Fusarium moniliforme secretes four endopolygalacturonases derived from a single gene product

Extracellular endopolygalacturonase, purified from the pathogenic fungus Fusarium moniliforme, consists of four molecular forms (38, 41·5, 45, and 48·5 kDa, respectively. Three forms (38, 41·5, and 45 kDa) were purified to homogencity by FPLC on a Mono S column followed by electroelution after SDS-PAGE. The N-terminal amino acid sequences of each of the three forms, and of a mixture containing all four forms were shown to be identical to that predicted from the nucleotide sequence of the endopolygalacturonase gene previously cloned from F. moniliforme. Enzymatic deglycosylation experiments revealed the presence of N-linked, high mannose oligosaccharide side-chains on all four forms of endo polygalacturonase. Hydrogen fluoride catalysed chemical deglycosylation of the polygalacturonase mixture yielded a single polypeptide with an apparent molecular mass of 36·2 kDa. Southern blot analysis, carried out at high stringency with an endopolygalacturonase-specific probe on genomic DNA digested with three different restriction enzymes, showed a single hybridizing restriction fragment in all three digests. A single 2·0 Mb chromosome hybridized with the endo polygalacturonase-specific probe, as shown by Southern blot analysis of F. moniliforme chromosomes separated by CHEF electrophoresis. Northern blot analysis revealed only one mRNA species 1350 nt encoding endo polygalacturonase. These data indicate that a single gene encodes the endopolygalacturonases of F. moniliforme.     

Use of random amplified polymorphic DNA (RAPD) to identify races 1, 2, 4 and 8 of Fusarium oxysporum f. sp. dianthi in Italy

The RAPD fingerprinting procedure was used in combination with pathogenicity assays on differential cultivars to characterize a representative collection of 72 Fusarium spp. isolates of different geographic origin collected from diseased carnation. In F. oxysporum f. sp. dianthi, isolates were grouped according to the physiologic race: group 1 included isolates of race 4; group 2 was formed by isolates of race 2 and single representatives of races 5 and 6; group 3 included isolates of races 1 and 8. No correlation was found between RAPD data and geographic origin of the isolates tested: representatives of race 2 isolated in Italy, Israel and Japan had the same amplification profile. Three isolates which showed a low level of pathogenicity on all carnation cultivars tested shared an identical amplification pattern and are probably saprophytic F. oxysporum. Finally, two F. redolens isolates from Japan and seven non-pathogenic isolates of F. proliferatum collected from diseased carnation in Italy, Israel and The Netherlands were clearly distinguishable according to their RAPD fingerprint. The results are discussed in relation to previous studies on the genetic diversity of F. oxysporum f. sp. dianthi and to the development of forma specialis- and pathotype-specific diagnostic tools.     

Residue level, persistence, and storage performance of citrus fruit treated with fludioxonil

The potential of postharvest dip treatments with fludioxonil (FLU) (a synthetic analogue of the bacterial metabolite of pyrrolnitrin), in controlling postharvest decay caused by Penicillium digitatum and Penicillium italicum of citrus fruit was investigated in comparison with the conventional fungicide imazalil (IMZ). The ultrastructural changes of fruit epicuticular wax was investigated as a function of water dip temperature, and the possible role of these changes was related to residue accumulation under FLU treatment. Residues retained by fruit were determined as a function of fungicide concentration, dip temperature, and fruit storage conditions. Scanning electron microscopy analysis revealed that fruit dipping in water at 30 or 40 degrees C did not cause differences in cuticular wax's ultrastructure in comparison to control fruit, while treatments at 50, 55, or 60 degrees C caused the disappearance of wax platelets, resulting in relatively homogeneous skin surface, due to partial "melting" of epicuticular wax. Residues of FLU in fruit treated at 20 or 50 degrees C were significantly correlated with the doses of fungicide applied. When equal amounts of fungicide were employed, the residue concentrations were notably higher (from 2.6- to 4-fold) in fruit treated at 50 degrees C than in fruit treated at 20 degrees C. The dissipation rate of FLU in "Salustiana" and "Tarocco" oranges was lower in fruit subjected to treatment at 50 degrees C. The minimal FLU concentration for almost complete decay control in artificially wounded fruit during 7-d storage at 20 degrees C was 400 mg/L active ingredient (ai) in fruit treated at 20 degrees C and 100 mg/L ai in fruit treated at 50 degrees C. Results on nonwounded Tarocco oranges subjected to 3 weeks of simulated quarantine conditions at 1 degrees C, plus 6 weeks of standard storage at 8 degrees C and an additional two weeks of simulated marketing period (SMP) at 20 degrees C revealed that almost complete decay control with FLU applications of 100 mg/L at 50 degrees C and 400 mg/L at 20 degrees C resulted in ca. 0.8 mg/kg FLU fruit residues, in agreement with results on wounded citrus fruit. When equal concentrations and temperatures were applied, FLU treatments were as effective as IMZ. In vitro trials showed a low sensitivity to FLU against P. digitatum and P. italicum isolates. MIC values for the complete inhibition of mycelium growth were >or=100 microg/mL, while ED(50) values ranged from 0.1 to 1 microg/mL for P. digitatum and from 1 to >100 microg/mL for P. italicum. The latter result suggests that care should be taken to avoid exclusive application of FLU in a sustainable program for management of fruit decay. However, integrating fungicide application and hot water dip may reduce the possibility of selecting fungicide-resistant populations of the pathogen, by increasing the effectiveness of the treatment.     

Residue levels and effectiveness of pyrimethanil vs imazalil when using heated postharvest dip treatments for control of Penicillium decay on citrus fruit

The influence of fungicide concentration and treatment temperature on residue levels of pyrimethanil (PYR) in comparison with the commonly used fungicide imazalil (IMZ) was investigated in orange fruits following postharvest dip treatments. The dissipation rate of PYR residues was recorded as a function of storage conditions. The fungicide efficacy against green and blue molds caused by Penicillium digitatum and Penicillium italicum, respectively, was evaluated on different citrus varieties following the fungicide application at 20 or 50 degrees C. Residue levels of PYR in Salustiana oranges were significantly correlated with the fungicide dosage, but residue concentrations were notably higher (ca. 13-19-fold) after treatment at 50 degrees C as compared to treatments at 20 degrees C. After treatment at temperatures ranging from 20 to 60 degrees C, PYR and IMZ residues in Salustiana oranges were significantly correlated with dip temperatures. Dissipation rates of PYR during storage were negligible in both Salustiana and Tarocco oranges. Results obtained on wounded, noninoculated Miho satsumas revealed that when treatments were performed at 50 degrees C, PYR or IMZ concentrations needed to achieve the complete control of decay were 8- and 16-fold less than by treatment at 20 degrees C. When fruits were inoculated with either P. digitatum or P. italicum, the application of 400 mg L(-1) PYR at 20 degrees C or 100 mg L(-1) PYR at 50 degrees C similarly reduced green and blue mold development. These results were corroborated by storage trials on Marsh grapefruits and Tarocco oranges. The lowest concentration of PYR required to achieve almost total protection of the fruit against decay accounted for 100 mg L(-1) at 50 degrees C and 400 mg L(-1) at 20 degrees C, respectively. Treatments did not affect fruit external appearance, flavor, and taste. It is concluded that postharvest PYR treatment represents an effective option to control green and blue mold in citrus fruit and that integration of fungicide applications and hot water dips may reduce the possibility of selecting fungicide-resistant populations of the pathogen, as a consequence of increased effectiveness of the treatment.     

Use of a complexation of tebuconazole with beta-cyclodextrin for controlling foot and crown rot of durum wheat incited by Fusarium culmorum

The methodology for the inclusion of tebuconazole (TBC) in beta-cyclodextrin (betaCD), spectroscopic characterization of the inclusion complex, and its activity for the control of a major soilborne disease of wheat caused by Fusarium culmorum are reported. Controlled release measured by chemical shift of the diagnostic protons H(3) and H(5) of betaCD confirmed stability of the complex at the solid state and in aqueous solution. Greenhouse and field experiments were conducted on durum wheat (Triticum durum cv. Prometeo) sown in substrate or in soil artificially infested with a virulent strain of F. culmorum. The inclusion complex betaCD-TBC, applied as seed dressing in combination or not with carboxymethylcellulose, reduced the disease incidence caused by F. culmorum and improved grain yield, showing effects that were generally comparable to those observed upon application of a commercial formulation of TBC. In the field experiment, only seed treatment with the inclusion complex betaCD-TBC allowed yield that was not different from that obtained from the uninoculated control. These results prove that by the use of the betaCD-TBC complex it is possible to obtain release of TBC and bioavailabilty of the fungicide without compromising its effectiveness.     

Development of real-time PCR systems based on SYBR? Green I and TaqMan? technologies for specific quantitative detection of Phoma tracheiphila in infected Citrus

Real-time PCR assays based on SYBR? Green I and TaqMan? technologies were developed for in planta detection and quantification of Phoma tracheiphila, the mitosporic fungus causing ‘mal secco’ disease on citrus. Primers and a hybridization probe were designed on the basis of the internal transcribed spacer (ITS) region of the nuclear rRNA genes. The real-time PCR assays were compared with a classic isolation method in two separate experiments carried out on 6 and 24 month-old sour orange seedlings, artificially inoculated with a conidial suspension of the pathogen. Both technologies made it possible to follow the progression of infection by P. tracheiphila, enabling detection and quantification of the target fungus prior to the development of symptoms. The detection limit was 10 copies of the cloned target sequence and 15 pg of genomic DNA extracted from fungal spores. The values of the cycle threshold (Ct) were linearly correlated with the concentration of the target DNA, indicating that the method is suitable as a qualitative and quantitative assay. The presence of non-target fungal DNA had no effect on the specificity of the assay, but resulted in a 10-fold reduction of sensitivity. Total inhibition of the reaction occurred when conidia of the target pathogen were mixed with an organic soil substrate before extracting DNA using the standard protocol, while an alternative purification kit resulted in a significant decrease in sensitivity. Compared to classic methods, real-time PCR proved faster and easier to perform and showed a higher sensitivity. These results suggest that real-time PCR, based on both chemistries, has a great potential for early diagnosis of ‘mal secco’ disease and for quantitative estimation of fungal growth within host tissue.     

Development of PCR Primers for a New Fusarium oxysporum Pathogenic on Paris Daisy (Argyranthemum frutescens L.)

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.     

Characterisation of <Emphasis Type="Italic">Phoma tracheiphila</Emphasis> by RAPD-PCR,microsatellite-primed PCR and ITS rDNA sequencing and development of specific primers for <Emphasis Type="Italic">in planta</Emphasis> PCR detection

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 (98–100% 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 10 pg of genomic DNA and 5 fg of the ITS target sequence.     

Recovery of Mutants Impaired in Pathogenicity After Transposition of Impala in Fusarium oxysporum f. sp. melonis

ABSTRACT The ability of transposon impala to inactivate genes involved in pathogenicity was tested in Fusarium oxysporum f. sp. melonis. Somatic excision of an impala copy inserted in the nitrate reductase-encoding niaD gene was positively selected through a phenotypic assay based on the restoration of nitrate reductase activity. Independent excision events were analyzed molecularly and shown to carry reinsertedimpala in more than 70% of the cases. Mapping of reinserted impala elements on large NotI-restriction fragments showed that impala transposes randomly. By screening 746 revertants on plants, a high proportion (3.5%) of mutants impaired in their pathogenic potential was recovered. According to the kinetics of wilt symptom development, the strains that were impaired in pathogenicity were clustered in three classes: class 1 grouped two strains that never induced Fusarium wilt symptoms on the host plant; class 2 and class 3 grouped 15 and 9 revertants which caused symptoms more than 50 and 30 days after inoculation, respectively. The first results demonstrate the efficiency of transposition in generating mutants affected in pathogenicity, which are usually difficult to obtain by classical mutagenesis, and open the possibility to clone the altered genes with impala as a tag.