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Abstract Thirty six isolates of Phoma tracheiphila from Italy, the causal agent of the mal secco disease on Citrus species, were characterised by different molecular tools in comparison with representative isolates of other phytopathogenic Phoma species. These included analysis of the distribution of RAPD and microsatellite markers and sequencing of the internal transcribed spacer (ITS) region of the nuclear rRNA genes. The results obtained with 12 RAPD primers (92 markers) and 7 microsatellite primers (56 markers) suggest that Italian isolates of P. tracheiphila are genetically homogeneous, leading to identical patterns upon amplification with all the tested primers. Accordingly, ITSI-5.8S-ITS2 sequences were highly conserved (98100% identity along a 544-characters alignment) among all the isolates of P. tracheiphila. A neighbor-joining analysis of ITS sequences of P. tracheiphila in comparison with those of other Phoma species, as well as with alignable sequences from anamorphic and teleomorphic taxa retrieved in BLAST searches, revealed a close relationship between P. tracheiphila and Leptosphaeria congesta. A pair of P. tracheiphila-specific primers was designed on the consensus sequence (555 residues) obtained from the alignment of the newly generated P. tracheiphila ITS sequences. A PCR-based specific assay coupled to electrophoretic separation of amplicons made it possible to detect P. tracheiphila in naturally infected Citrus wood tissue collected from both symptomatic and symptomless plants. The limit of detection was 10pg of genomic DNA and 5fg of the ITS target sequence. [ABSTRACT FROM AUTHOR] more...

Abstract Random amplification of polymorphic DNA (RAPD-PCR) analysis was conducted on 48 isolates of Fusarium oxysporum f. sp. radicis-lycopersici ( F.o.r.l. ) from different geographic regions, representing all known vegetative compatibility groups (VCGs) except VCG 0097 and VCG 0099 and on eight isolates of F.oxysporum f. sp. lycopersici ( F.o.l. ), representing VCGs 0030, 0031, 0032 and 0033. Upon UPGMA (unweighted pair-group method with arithmetic averages) analysis of 86 RAPD-PCR markers generated by 16 informative primers and 44 markers obtained with eight microsatellite primers, a close relatedness was evident for F.o.r.l. isolates in VCGs 0090, 0092, 0096, and, to a lesser extent, for those in VCG 0093. Representatives of VCG 0091 formed a distinct group, while F.o.r.l. isolates in VCGs 0094 and 0098 were not distinguishable by the tested markers, most of which were also shared by F.o.l. isolates belonging to VCGs 0031 and 0033. F.o.l. isolates in VCGs 0030 and 0032 shared most of the molecular markers. The correlation between RAPD-PCR and microsatellite genetic distance was highly significant ( R 2 = 0.77; P by Mantel test < 0.001). The molecular variability observed in both formae speciales is discussed in relation to the development of F.o.r.l. - and F.o.l. -specific diagnostic tools. [ABSTRACT FROM AUTHOR] more...

The inverse PCR technique was applied to clone genomic DNA flanking insertion sites of sequences homologous to the transposable element Fot1 in the genome of a new pathogenic isolate of Fusarium oxysporum obtained from wilted Argyranthemum frutescens (Paris daisy). Based on the genomic flanking regions, a primer was designed which when paired to a second primer matching the Fot1 sequence allowed detection of this pathogen by PCR. The primer pair Mg5/Mg6 could specifically identify nine tested isolates of F. oxysporum from A. frutescens, when fungal genomic DNA was used as template. Moreover, the primer pair Mg5/Mg6 allowed successful detection of the pathogen in stem and root tissue from asymptomatic plants that were artificially inoculated with a representative isolate of F. oxysporum from A. frutescens. [ABSTRACT FROM AUTHOR] more...