Decreasing global transcript levels over time suggest that phytoplasma cells enter stationary phase during plant and insect colonization.

To highlight different transcriptional behaviors of the phytoplasma in the plant and animal host, expression of 14 genes of "Candidatus Phytoplasma asteris," chrysanthemum yellows strain, was investigated at different times following the infection of a plant host (Arabidopsis thaliana) and two insect vector species (Macrosteles quadripunctulatus and Euscelidius variegatus). Target genes were selected among those encoding antigenic membrane proteins, membrane transporters, secreted proteins, and general enzymes. Transcripts were detected for all analyzed genes in the three hosts; in particular, those encoding the antigenic membrane protein Amp, elements of the mechanosensitive channel, and two of the four secreted proteins (SAP54 and TENGU) were highly accumulated, suggesting that they play important roles in phytoplasma physiology during the infection cycle. Most transcripts were present at higher abundance in the plant host than in the insect hosts. Generally, transcript levels of the selected genes decreased significantly during infection of A. thaliana and M. quadripunctulatus but were more constant in E. variegatus. Such decreases may be explained by the fact that only a fraction of the phytoplasma population was transcribing, while the remaining part was aging to a stationary phase. This strategy might improve long-term survival, thereby increasing the likelihood that the pathogen may be acquired by a vector and/or inoculated to a healthy plant.

Detection of Flavescence Dorée Phytoplasma in Grapevine by Reverse-Transcription PCR.

Flavescence dorée (FD) is the most serious phytoplasma disease of grapevine. This report describes a novel method of detecting FD phytoplasma based on reverse-transcription polymerase chain reaction (RT-PCR) on 16S ribosomal RNA (16SrRNA) which will greatly improve mass screening of infected grapevines. A rapid protocol for extracting sap from whole leaves or midveins and successive one-tube amplification by RT-PCR was applied to grapevine samples with or without symptoms collected from different areas of Piedmont (northwestern Italy). Results were compared with those obtained using one of the current diagnostic methods that utilizes nested PCR on phytoplasma DNA-enriched preparations. A Cohen's kappa index of 0.76 indicated a substantial agreement between the two sets of results. The RT-PCR method has the advantage of being a rapid, reliable, and sensitive assay for large-scale screening of grapevines.

Characterization of Four Viral Species Belonging to the Family Potyviridae Isolated from Ranunculus asiaticus.

ABSTRACT Four different viral species were isolated from diseased Ranunculus asiaticus plants growing in Imperia Province (Italian Riviera-Liguria Region). Infected plants exhibited mosaic symptoms and growth abnormalities. The viruses were mechanically inoculated to a range of herbaceous hosts and differentiated biologically. Long flexuous particles were present in leaf dip extracts observed by electron microscopy. A general protocol for the amplification of potyvirus genome fragments through reverse transcription-polymerase chain reaction generated products that were cloned and sequenced. Sequence and phylogenetic analysis suggested that three of these isolates could be considered new viral species belonging to the genus Potyvirus. The fourth isolate is a new member of the genus Macluravirus. Purified virus was used as antigen to produce a specific polyclonal antiserum in rabbit; serological features were established through double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA), antigen coated plate (ACP)-ELISA, and western blot analysis. DAS-ELISA was highly specific for each virus isolate, whereas some cross-reactivity was shown in ACP-ELISA and western blot analysis. Aphid transmission by Myzus persicae was demonstrated in a controlled environment for each of the four viral isolates, whereas no transmission through seed was observed.

Expression of chloramphenicol acetyltransferase in Bacillus subtilis under the control of a phytoplasma promoter.

A cloned putative promoter region upstream of the 16S rRNA gene of the western X-disease phytoplasma was inserted behind the promoterless chloramphenicol acetyltransferase gene of plasmid pPL603. The DNA construct was used to transform Bacillus subtilis cells. The transformants were assayed for chloramphenicol acetyltransferase activity, showing that the phytoplasma promoter is efficiently expressed in a B. subtilis background.

Diversity of phytoplasmas isolated from insects, determined by a DNA heteroduplex mobility assay and a length polymorphism of the 16S-23S rDNA spacer region analysis.

Two techniques were developed for the analysis of non-cultivable mollicutes in insects. The first was aimed at detecting organisms belonging to undiscovered groups within the phytoplasma clade. After prescreening by polymerase chain reaction with phytoplasma-specific primers, nucleic acids from 54 positive samples were amplified using phytoplasma-specific fluorescein-labelled primers flanking the 16S-23S rDNA spacer region, which is variable in length among the phytoplasmas. The sizes of all the detected products were only those expected for already-described phytoplasma subclades. It was also shown that a single leafhopper might carry different phytoplasmas, at similar or very different relative concentrations. The second technique, based on the heteroduplex mobility assay, was designed for the detection of organisms phylogenetically similar to phytoplasmas but not recognized by the specific primer pair. As a result, signals generated by ribosomal DNA of organisms which appear to be closely related but not identical to phytoplasmas were detected.

Sequence analysis of domains III and IV of the 23S rRNA gene of verticillate streptomycetes.

Domains III and IV of the 23S rRNA gene of 25 strains of verticillate streptomycetes were sequenced. None of the sequences was identical to any other, with regions of variability being restricted to parts of helices 54 and 64. No relationships were detected between the similarities in the sequence and the assignment to phenetic clusters as defined by the numerical taxonomy studies. Limited agreement was also found between similarity of the sequences and DNA-DNA homology values. However, species (> 70% DNA-DNA homology values)-specific diagnostic oligonucleotides generally could be defined, except for Streptomyces baldaccii. Therefore the determination of the 23S rRNA sequence may be of greater value for fingerprinting individual strains than for taxonomic or identification purposes.

Development of a PCR test for the detection of Curtobacterium flaccumfaciens pv. flaccumfaciens.

A chromosomal DNA library of the bacterial pathogen of bean, Curtobacterium flaccumfaciens pv.flaccumfaciens NCPPB 559 was constructed in the plasmid pGEM-7Zf(+). Several clones were identified that hybridised to all Curtobacterium flaccumfaciens pathovars including: C. f betae, C. f flaccumfaciens, C. f oortii, C. f. poinsettiae and, in addition, to some strains of Clavibacter michiganensis subsp. insidiosus and Clavibacter michiganensis subsp. One of these clones (pPMP-26), after subsequent digestion with restriction endonucleases EcoRI/SacI, yielded a fragment of approximately 0.2 Kb (pPMP-26D) that hybridised specifically to C. f flaccumfaciens and not to any of the other plant pathogenic members of the order Actinomycetales or any of the other prokaryotic bean pathogens tested. This fragment was subcloned and sequenced, analysis of the resultant 198 bp sequence showed that no significant homology existed with any other sequence currently deposited in public databases. Further analysis of these data facilitated the design of PCR primers which were subsequently tested against a wide range of plant pathogenic actinomycetes and other prokaryotic bean pathogens. Results show that these primers are highly specific for all strains of C. f flaccumfaciens with no cross-reaction to strains from any other bacterial taxa tested.