Induction of cutinolytic esterase activity during saprophytic growth of cucurbit pathogens, Fusarium solani f. sp. cucurbitae races one and two (Nectria haematococca MPI and MPV, respectively).

Cutins from fruit of Cucurbita maxima and Cucurbita moschata cultivars, apple and a C(16) alcohol (hexadecanol) were used to induce cutinolytic esterase activity during saprophytic growth of strains of the two cucurbit pathogens, Fusarium solani f. sp. cucurbitae, race 1 (Nectria haematococca mating population (MPI) and F. solani f. sp. cucurbitae, race 2 (MPV). Four strains of MPV and 11 strains of MPI were were included in the study. Although we were primarily interested in the two cucurbit pathogens (MPI and MPV), six strains of the pea pathogen F. solani f. sp. pisi (MPVI) were included to provide a comparison since most of the knowledge on cutinase activity in N. haematococca has come from a study of that group. Cutinolytic esterase was induced in all strains from both MPV and MPVI but was not detected in any of the 11 strains from MPI regardless of the induction conditions. The amount of cutinolytic esterase activity induced in the MPV strains differed according to the strain and both the source and the amount of cutin used in the induction medium. Information on the influence of cutin source and pH on the induction of cutinolytic esterase activity during saprophytic growth of strains from MPV demonstrates that the gene is regulated differently from that in MPVI.

Effect of disruption of a cutinase gene (cutA) on virulence and tissue specificity of Fusarium solani f. sp. cucurbitae race 2 toward Cucurbita maxima and C. moschata.

A 3.9-kb genomic DNA fragment from the cucurbit pathogen Fusarium solani f. sp. cucurbitae race 2 was cloned. Sequence analysis revealed an open reading frame of 690 nucleotides interrupted by a single 51-bp intron. The nucleotide and predicted amino acid sequences showed 92 and 98% identity, respectively, to those of the cutA gene of the pea pathogen F. solani f. sp. pisi. A gene replacement vector was constructed and used to generate cutA- mutants that were detected with a polymerase chain reaction (PCR) assay. Seventy-one cutA- mutants were identified among the 416 transformants screened. Vector integration was assessed by Southern analysis in 23 of these mutants. PCR and Southern analysis data showed the level of homologous integration was 14%. Disruption of the cutA locus in mutants was confirmed by RNA gel blot hybridization. Neither virulence on Cucurbita maxima cv. Delica at any of six different inoculum concentrations, nor pathogenicity on intact fruit of four different species or cultivars of cucurbit or hypocotyl tissue of C. maxima cv. Crown, was found to be affected by disruption of the cutA gene.

Differentiation of Fusarium solani f. sp. cucurbitae races 1 and 2 by random amplification of polymorphic DNA.

We have used a PCR-based technique, involving the random amplification of polymorphic DNA (RAPD), to assess genome variability between 21 isolates from F. solani f. sp. cucurbitae races 1 and 2. Based on RAPD marker patterns the isolates fell into two distinct groups corresponding to mating populations MPI and MPV. Four isolates that could not be assigned to one or other mating population by traditional means were distinguished by RAPD patterns. Seven polymorphic RAPD products were used to probe Southern blots of MPI and MPV genomic DNA. Six of the seven probes hybridized to single-copy sequences and five of the seven probes showed specificity for one or other mating population. We suggest that not only is the technique a rapid and reliable tool for isolate-typing of fungi but it also provides a rapid method for obtaining species- or race-specific hybridization probes.