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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.

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Fusarium head blight (FHB, scab) causes severe yield and quality losses, but the most serious concern is the mycotoxin contamination of cereal food and feed. The cultivation of resistant varieties may contribute to integrated control of this fungal disease. Breeding for FHB resistance by conventional selection is feasible, but tedious and expensive. The aim of this work was to detect QTLs for combined type I and type II resistance against FHB and estimate their effects in comparison to the QTLs identified previously for type II resistance. A population of 364, F1 derived doubled-haploid (DH) lines from the cross 'CM-82036' (resistant)/'Remus' (susceptible) was evaluated for components of FHB resistance during 2 years under field conditions. Plants were inoculated at anthesis with a conidial suspension of Fusarium graminearum or Fusarium culmorum. The crop was kept wet for 20 h after inoculation by mist-irrigation. Disease severity was assessed by visual scoring. Initial QTL analysis was performed on 239 randomly chosen DH lines and extended to 361 lines for putative QTL regions. Different marker types were applied, with an emphasis on PCR markers. Analysis of variance, as well as simple and composite interval mapping, revealed that two genomic regions were significantly associated with FHB resistance. The two QTLs on chromosomes 3B (Qfhs.ndsu-3BS) and 5A (Qfhs.ifa-5A) explained 29 and 20% of the phenotypic variance, respectively, for visual FHB severity. Qfhs.ndsu-3BS appeared to be associated mainly with resistance to fungal spread, and Qfhs.ifa-5A primarily with resistance to fungal penetration. Both QTL regions were tagged with flanking SSR markers. These results indicate that FHB resistance was under the control of two major QTLs operating together with unknown numbers of minor genes. Marker-assisted selection for these two major QTLs appears feasible and should accelerate the development of resistant and locally adapted wheat cultivars.

A method has been developed to amplify the 16s rRNA gene of plant-pathogenic mycoplasma-like organisms (MLOs) from infected plant material using the polymerase chain reaction (PCR). The procedure is dependent on the presence of a BcZI restriction site in the 16s rDNA of chloroplasts but not in that of the MLOs. This difference permits the specific amplification of the 16s rDNA of the MLOs from BcZI-digested total DNA from infected plants using primers from conserved regions of this gene. In this study 16s rDNA was obtained from 52 MLO isolates from herbaceous dicots and monocots as well as woody plants. Digestion of the 16s rRNA genes using A M endonuclease revealed seven restriction patterns, which were used to group the isolates examined. Group I, which is also characterized by the presence of two KpnI sites, consisted of 31 isolates, most of which are from herbaceous dicots. Isolates assigned to groups I1 to VI were mostly from woody plants, while the isolates of group VII were from monocots or obtained from a leafhopper. The restriction patterns varied little within groups; however, four group I isolates and one group IV isolate differed slightly from the typical patterns of these groups as a result of a deletion or a slight shift of one restriction site. The groupings uncovered by Ah1 restriction were also obtained by digesting the 16s rDNA with RsaI endonuclease. However, some atypical patterns were observed within group V isolates. The groups described on the basis of restriction digest data were supported by sequence analysis. With one exception, the 16s rDNA of isolates within the same group exhibited 97.8 to 99-5 YO homology while those of different groups showed 89.6 to 92.0% homology.

Background: Phytoplasmas are insect-transmitted, uncultivable bacterial plant pathogens that cause diseases in hundreds of economically important plants. They represent a monophyletic group within the class Mollicutes (trivial name mycoplasmas) and are characterized by a small genome with a low GC content, and the lack of a firm cell wall. All mycoplasmas, including strains of 'Candidatus (Ca.) Phytoplasma asteris' and 'Ca. P. australiense', examined so far have circular chromosomes, as is the case for almost all walled bacteria.

Primers designed from sequences of the gene encoding the elongation factor Tu (tuf gene) of several culturable mollicutes amplified most of the tuf gene from phytoplasmas of the aster yellows, stolbur and Xdisease groups. About 85% of the tuf gene from two aster yellows strains and a tomato stolbur phytoplasma was sequenced. The nucleotide sequence similarity between these related phytoplasmas was between 87.8 and 97G0/o, whereas the homology with other mollicutes was 663-7207 %. The similarity of the deduced amino acid sequence was significantly higher, ranging from 960 to 994% among the phytoplasmas and 785% to 833% between phytoplasmas and the culturable mollicutes examined. From the nucleotide sequences of the phytoplasma strains, two pairs of primers were designed; one amplified the phytoplasmas of most phylogenetic groups that were established, the other was specific for the aster yellows and stolbur groups. The phytoplasmas of the various groups that were amplified could be distinguished by RFLP analysis using Sau3A1, AIul and Hpall. The aster yellows group could be divided into five Sau3Al RFLP groups. These results showed that the tuf gene has the potential to be used to differentiate and classify phytoplasmas. Southern blot analysis revealed that the tuf gene is present as a single copy.1 Keywords : phytoplasmas, differentiation, classification, elongation factor, PCR INTRODUCTIONPhytoplasmas, formerly called mycoplasma-like organisms (MLOs), are plant-pathogenic prokaryotes of the class Mollicutes that cannot be cultured under axenic conditions. The inability to culture phytoplasmas has made it difficult to characterize these pathogens. Only recently, by the introduction of molecular methods into plant mycoplasmology, has it become possible to determine the phylogenetic and taxonomic relationships of the phytoplasmas to each other and to other prokaryotes. Currently, classification is based on sequence analysis of the 16s rRNA gene (Lim & Sears, 1989 ;Kuske & Kirkpatrick, 1992 ;Namba et al., 1993 ;Gundersen et al., 1994; Schneider et al., 1995b). This gene is present in all prokaryotes and the conserved and variable regions make it suitable for phylogenetic classifications. Other phytoplasma genes or DNA regions that have been used for classificationThe GenBank accession numbers for the sequences reported in this paper are L46368 ( M Y ) , L46369 (KV) and L46370 (STOLF).are the ribosomal protein genes rpl2.2 and rps3 (Lim & Sears, 1992;Gundersen et al., 1994; Toth et al., 1994) and the 16S/23S rRNA spacer region Schneider et al., 1995b). The latter are considerably more variable than the 16s rRNA gene but phylogenetic analysis of all three sequence categories has resulted in a similar classification of the phytoplasmas in relation to each other and to other mollicutes (Lim & Sears, 1989Kuske & Kirkpatrick, 1992;Namba et al., 1993;Gundersen et al., 1994; Toth et al., 1994).Although the relationship between the phylogenetic classification of the phytoplasmas and pathological and other biological tra...

The chromosome sequence of “Candidatus Phytoplasma australiense” (subgroup tuf-Australia I; rp-A), associated with dieback in papaya, Australian grapevine yellows in grapevine, and several other important plant diseases, was determined. The circular chromosome is represented by 879,324 nucleotides, a GC content of 27%, and 839 protein-coding genes. Five hundred two of these protein-coding genes were functionally assigned, while 337 genes were hypothetical proteins with unknown function. Potential mobile units (PMUs) containing clusters of DNA repeats comprised 12.1% of the genome. These PMUs encoded genes involved in DNA replication, repair, and recombination; nucleotide transport and metabolism; translation; and ribosomal structure. Elements with similarities to phage integrases found in these mobile units were difficult to classify, as they were similar to both insertion sequences and bacteriophages. Comparative analysis of “Ca. Phytoplasma australiense” with “Ca. Phytoplasma asteris” strains OY-M and AY-WB showed that the gene order was more conserved between the closely related “Ca. Phytoplasma asteris” strains than to “Ca. Phytoplasma australiense.” Differences observed between “Ca. Phytoplasma australiense” and “Ca. Phytoplasma asteris” strains included the chromosome size (18,693 bp larger than OY-M), a larger number of genes with assigned function, and hypothetical proteins with unknown function.