PREPARATION OF B-CYCLODEXTRIN-THIABENDAZOLE-PIPERONYL BUTOXIDE SUPRAMOLECULAR COMPLEX AND ITS ACTIVITY AGAINST BLUE AND GREEN MOULD DECAY ON INOCULATED SATSUMA FRUIT.

The supramolecular complex of ß-cyclodextrin-thiabendazole-piperony butoxide (ßCD-TBZ/PBO) was prepared and its structure was characterized by 1H NMR. Additionally, the antifungal activity of ßCD-TBZ/PBO was investigated in comparison with the commercially available thiabendazole (TBZ) fungicide by in vitro tests and on artificially inoculated 'Okitsu' satsuma fruit dipped in water at 20 degrees C or at 50 degrees C to control postharvest blue (Penicillium italicum) and green mould (P. digitatum). ß-CD-TBZ/PBO is stable for several months when stored as powder in a dark bottle. At pH 7.0 the water solubility of the ßCD-TBZ/PBO complex was consistently higher than free TBZ. Water dip at 20 degrees C did not affect decay incidence caused by blue mould but favoured the development of green mould during 4-8 days of storage at 20 degrees C with respect to untreated (control) fruit. Water at 50 degrees C effectively reduced the incidence of blue mould and totally suppressed green mould during the first 4 days but lost its efficacy afterwards. By contrast, both TBZ and ßCD-TBZ/PBO had a lasting effect and were equally effective in controlling green and blue mould decay when applied at 20 degrees C and 60 mg L(-1) active ingredient (a.i.). When applied at 50 degrees C and 20 mg L(-1) a.i. the activity of the complex against blue mould was inferior than the corresponding treatment with TBZ. In vitro assays revealed a significant effectiveness of ßCD-TBZ/PBO complex at low concentration compared to commercial formulation of TBZ.

Comparative analysis of proteome changes induced by the two spotted spider mite Tetranychus urticae and methyl jasmonate in citrus leaves.

Citrus plants are currently facing biotic and abiotic stresses. Therefore, the characterization of molecular traits involved in the response mechanisms to stress could facilitate selection of resistant varieties. Although large cDNA microarray profiling has been generated in citrus tissues, the available protein expression data are scarce. In this study, to identify differentially expressed proteins in Citrus clementina leaves after infestation by the two-spotted spider mite Tetranychus urticae, a proteome comparison was undertaken using two-dimensional gel electrophoresis. The citrus leaf proteome profile was also compared with that of leaves treated over 0-72h with methyl jasmonate, a compound playing a key role in the defense mechanisms of plants to insect/arthropod attack. Significant variations were observed for 110 protein spots after spider mite infestation and 67 protein spots after MeJA treatments. Of these, 50 proteins were successfully identified by liquid chromatography-mass spectrometry-tandem mass spectrometry. The majority constituted photosynthesis- and metabolism-related proteins. Five were oxidative stress associated enzymes, including phospholipid glutathione peroxidase, a salt stressed associated protein, ascorbate peroxidase and Mn-superoxide dismutase. Seven were defense-related proteins, such as the pathogenesis-related acidic chitinase, the protease inhibitor miraculin-like protein, and a lectin-like protein. This is the first report of differentially regulated proteins after T. urticae attack and exogenous MeJA application in citrus leaves.

Influence of post-harvest treatments with fludioxonil and soy lecithin co-application in controlling blue and grey mould and fludioxonil residues in Coscia pears.

The residue levels of fludioxonil (FLU) were determined in Coscia pear following a 1-, 2- or 4-min dip in an aqueous mixture of FLU containing 300 or 100 mg l(-1) (active ingredient, a.i.) at 20 and 50 degrees C, respectively, with or without 2% soy lecithin. The efficacy of heat treatment with water and FLU mixtures was investigated on artificially inoculated pears for the control of post-harvest decay caused by blue (Penicillium expansum Link) and grey (Botrytis cinerea Pers. ex Fr.) mould. Treatment with 300 mg l(-1) FLU at 20 degrees C increased residues significantly when treatment time rose from 1 to 2 min; no further increase was recorded when dip time raised from 2 to 4 min. FLU residue rates were unaffected by treatment time when 300 mg l(-1) a.i. was applied in combination with lecithin at 20 degrees C. While treatment with 100 mg l(-1) a.i. at 50 degrees C for 1 and 2 min resulted in similar residue levels, significantly higher residues were detected when dip time increased from 1 to 4 min. Co-application of lecithin significantly decreased FLU residues with respect to fruit treated with FLU alone. Treatments with FLU at 20 or 50 degrees C effectively controlled decay over 10 days of incubation. While co-application of lecithin did not affect the efficacy of FLU at 300 mg l(-1)and 20 degrees C, treatment efficacy decreased when lecithin was applied in combination with 100 mg l(-1) FLU and 50 degrees C for 4 min and to a greater extent when dip time was 1-2 min.

First Report of Fusarium oxysporum f. sp. canariensis Causing Fusarium Wilt on Phoenix canariensis in Sardinia, Italy.

During the summer of 2004, severe symptoms of wilt were observed on 25-year-old plants of Canary Island Date Palm (Phoenix canariensis Hort. ex Chabaud) located at the seafront of Poetto Beach in the metropolitan area of Cagliari, southern Sardinia, Italy. Symptoms consisted of one-sided leaflet dieback of fronds, necrotic and brown streaking on the lower rachis base of older leaves, and necrosis of vascular bundles. Of 300 palms, there were 90 plants that were symptomatic and at least 4 were dead. Fusarium oxysporum Schlecht. emend. Snyder & Hansen has been consistently isolated from surface-sterilized petioles of symptomatic leaves sampled from affected palms. The opportunistic pathogen Gliocladium vermoesenii (Biourge) Thom was frequently associated with F. oxysporum in diseased samples, confirming previous reports of a disease complex between these two fungi (1). Five F. oxysporum isolates collected from different symptomatic plants were analyzed with a polymerase chain reaction (PCR)-based assay with the F. oxysporum f. sp. canariensis-specific primers HK66 + HK67 (2). The thermocycling schedule was as follows: initial denaturation at 94°C for 5 min, 35 cycles each of 1 min at 94°C, 1 min at 62°C, 1 min and 30 s at 72°C, followed by a final extension at 72°C for 5 min. A 567-bp PCR product of the expected size was obtained from all tested F. oxysporum isolates, allowing their identification as F. oxysporum f. sp. canariensis. This disease was previously reported from other Italian regions (Sicily, Marche, and Liguria), but its presence in Sardinia should be considered carefully since it represents a serious threat to ornamental palms, which are abundant all over the island. The source of this outbreak may be related to the importation of seedlings from areas where F. oxysporum f. sp. canariensis is widely established. References: (1) H. D. Ohr. Pink rot (Gliocladium Blight). Pages 24-25 in: Diseases and Disorders of Ornamental Palms. A. R. Chase and T. K. Broschat, eds. The American Phytopathological Society, St. Paul, MN, 1991. (2) T. R. Plyler et al. Phytopathology 89:407, 1999.

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.

Transposition of the autonomous Fot1 element in the filamentous fungus Fusarium oxysporum.

Autonomous mobility of different copies of the Fot1 element was determined for several strains of the fungal plant pathogen Fusarium oxysporum to develop a transposon tagging system. Two Fot1 copies inserted into the third intron of the nitrate reductase structural gene (niaD) were separately introduced into two genetic backgrounds devoid of endogenous Fot1 elements. Mobility of these copies was observed through a phenotypic assay for excision based on the restoration of nitrate reductase activity. Inactivation of the Fot1 transposase open reading frame (frameshift, deletion, or disruption) prevented excision in strains free of Fot1 elements. Molecular analysis of the Nia+ revertant strains showed that the Fot1 element reintegrated frequently into new genomic sites after excision and that it can transpose from the introduced niaD gene into a different chromosome. Sequence analysis of several Fot1 excision sites revealed the so-called footprint left by this transposable element. Three reinserted Fot1 elements were cloned and the DNA sequences flanking the transposon were determined using inverse polymerase chain reaction. In all cases, the transposon was inserted into a TA dinucleotide and created the characteristic TA target site duplication. The availability of autonomous Fot1 copies will now permit the development of an efficient two-component transposon tagging system comprising a trans-activator element supplying transposase and a cis-responsive marked element.

Detection of Fusarium oxysporum f. sp. dianthi in Carnation Tissue by PCR Amplification of Transposon Insertions.

ABSTRACT Strains of the carnation wilt pathogen, Fusarium oxysporum f. sp. dianthi, can be distinguished by DNA fingerprint patterns, using the fungal transposable elements Fot1 and impala as probes for Southern hybridization. The DNA fingerprints correspond to three groups of F. oxysporum f. sp. dianthi strains: the first group includes isolates of races 1 and 8; the second group includes isolates of races 2, 5 and 6; and the third group includes isolates of race 4. Genomic DNAs flanking race-associated insertion sites of Fot1 (from races 1, 2, and 8) or impala (from race 4) were amplified by the inverse polymerase chain reaction (PCR) technique. These regions were cloned and sequenced, and three sets of primers overlapping the 3' or 5' end of the transposon and its genomic insertion were designed. Using fungal genomic DNA as template in PCR experiments, primer pairs generated amplification products of 295, 564 and 1,315 bp, corresponding to races 1 and 8; races 2, 5, and 6; and race 4, respectively. When multiplex PCR was performed with genomic DNA belonging to races 1 and 8, 2, or 4, single amplimers were generated, allowing clear race determination of the isolate tested. PCR was successfully performed on DNA extracted from susceptible carnation cv. Indios infected with isolates representative of races 1, 2, 4, and 8.

Transformants of Trichoderma longibrachiatum Overexpressing the beta-1,4-Endoglucanase Gene egl1 Show Enhanced Biocontrol of Pythium ultimum on Cucumber.

ABSTRACT Nine transformants of Trichoderma longibrachiatum with extra copies of the egl1 gene were studied for mitotic stability, endoglucanase production, and biocontrol activity against Pythium ultimum on cucumber seedlings. The transformants showed a significantly higher level of expression of the egl1 gene in comparison to the wild type under both inducing and noninducing growth conditions. Transformants with the egl1 gene under the control of a constitutive promoter had the highest enzymatic activity. Both the endoglucanase activity and the transforming sequences were stable under nonselective conditions. When applied to cucumber seeds sown in P. ultimum-infested soil, T. longibrachiatum transformants with increased inducible or constitutive egl1 expression generally were more suppressive than the wild-type strain.