible explanations for the occurrence of more than one band may be infection of the host plant by different phytoplasmas, the presence of more than one chromosome in the same organism, or the presence of large extrachromosomal DNA elements.

Oligodeoxynucleotides as probes forin situhybridization with transmission electron microscopy to specifically localize phytoplasma in plant cells

Phytoplasmas are plant-pathogenic mollicutes restricted to phloem. They belong to several groups in a unique phylogenetic clade. Non-related phytoplasmas may infect the same plant species, often with similar symptoms. Hence methods are needed to specifically localize phytoplasmas and to study their multiplication and movement in their hosts. Conditions for post-embeddingin situhybridization (ISH) with transmission electron microscopy using oligodeoxynucleotides as probes for labelling of phytoplasmas in plant tissues have been searched. Sections of acrylic resin-embedded tissues of phytoplasma-infected periwinkle were submitted to ISH using digoxigenin or biotin-labelled oligoprobes (22 mers). These probes were the complementary sequences of primers used in group-specific polymerase chain reaction (PCR) amplification of 16S rDNA of stolbur and elm yellows phytoplasma, respectively. Together with preliminary digestion with pepsin, differentin situdenaturation conditions and formamide concentrations were tested. The grids were incubated in the hybridization mixture at 37°C overnight. Detection of hybridized material was performed with gold immunocytochemistry. Specificity of labelling was checked with appropriate controls. Stringency conditions could be found to ensure specific hybridization with such short probes. A specific labelling was obtained for stolbur phytoplasma on groups of mature as well as senescent phytoplasma cells. The results show that oligonucleotides may be used as probes for phytoplasma identification in post-embedding ISH with electron microscopy.

Oncopsis alni (Schrank) (Auchenorrhyncha: Cicadellidae) as a Vector of the Alder Yellows Phytoplasma of Alnus glutinosa (L.) Gaertn.

Alder yellows phytoplasma was detected by PCR in Alnus glutinosa trees in the Palatine and Mosel areas of Germany. The restriction profiles obtained by TaqI and AluI digestion of a PCR amplified ribosomal DNA fragment from this phytoplasma and a periwinkle isolate of alder yellows from Italy (ALY) could not be distinguished while elm yellows isolates from Europe and North America led to different fragment patterns. Different restriction profiles for ALY and the German alder phytoplasma were obtained by TruI digestion of a non-ribosomal DNA fragment. Phloem feeding insects were collected from infected alder trees. Phytoplasmas of the elm-yellows group were detected by PCR in psyllids and the leafhopper Oncopsis alni. These pathogens were indistinguishable from the phytoplasma found in alder. Only O. alni was able to transmit the pathogen to healthy alder seedlings. Thus, it is the first insect known to transmit this phytoplasma. This leafhopper could be responsible for the ubiquitous infection of Alnus glutinosa due to its close association with alder and its wide distribution in Europe.

Development of Cloned DNA Probes for Phytoplasma Associated with Loofah Witches’ Broom

AbstractDNA was isolated from periwinkle (Catharanihus roseus) infected with a phytoplasma that originated in loofah witches’ broom affected by loofah. Cloned DNA inserts from six LfWB‐phytoplasma specific recombinant plasmids were not only labelled with digoxigenin, but also used as probes. Probes hybridized with DNA derived from LfWB‐phytoplasma affected periwinkle and loofah, but not with DNA from healthy plants or plants infected with phytoplasmas associated with elm yellows, red bird cactus, peanut witches’ broom, paulownia witches’ broom, Ipomoea obscura witches’ broom, aster yellows (two isolates), and sweet potato witches’ broom obtained with DNA from different phytoplasmas experimentally maintained in periwinkle. The probes could detect LfWB‐phytoplasma DNA with as little as 16 ng and 32 ng of DNA from periwinkle and loofah, respectively. The method proposed herein provides a means for specifically detecting and identifying of loofah witches’ broom phytoplasma, as well as confirming the notion that this phytoplasma represents a distinct strain cluster,

Two Groups of Phytoplasmas from Japan Distinguished on the Basis of Amplification and Restriction Analysis of 16S rDNA.

Phytoplasmas (mycoplasmalike organisms, MLOs) associated with mitsuba (Japanese hone-wort) witches'-broom (JHW), garland chrysanthemum witches'-broom (GCW), eggplant dwarf (ED), tomato yellows (TY), marguerite yellows (MY), gentian witches'-broom (GW), and tsu-wabuki witches'-broom (TW) in Japan were investigated by polymerase chain reaction (PCR) amplification of DNA and restriction enzyme analysis of PCR products. The phytoplasmas could be separated into two groups, one containing strains JHW, GCW, ED, TY, and MY, and the other containing strains GW and TW, corresponding to two groups previously recognized on the basis of transmission by Macrosteles striifrons and Scleroracus flavopictus, respectively. The strains transmitted by M. striifrons were classified in 16S rRNA gene group 16SrI, which contains aster yellows and related phytoplasma strains. Strains GW and TW were classified in group 16SrIII, which contains phytoplasmas associated with peach X-disease, clover yellow edge, and related phytoplasmas. Digestion of amplified 16S rDNA with HpaII indicated that strains GW and TW were affiliated with subgroup 16SrIII-B, which contains clover yellow edge phytoplasma. All seven strains were distinguished from other phytoplasmas, including those associated with clover proliferation, ash yellows, elm yellows, and beet leafhopper-transmitted virescence in North America, and Malaysian periwinkle yellows and sweet potato witches'-broom in Asia.

Identification and characterization of the phytoplasma associated with elm yellows in southern Italy and its relatedness to other phytoplasmas of the elm yellows group

SummaryIn the neighbouring regions Basilicata, Campania, and Calabria of southern Italy, diseased trees of European field elm (Ulmus minor) were examined for phytoplasmal infection using polymerase chain reaction (PCR) technology. All affected trees examined tested positively. Using a primer pair specific for the EY phytoplasma group and restriction fragment length polymorphism (RFLP) analysis of PCR‐amplified ribosomal DNA, the organism detected was identified as the elm yellows (EY) phytoplasma. RFLP analysis of PCR‐amplified ribosomal DNA was also employed to attempt differentiation within the EY group which includes, in addition to the EY agent, phytoplasmas infecting Rubus, alder, eucalypts, Spanish broom, and grapevine. Following separate digestion with AluI, RsaI, Sau3AI, MseI, HhaI, and KpnI, all PCR‐products from EY‐group phytoplasmas examined had similar RFLP profiles. When the same ribosomal DNA fragments were digested with TaqI restriction endonuclease, three different restriction profiles were detected among the EY‐group phytoplasmas. These profiles represented, respectively, (1) the EY phytoplasma (2) the phytoplasmas causing rubus stunt and being associated with alder yellows, spartium witches broom, and eucalyptus little leaf, and (3) the flavescence dorée phytoplasma. RFLP analysis using TaqI endonuclease enabled for the first time the differentiation of the phytoplasmas associated with alder yellows, eucalyptus little leaf, and spartium witches broom from the EY agent.

Detection and differentiation of grapevine yellows phytoplasmas belonging to the elm yellows group and to the stolbur subgroup by PCR amplification of non-ribosomal DNA

Primer pairs were designed from a cloned DNA probe of a strain of flavescence doree (FD) phytoplasma and from a cloned DNA probe of a strain of stolbur phytoplasma. Among an array of reference phytoplasma strains maintained in periwinkle, pair FD9f/r amplified a 1.3 kb DNA fragment only with phytoplasma strains of elm yellows (EY) group, i.e. two strains of FD and two strains of EY. Tru9I restriction analysis of the fragment amplified by FD9f/r revealed a diversity among EY-group phytoplasmas. The FD strains differed from the strains isolated from elm. The profile of the phytoplasmas infecting the grapevine samples from Catalonia and most of the samples from Northern Italy were identical to that of a FD strain. Three other profiles were detected in grapevine from Palatinate, in Germany.

DNA probes and PCR primers for the detection of a phytoplasma associated with peanut witches'-broom

EcoRI restriction fragments of genomic DNA from the phytoplasma associated with peanut witches'-broom (PNWB) were cloned in plasmid pGEM-3Zf(+). Cloned inserts from seven PNWB-phytoplasma-specific recombinant plasmids and two subcloned plasmids were excised with restriction enzymes, labeled with digoxigenin, and used as probes. Probe PNWB281 and its derivative subclones PNWB281-4 and PNWB281-5 hybridized with DNA from PNWB-phytoplasma infected peanut and periwinkle specifically but not with DNA from healthy plants or plants infected with phytoplasmas associated with sweetpotato witches'-broom (SPWB), loofah, Ipomoea obscura, and paulownia witches'-broom, elm and aster yellows, rice yellow dwarf, and bamboo little leaf disease. Six other probes hybridized with DNA derived from PNWB and SPWB-phytoplasma-affected periwinkle but not with DNA from healthy plants or plants infected with other phytoplasmas mentioned. In Southern hybridizations, four of the nine cloned and subcloned probes could differentiate the PNWB-phytoplasma from SPWB-phytoplasma. Three primer pairs for PCR were synthesized according to the partial sequences at both ends of the cloned inserts and were able to distinguish PNWB-phytoplasma from SPWB-phytoplasma by using PCR for the first time. A minimum of 1 pg and 10 pg of total DNA from diseased periwinkle and peanut, respectively, was sufficient to amplify the specific PNWB-phytoplasma PCR fragments, allowing the detection of PNWB-phytoplasma DNA from healthy-looking periwinkle plants two weeks after graft inoculation.

Generation and characterization of monoclonal antibodies to Flavescence dorée phytoplasma: Serological relationships and differences in electroblot immunoassay profiles of Flavescence dorée and elm yellows phytoplasmas

Eleven stable hybridoma cell lines secreting monoclonal antibodies specific for FD-phytoplasma, the pathogenic agent of grapevine Flavescence doree, were produced by fusing a non-secreting myeloma cell line with spleen cells from Balb/c mice immunized with Flavescence doree phytoplasma purified by immunoaffinity. These monoclonal antibodies were characterized for their recognition of phytoplasma proteins by western blot. Six of eleven reacted specifically in ELISA and immunoblotting with Elm-yellows phytoplasma. These antibodies did not react either in ELISA or in western blot with preparations from periwinkles infected with phytoplasmas that cause GYU (Grapevine Yellows from Udine), AP (Apple Proliferation), EAY (European Aster Yellows) and StolC (Stolbur from France). Two of these hybridoma lines were used routinely for the immunodiagnosis of Flavescence doree phytoplasma in diseased grapevines.

Phytoplasma-specific PCR primers based on sequences of the 16S-23S rRNA spacer region.

In order to develop a diagnostic tool to identify phytoplasmas and classify them according to their phylogenetic group, we took advantage of the sequence diversity of the 16S-23S intergenic spacer regions (SRs) of phytoplasmas. Ten PCR primers were developed from the SR sequences and were shown to amplify in a group-specific fashion. For some groups of phytoplasmas, such as elm yellows, ash yellows, and pear decline, the SR primer was paired with a specific primer from within the 16S rRNA gene. Each of these primer pairs was specific for a specific phytoplasma group, and they did not produce PCR products of the correct size from any other phytoplasma group. One primer was designed to anneal within the conserved tRNA(Ile) and, when paired with a universal primer, amplified all phytoplasmas tested. None of the primers produced PCR amplification products of the correct size from healthy plant DNA. These primers can serve as effective tools for identifying particular phytoplasmas in field samples.