The phylogenetic relationships of 17 phytopathogenic mycoplasmalike organisms (MLOs) representing seven major taxonomic groups established on the basis of MLO 16s ribosomal DNA (rDNA) restriction patterns were examined by performing a sequence analysis of the 16s rDNA gene. The sequence data showed that the MLOs which we examined are members of a relatively homogeneous group that evolved monophyletically from a common ancestor. In agreement with results obtained previously with other MLOs, our results also revealed that the organisms are more closely related to AchoZepZasma Zaidhwii and other members of the anaeroplasma clade than to any other mollicutes. A phylogenetic tree based on 16s rDNAs showed that the MLOs which we examined can be divided into the following five primary clusters: (i) the aster yellows strain cluster; (ii) the apple proliferation strain cluster; (iii) the western-X disease strain cluster; (iv) the sugarcane white leaf strain cluster; and (v) the elm yellows strain cluster. The aster yellows, western-X disease, and elm yellows strain clusters were divided into two subgroups each. MLOs whose 16s rDNA sequences have been determined previously by other workers can be placed in one of the five groups. In addition to the overall division based on 16s rDNA sequence homology data, the primary clusters and subgroups could be further defined by a number of positions in the 16s rDNAs that exhibited characteristic compositions, especially in the variable regions of the gene.Nonhelical phytopathogenic mollicutes, which are most often referred to as mycoplasmalike organisms (MLOs), are wall-less, nonculturable prokaryotes that cause diseases in several hundred plant species (23). The first attempts to differentiate and classify these organisms were based on symptoms, host ranges, and insect vector relationships (4, 9, 21).The phylogenetic interrelatedness of the MLOs was poorly understood until recently, when molecular methods were introduced into plant mycoplasmology. Many MLO strains were differentiated and partially characterized by dot and Southern hybridization techniques, as well as by serological techniques. Most of this work has been reviewed by Kirkpatrick (10) and Lee and Davis (16). Closely related MLOs belonging to the aster yellows strain cluster could be classified by Southern blot analysis (17).Neither serological methods nor nucleic acid hybridization experiments performed with randomly cloned DNA fragments have revealed the phylogenetic or taxonomic positions of MLOs in relation to each other and to other microorganisms. In contrast, the 16s rRNA gene is a universal characteristic in prokaryotes and has both conserved and sufficiently variable regions. This gene is therefore suitable for phylogenetic and taxonomic classifications at various levels, including intrageneric differentiation (30,34 yellows agent (strain SAY), and the western-X disease agent (strain WX), as well as several Japanese MLOs, showed that these organisms exhibit levels of sequence homology of at least 89.4%. Thus...

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.

Restriction fragment length polymorphism (RFLP) analysis of PCR-amplified ribosomal DNA and Southern blot hybridization using cloned chromosomal DNA fragments from the apple proliferation (AP) phytoplasma as probes were used to investigate the genetic relationship of the California peach yellow leaf roll (PYLR) agent with phytoplasmas causing fruit tree diseases in Europe. This comparison showed that the California PYLR phytoplasma is closely related to apple proliferation (AP), pear decline, and European stone fruit yellows phytoplasmas and that it is a member of the phylogenetic AP group. The PYLR agent could clearly be distinguished from the AP and European stone fruit yellows phytoplasmas by Southern blot hybridization with DNA fragments from the AP phytoplasma and by RFLP analysis of ribosomal DNA employing SspI, BsaAI, and RsaI restriction endonucleases. However, the PYLR phytoplasma was indistinguishable from the pear decline agent by RFLP analysis of PCR-amplified ribosomal DNA.

Fluorescence microscopy and PCR as well as graft transmissioti experitnenls were employed to examine the persistetice of the European stone fruit phytoplasma in the stem of various Prunus taxa during the dortnant season. Both approaches revealed that the phytoplasma pathogen persists in the aerial parts of the trees in all stone fruit taxa examined.

Genetic homogeneity o! the mycoplasmalike organisms (MLOs) associated with apple proliferadon (AP) tjisea,sc was exaBiuied by comparing samples collected in south-western Germany, hrance, Italy, and Romania hv Southern bSot analysis. The matena! cxainmed consssied ot rwo penwinkle-maintaified strains of the AP-MLO, samples from witches' brooms of 48 symptomatic trees, and root samples from 14 latently infected trees. Six cloned chromosomal DNA fragments of one oi the perjwmkk'-maintained strains were used as probes. These probes, which were specific for the AP-MLO and other European fruit MLOs, hybridized with DNA of all samples which indicates thai ihe MLOs examined were closely related with one another. However, restriction fragment length polymorphism was obsen^ed among the samples. Four bybridiEation patterns occurred which were interrelated by common bands. Therefore, the AP ageni can be regarded as a taxonomic entit)". The variability among the isolates seems not to be due to their geographical origin but might be related to pathological traits. Zusammenfassiing Restriktionsfragnientlangenpolymorphimus beim Erreger der ApfeitriebsuchtZur Untersuchung dcr geneaschcn Homogenitat dcr mit der Aptehnebsucht assoznerten mycoplasmaahnlicheii Org,inismen (MLOs) wurden 48 Hexeiibesenproben von siclitbar kranken Baumen und 14 Wurxelproben von latent bcfallenen Baumeo in Deutschland, Frankreich, Itahen und Rumanian gesammek und durch Southern Blot-Analyse miteinander vergbchen. Auch 2 auf Catharanthus ?'osi'us iibertragcne Stamme dcs Triebsuchterregers wurden in die Untt'rsuchungen einbezogen. Als Sonden dienten 6 klonierte chromosomale DNA-Fragmente emes dcr auf C. ?-oseus gehaltenen Stamme. Dtese Sonden waren spezifiscii fiir das AP-MLO und andere europaische Obstmycoplasmen-Sie hybridisierten mit alien urstcr such ten DNA-Proben, was ein Nachwcis fiJr ihre enge genetische Verv^'andtsciiaft isi.. Zwischeo den Isolates war jedoch ein Restriktionsfragmentlangenpolymorphismus iestziistellcn. Insgesanit traten 4 verschiedene Hybridisierungsmuster auf, die durch geniclnsame Banden cng mitcmaiider m Verbmdung standen. Der Erreger der Apfckricbsucht kaiin daher als taxonomische Einheit betrachtet wcrden. Die Variabiliciit zwischen den Isolaten scheint mcht auf ihre geographische Herkunft zuriJckzuiuhrcn zy sein, konnte jedoch mit der Symptomauspritgung in Vcrbindueg stchen.