Twenty strains of the ESFY phytoplasma, which on the basis of graft-inoculation experiments greatly differ in aggressiveness, were examined by sequence analyses of several PCR-amplified non-ribosomal genes in order to identify molecular markers linked to virulence. These strains, which were maintained in P. insititia rootstock St. Julien GF 655/2 were indistinguishable with techniques for routine phytoplasma differentiation and characterization such as sequence and RFLP analyses of PCR-amplified rDNA. Also, the virulent ESFY strains maintained in periwinkle, namely GSFY1, GSFY2 and ESFY1, as well as an avirulent strain of the same phytoplasma, maintained in apricot, which was identified in recovered apricot trees in France and used there as a cross protecting agent, were included in the work for comparison. For PCR amplification, primers were designed from a number of genes distributed over the chromosome of the closely related apple proliferation phytoplasma strain AT. Visible PCR products were only obtained with primer pairs derived from the tuf gene which encodes the elongation factor Tu (EF-Tu), rps C ( rps 3) gene encoding the ribosomal protein S3, tly C gene which encodes a hemolysin known as a membrane-damaging agent and important virulence factor of many bacteria, the imp and fol genes encoding an immunodominant membrane protein and an enzyme involved in the folate biosynthesis, respectively. Nucleotide sequence comparisons revealed that the highest genomic variability occurred within the imp gene sequence with dissimilarity values ranging from 0.2 to 4.6%. For the remaining genes, the strains examined proved to be identical or nearly identical. Within the tuf gene, an extra Taq I site known to occur in strain GSFY1 was not identified in other strains. The genetic differences observed among the strains examined are neither suitable markers for strain differentiation nor linked to pathological traits. Keywords : European stone fruit yellows, strain virulence, 16SrX group, tly C gene, Prunus spp.

Pinus silvestris and Pinus halepensis trees grown in Germany and Spain, respectively, showing abnormal shoot branching, dwarfed needles and other symptoms were examined for the presence of plant-pathogenic mollicutes (phytoplasmas). While phytoplasmas could not be detected unambiguously with microscopical methods, PCR amplification using universal phytoplasma primers yielded positive results. Samples collected from symptomatic and non-symptomatic plant parts of both symptomatic Pinus silvestris and Pinus halepensis trees tested positive. Also, surrounding non-symptomatic trees proved to be phytoplasma-infected. Comparisons revealed that the 16S rRNA gene sequences of the phytoplasmas identified in Pinus silvestris and Pinus halepensis were nearly identical. However, the pine phytoplasma is only distantly related to other phytoplasmas. The closest relatives are members of the palm lethal yellowing and rice yellow dwarf groups and 'Candidatus Phytoplasma castaneae', which share between 94.5 and 96.6 % 16S rRNA gene sequence similarity. From these data it can be concluded that the phytoplasmas identified in the two Pinus species represent a coherent but discrete taxon; it is proposed that this taxon be distinguished at putative species level under the name 'Candidatus Phytoplasma pini'.

Aster yellows (AY) group (16SrI) phytoplasmas are associated with over 100 economically important diseases worldwide and represent the most diverse and widespread phytoplasma group. Strains that belong to the AY group form a phylogenetically discrete subclade within the phytoplasma clade and are related most closely to the stolbur phytoplasma subclade, based on analysis of 16S rRNA gene sequences. AY subclade strains are related more closely to their culturable relatives, Acholeplasma spp., than any other phytoplasmas known. Within the AY subclade, six distinct phylogenetic lineages were revealed. Congruent phylogenies obtained by analyses of tuf gene and ribosomal protein (rp) operon gene sequences further resolved the diversity among AY group phytoplasmas. Distinct phylogenetic lineages were identified by RFLP analysis of 16S rRNA, tuf or rp gene sequences. Ten subgroups were differentiated, based on analysis of rp gene sequences. It is proposed that AY group phytoplasmas represent at least one novel taxon. Strain OAY, which is a member of subgroups 16SrI-B, rpI-B and tufI-B and is associated with evening primrose (Oenothera hookeri) virescence in Michigan, USA, was selected as the reference strain for the novel taxon ‘Candidatus Phytoplasma asteris’. A comprehensive database of diverse AY phytoplasma strains and their geographical distribution is presented.

Apple proliferation (AP), pear decline (PD) and European stone fruit yellows (ESFY) are among the most economically important plant diseases that are caused by phytoplasmas. Phylogenetic analyses revealed that the 16S rDNA sequences of strains of each of these pathogens were identical or nearly identical. Differences between the three phytoplasmas ranged from 1.0 to 1.5% of nucleotide positions and were thus below the recommended threshold of 2.5% for assigning species rank to phytoplasmas under the provisional status 'Candidatus'. However, supporting data for distinguishing the AP, PD and ESFY agents at the species level were obtained by examining other molecular markers, including the 16S-23S rDNA spacer region, protein-encoding genes and randomly cloned DNA fragments. The three phytoplasmas also differed in serological comparisons and showed clear differences in vector transmission and host-range specificity. From these results, it can be concluded that the AP, PD and ESFY phytoplasmas are coherent but discrete taxa that can be distinguished at the putative species level, for which the names 'Candidatus Phytoplasma mali', 'Candidatus Phytoplasma pyri' and 'Candidatus Phytoplasma prunorum', respectively, are proposed. Strains AP15R, PD1R and ESFY-G1R were selected as reference strains. Examination of available data on the peach yellow leaf roll (PYLR) phytoplasma, which clusters with the AP, PD and ESFY agents, confirmed previous results showing that it is related most closely to the PD pathogen. The two phytoplasmas share 99.6% 16S rDNA sequence similarity. Significant differences were only observed in the sequence of a gene that encodes an immunodominant membrane protein. Until more information on this phytoplasma is available, it is proposed that the PYLR phytoplasma should be regarded as a subtype of 'Candidatus Phytoplasma pyri'.

The trivial name 'phytoplasma' has been adopted to collectively name wall-less, non-helical prokaryotes that colonize plant phloem and insects, which were formerly known as mycoplasma-like organisms. Although phytoplasmas have not yet been cultivated in vitro, phylogenetic analyses based on various conserved genes have shown that they represent a distinct, monophyletic clade within the class Mollicutes. It is proposed here to accommodate phytoplasmas within the novel genus 'Candidatus (Ca.) Phytoplasma'. Given the diversity within 'Ca. Phytoplasma', several subtaxa are needed to accommodate organisms that share <97.5% similarity among their 16S rRNA gene sequences. This report describes the properties of 'Ca. Phytoplasma', a taxon that includes the species 'Ca. Phytoplasma aurantifolia' (the prokaryote associated with witches'-broom disease of small-fruited acid lime), 'Ca. Phytoplasma australiense' (associated with Australian grapevine yellows), 'Ca. Phytoplasma fraxini' (associated with ash yellows), 'Ca. Phytoplasma japonicum' (associated with Japanese hydrangea phyllody), 'Ca. Phytoplasma brasiliense' (associated with hibiscus witches'-broom in Brazil), 'Ca. Phytoplasma castaneae' (associated with chestnut witches'-broom in Korea), 'Ca. Phytoplasma asteris' (associated with aster yellows), 'Ca. Phytoplasma mali' (associated with apple proliferation), 'Ca. Phytoplasma phoenicium' (associated with almond lethal disease), 'Ca. Phytoplasma trifolii' (associated with clover proliferation), 'Ca. Phytoplasma cynodontis' (associated with Bermuda grass white leaf), 'Ca. Phytoplasma ziziphi' (associated with jujube witches'-broom), 'Ca. Phytoplasma oryzae' (associated with rice yellow dwarf) and six species-level taxa for which the Candidatus species designation has not yet been formally proposed (for the phytoplasmas associated with X-disease of peach, grapevine flavescence dorée, Central American coconut lethal yellows, Tanzanian lethal decline of coconut, Nigerian lethal decline of coconut and loofah witches'-broom, respectively). Additional species are needed to accommodate organisms that, despite their 16S rRNA gene sequence being >97.5% similar to those of other 'Ca. Phytoplasma' species, are characterized by distinctive biological, phytopathological and genetic properties. These include 'Ca. Phytoplasma pyri' (associated with pear decline), 'Ca. Phytoplasma prunorum' (associated with European stone fruit yellows), 'Ca. Phytoplasma spartii' (associated with spartium witches'-broom), 'Ca. Phytoplasma rhamni' (associated with buckthorn witches'-broom), 'Ca. Phytoplasma allocasuarinae' (associated with allocasuarina yellows), 'Ca. Phytoplasma ulmi' (associated with elm yellows) and an additional taxon for the stolbur phytoplasma. Conversely, some organisms, despite their 16S rRNA gene sequence being <97.5% similar to that of any other 'Ca. Phytoplasma' species, are not presently described as Candidatus species, due to their poor overall characterization.

Bermuda grass white leaf (BGWL) is a destructive, phytoplasmal disease of Bermuda grass (Cynodon dactylon). The causal pathogen, the BGWL agent, differs from other phytoplasmas that cluster in the same major branch of the phytoplasma. phylogenetic clade in < 2.5% of 16S rDNA nucleotide positions, the threshold for assigning species rank to phytoplasmas under the provisional status 'Candidatus'. Thus, the objective of this work was to examine homogeneity of BGWL isolates and to determine whether there are, in addition to 16S rDNA, other markers that support delineation of the BGWL agent at the putative species level. Phylogenetic analyses revealed that the 16S rDNA sequences of BGWL strains were identical or nearly identical. Clear differences that support separation of the BGWL agent from related phytoplasmas were observed within the 16S-23S rDNA spacer sequence, by serological comparisons, in vector transmission and in host-range specificity. From these results, it can be concluded that the BGWL phytoplasma is a discrete taxon at the putative species level, for which the name 'Candidatus Phytoplasma cynodontis' is proposed. Strain BGWL-C1 was selected as the reference strain. Phytoplasmas that are associated with brachiaria white leaf, carpet grass white leaf and diseases of date palms showed 16S rDNA and/or 16S-23S rDNA spacer sequences that were identical or nearly identical to those of the BGWL phytoplasmas. However, the data available do not seem to be sufficient for a proper taxonomic assignment of these phytoplasmas.

In nature, only a minority of alder trees infected by the alder yellows phytoplasma develop symptoms. To elucidate the reason for this phenomenon, healthy alder seedlings were inoculated with scion wood from five infected but differently affected trees. The symptoms developed by the inoculated plants largely corresponded to disease severity of the inoculum sources. Inoculation with tissue from three of four severely affected trees resulted in severe disease of the recipient trees as expressed by reduced vigour, sparse foliage and little leaves. Inoculum from one of the severe sources caused milder symptoms. No symptoms were developed when the inoculum was collected from a vigorous, non-symptomatic tree. From these results it can be concluded that the alder yellows phytoplasma is pathogenic to alder but that avirulent and low virulent strains do occur.

Twenty strains of the European stone fruit yellows (ESFY) phytoplasma showed great differences in virulence when examined by graft inoculation of trees on peach, peach hybrid GF 677 and P.‘Marianna’ GF 8/1 rootstocks. The most virulent strains killed all trees on peach rootstocks whereas the mild strains did not cause mortality but induced only mild foliar symptoms and slightly reduced vigour. Virulence often depended on the pathogen–scion combination and was in several cases most severe when the scion consisted of the original host of the pathogen. To examine resistance in stone fruits, trees on a total of 23 rootstocks were inoculated with the ESFY strains. Trees on the Prunusdomestica stocks Ackermann’s, Brompton and P 1275 and on Prunuscerasifera stock Myrabi were little affected. Slightly more damage occurred in trees on rootstocks GF 677, GF 8–1, and the P.insititia stocks St Julien A and St Julien GF 655/2. Ishtara, P.cerasifera stock Myrobalan, and peach rootstocks Higama and GF 305 were shown to be moderately susceptible and a high susceptibility was found in trees on peach rootstocks Montclar, peach seedling, Rutgers Red Leaf, and Rubira, on apricot seedlings and St Julien 2. Of flowering cherry trees on various rootstocks, the least susceptible were those on Gisela 3 and F 12/1 whereas Gisela 1, Weihroot 158 and Gisela 5 were more affected. Phytoplasmas were detected by either DAPI (4′‐6‐diamidino‐2‐phenylindole) staining or polymerase chain reaction in all rootstocks and scions tested. However, detection frequency and phytoplasma concentrations were usually lower in the more tolerant hosts than in susceptible genotypes.

A physical map of the European stone fruit yellows phytoplasma strain GSFY1 chromosome was constructed using PFGE-purified genomic DNA from diseased tobacco and tomato plants. The map was generated with single and double digestions of the chromosome with SmaI, BssHII, ApaI, BamHI and XhoI restriction endonucleases and the fragments were resolved by PFGE. Reciprocal double digestions were used to locate 26 restriction sites on the chromosome. Southern blot analysis was also used to assist in the arrangement of the contiguous restriction fragments obtained. From the restriction fragments generated by double digestion, the circular chromosome was calculated to be approximately 635 kb. Loci of two rRNA operons, the operon containing the tuf gene, genes encoding an immunodominant membrane protein and a putative nitroreductase, and randomly cloned DNA fragments IH184 and AT67 were placed on the map. Digestion of chromosomal DNA of strain GSFY1 with MluI gave a complex restriction pattern, suggesting that this isolate consists of a population with heterogeneity with respect to MluI restriction sites. The GSFY1 physical map was different from that of the closely related apple proliferation phytoplasma but the genetic arrangement was similar.

Seventy phytoplasma isolates, including 10 previously characterized reference strains, of the aster yellows group were examined by RFLP analysis of PCR-amplified rDNA and RFLP and sequence analysis of the tuf gene. On the basis of rDNA restriction profiles, seven previously proposed 16S rDNA subgroups (16SrI-A, -B, -C, -D, -E, -F and -K) were recognized in the material examined. In addition, three new subgroups that differ in the RFLP profiles were identified and designated 16SrI-L, 16SrI-M and 16SrI-N. Of the two types of rDNA sequences used, an 1800 bp fragment comprising the entire 16S rRNA gene and the 16S-23S rDNA spacer region proved more suitable for AY-group phytoplasma differentiation than a 1240 bp fragment of the 16S rRNA gene. Many differences in the rDNA profiles between the subgroups could be explained by sequence heterogeneity of the two phytoplasmal rRNA operons. The subgroups delineated by RFLP analysis of a 940 bp tuf gene fragment are consistent with subgroups defined on the basis of rDNA sequences. However, subgroups 16SrI-D, -L and -M showed the same tuf gene restriction profiles as subgroup 16SrI-B. This result was confirmed by sequence analysis in which these subgroups differed slightly in their tuf gene sequence, when compared with members of subgroup 16SrI-B. On the basis of combined analyses of rDNA and tuf gene sequences and in view of pathological aspects, the taxonomic distinction of AY-subgroups 16SrI-A, -B, -C, -D, -E, -F, -K and -N appears to be substantial.

A physical map of the apple proliferation phytoplasma strain AT chromosome was constructed from genomic DNA extracted from diseased tobacco plants. The map was generated with single and double digestions of the chromosome with BssHII, SmaI, MluI, and ApaI restriction endonucleases and resolving the fragments by pulsed-field gel electrophoresis. Partial digestion and Southern blot analysis were used to assist in the arrangement of the 14 contiguous restriction fragments obtained. From the restriction fragments generated by double digestions, the size of the circular chromosome was calculated to be approximately 645 kb. Locations of the two rRNA operons, the operon including the fus and tuf genes, and three other genes were placed on the map. Genome sizes and BssHII restriction profiles of apple proliferation strain AP15 and the pear decline and European stone fruit yellows phytoplasmas were different from that of strain AT.

Pear trees showing pear decline symptoms and yellows-diseased trees of apricot, peach and almond, all collected in Bulgaria, were examined for phytoplasma infection using polymerase chain reaction technology. From diseased pear and stone fruit trees, the target DNA could be amplified with both universal phytoplasma primers and primers specific for fruit trees phytoplasmas of the apple proliferation group. Restriction fragment length polymorphism analysis revealed that pear and stone fruit trees were infected with the pear decline and European stone fruit yellows phytoplasmas, respectively. This is the first report on the occurrence of these phytoplasmas in Bulgaria.

An immunodominant membrane protein (IMP) of the apple proliferation (AP) phytoplasma was detected in preparations from AP-diseased periwinkle plants using monoclonal and polyclonal antibodies to the AP agent. Following isolation from Western blots and partial sequencing, degenerate oligonucleotides derived from the IMP sequence were used as probes to identify a DNA fragment containing the ORF encoding the IMP. Complete sequencing and subsequent analysis of the cloned DNA fragment revealed the presence of two ORFs, predicted to encode proteins with molecular masses of 25 kDa (P-318A) and 19 kDa (P-318B). Whilst database searches failed to assign a possible function to P-318A, analysis of P-318B predicted an amphiphilic membrane protein with a positively charged N-terminal portion, followed by a hydrophobic segment forming an alpha-helix, and a hydrophilic C-terminal part located outside of the cell. The amphiphilic nature of P-318B was confirmed by its solubility in Triton X-114. The gene encoding P-318B was expressed in Escherichia coli and the resulting protein was used to immunize rabbits. The antiserum obtained reacted specifically with P-318B. The same protein was also detected by an antiserum raised against antigen preparations from AP-diseased plants. The P-318B antiserum did not react with antigen preparations from plants infected with the closely related pear decline phytoplasma. However, in Southern hybridization studies, the gene encoding the IMP hybridized to genomic fragments of the pear decline and European stone fruit yellows phytoplasmas. It also showed significant sequence similarity to a gene encoding an antigenic membrane protein of the sweet potato witches' broom phytoplasma, but not to a gene encoding a major immunogenic membrane protein of an aster yellows group phytoplasma. Since it appears that most phytoplasmas possess a major immunogenic membrane protein which may have a function in pathogenesis, this work may be a basis for further studies on fundamental aspects of host-pathogen interactions. It also describes a new approach to obtain suitable immunogens to produce specific antibodies for detection and characterization of the non-culturable phytoplasmas.

Phytoplasma decline severely attacks Populus nigra cv Italica in France where it creates a risk for the future of this species in urban areas. The disease agent is transmitted by leafhoppers living exclusively on poplar. Rhytidodus decimusquartus is the main vector of the disease. Phytoplasmas have been detected on P. nigra cv Italica by polymerase chain reaction. The disease has been reported in several countries in Europe. However the symptoms are not so severe in Germany as in France. The amplification obtained using only one primer pair is sufficient to detect phytoplasma in France. The detection of the German isolates often requires the successive use of two primer pairs and slight differences have been suggested in their DNA nucleotide sequences. Indirect prophylactic methods against the vector may be considered, although none of them allows complete eradication of the disease.

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, ≈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.

Genes coding for elongation factors G (fus) and Tu (tuf) of the non-culturable apple proliferation (AP) phytoplasma were cloned and sequenced. Arrangement of these genes and identification of the ribosomal protein gene rps7 upstream of the fus gene suggest a transcriptional organization similar to that of the streptomycin operon of Escherichia coli and other bacteria. The fus and tuf genes from other tested phytoplasmas were found to be similarly linked as in the AP agent. Thus, it is likely that they show a similar chromosomal arrangement. This organization would be in contrast to that of the phylogenetically distinctly different culturable mollicutes of the genus Mycoplasma in which the tuf and fus genes are separately transcribed.

The pear decline, European stone fruit yellows and rubus stunt agents as well as the phytoplasmas causing Picris echioides (bristly oxtongue) yellows and cotton (Gossypium hirsutum) phyllody, respectively, were transmitted from naturally infected plants to the experimental host Catharanthus roseus (periwinkle) via dodder (Cuscuta spp.) bridges. The identities of the dodder-transmitted phytoplasmas were confirmed by restriction length fragment polymorphism analysis of polymerase chain reaction-amplified ribosomal DNA. On the basis of restriction profiles the cotton phyllody agent could be differentiated from the phytoplasma causing faba bean phyllody, a disease previously thought to be induced by the same organism as cotton phyllody.

In Europe, phytoplasmas cause severe yellows, witches'-broom and decline diseases of trees and shrubs grown in urban areas. Spanish broom (Spartium junceum) is affected by the spartium witches'-broom disease which is associated with phytoplasmas,of the apple proliferation (AP) and elm yellows (EY) groups. Broom (Sarothamnus scoparius) and buckthorn (Rhamnus catharticus) are affected by lethal witches'-broom diseases whose causal agents are distinct members of the AP group. An important disease of elm (Ulmus minor and others) is elm yellows, which is caused by the EY phytoplasma. Alder (Alnus glutinosa and others) and eucalyptus (Eucalyptus spp.) are affected by alder yellows and eucalyptus little-leaf disease, respectively, both induced by phytoplasmas closely related to the EY agent. Yellows diseases associated with phytoplasmas of the aster yellows group affect several species of poplar including Populus nigra 'Italica', P. alba, and P. tremula. Ornamental apples (Malus spp.) and flowering cherry (Prunus serrulata) are affected by apple proliferation and European stone fruit yellows, respectively. Other trees and shrubs affected by phytoplasmoses include black locust (Robinia pseudoacacia), hazel (Corylus maxima and C. colurna), northern red oak (Quercus rubra), hawthorn (Crataegus monogina) and weeping-willow (Salix babylonica). However, the identity of the phytoplasmas infecting these species has never been determined.

Heteroduplex mobility analysis (HMA) was performed to distinguish French and German isolates of phytoplasmas from Populus nigra cv Italica witches' broom. The French isolate was similar to the phytoplasma responsible for the European aster yellows while the German isolate was different but closely related to it. When phytoplasmas inducing similar 'stolbur' symptoms in tomato were compared to HMA, a high degree of genetic differences was observed among the reference stolbur C (StC) isolate, the European aster yellows and the other phytoplasmas inducing stolbur or big bud symptoms in tomato. Pseudo-stolbur B and D from Brazil were differentiated from the reference St C using this method.