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Applied and Environmental Microbiology, April 2008, p. 2248-2253, Vol. 74, No. 7
0099-2240/08/$08.00+0 doi:10.1128/AEM.01998-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.
Inhibition of Species of the Aspergillus Section Nigri and Ochratoxin A Production in Grapes by Fusapyrone
Mara Favilla,1
Michelangelo Pascale,1
Alessandra Ricelli,1
Antonio Evidente,2
Carmine Amalfitano,2 and
Claudio Altomare1*
Institute of Sciences of Food Production, CNR, 70125 Bari, Italy,1
Dipartimento di Scienze del Suolo, della Pianta, dell'Ambiente, e delle Produzioni Animali, Università di Napoli Federico II, 80055 Portici, Italy2
Received 31 August 2007/
Accepted 30 January 2008
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ABSTRACT
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Fusapyrone (FP), an antifungal natural compound, was tested against the three main ochratoxigenic species of the Aspergillus section Nigri. The MICs at 24 h were 6.0, 11.6, and 9.9 µg/ml for Aspergillus carbonarius, Aspergillus tubingensis, and Aspergillus niger, respectively. Strong inhibition of growth and morphological changes were still observed at half the MIC after 7 days. The application of a 100 µg/ml FP solution in a laboratory assay on artificially inoculated grapes resulted in a significant reduction (up to 6 orders of magnitude) of A. carbonarius CFU counts. Dramatic reductions of the ochratoxin A (OTA) content, compared to the content of the positive control (average amount of OTA, 112.5 ng/g of grape; three experiments), were obtained with the application of either 100 or 50 µg/ml of FP (0.6 or 5.1 ng/g of grape, respectively).
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INTRODUCTION
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Ochratoxin A (OTA) is a mycotoxin with nephrotoxic, teratogenic, immunosuppressive, and carcinogenic properties (38) that has been classified by the International Agency for Research on Cancer as a possible human carcinogen (group 2B) (22). OTA was first isolated from moldy cornmeal in South Africa (37). Subsequently, OTA has been found in a number of agricultural commodities and foodstuffs, including cereals, coffee beans, and beer, as a by-product of contamination with fungi of the genera Aspergillus and Penicillium, mainly Aspergillus ochraceus (also known as Aspergillus alutaceus) and Penicillium verrucosum. In recent years, there has been a growing interest in the occurrence of OTA in grapes and grape derivatives. In particular, concern has been raised for OTA contamination of wine grapes, due to the large and increasing consumption of wine and the economical relevance of wine industry (5, 11, 36, 41). Several reports have indicated that members of the Aspergillus section Nigri, the so-called black aspergilli, are the dominant ochratoxigenic species on wine grapes worldwide (7, 10, 13, 27). Among these species, Aspergillus carbonarius seems to be the most important source of OTA because of the high proportion of producing strains and high amounts produced (6, 30). A correlation has been found between the occurrence of OTA in wines and both wine color and geographic area of production. The risk of OTA contamination and the concentration of toxin are higher in red wines and in wines made with grapes from Mediterranean and subtropical areas (29, 41). There are evidences that grapes are already contaminated with OTA before harvest (6, 9, 28, 34), although its concentration may increase substantially in the time between harvest and alcoholic fermentation (19, 41). OTA is a rather stable molecule (33), and it does not undergo substantial degradation in the course of the technological process of winemaking (1, 12). Nevertheless, experiments of microvinification aimed at studying the fate of OTA during winemaking have shown that the levels of OTA drop by around 90% from must to wine, mostly as a result of its adsorption onto biomass and pomace (residual solid parts) surfaces (1, 12, 26, 32). In spite of this drop, severe infestations of raw materials with black aspergilli may result in the contamination of the final product with OTA at levels above the allowed limits (2 µg/liter in the European Union). Therefore, management of the sanitary state of grapes is a critical point in a strategy aimed at the prevention of OTA occurrence in wine. Unfortunately, very few chemical pesticides seem to be effective (27, 36). In addition, the intensive use of these compounds may cause different important drawbacks, such as a loss of natural competitors, onset of resistant pathogen populations, and the presence of residues in the products and in the environment. In this context, the availability of alternative methods to control black aspergilli would be highly desirable.
A relatively novel and promising field of study is the application of antimicrobial compounds of microbial origin as an alternative to synthetic pesticides (35). Fusapyrone (FP) is a bioactive metabolite produced by the fungus Fusarium semitectum (16). Structurally, FP consists of a highly functionalized aliphatic chain and a 4-deoxy-β-xylo-hexopyranosyl C-glycosyl moiety bound to the C-6 of the 2-pyrone ring (Fig. 1). FP exhibited considerable antifungal activity against several plant pathogenic, mycotoxigenic, and human pathogenic filamentous fungi, including Aspergillus flavus, Aspergillus parasiticus, Aspergillus niger, and Aspergillus fumigatus (3). The inhibitory activity of FP to A. flavus and A. parasiticus was similar to that of the antibiotic nystatin in disk diffusion assays (3) and higher than that of the fungicides benomyl and dicloran on A. parasiticus in broth dilution assays (4). Interestingly, FP was found to be ineffective toward yeasts isolated from plants and fruits and not toxic to Artemia salina (brine shrimp), a common invertebrate model in ecotoxicological testing (3, 4). In consideration of these features, we examined the ability of FP to inhibit the growth of the main ochratoxigenic species of Aspergillus section Nigri, viz., A. carbonarius, A. niger, and Aspergillus tubingensis, and to prevent fungal colonization and OTA production in grapes.
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Fusapyrone.
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FP was purified from crude corn culture extracts of F. semitectum by SiO2 medium-pressure (20 bar) column chromatography and preparative SiO2 thin-layer chromatography, following the method described previously by Evidente et al. (16). The purity of FP as determined by high-pressure liquid chromatography was higher than 97% (17).
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Inhibitory activity of FP to ochratoxigenic Aspergillus spp.
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The antifungal activity of FP was tested against 16 Aspergillus isolates belonging to three different ochratoxigenic species, viz., A. carbonarius, A. niger, and A. tubingensis. The test isolates were obtained from the collection of the Institute of Sciences of Food Production, Bari, Italy, and were originally isolated from grapes in several countries of the Mediterranean basin (Table 1). The MIC of FP to ochratoxigenic aspergilli was determined by a broth dilution method (18). Sterile Czapek Dox broth (Difco, Detroit, MI), at pH 7.0 (35 g of lyophilized medium dissolved in 1 liter of 0.05 M phosphate buffer, pH 7.0, and sterilized in the autoclave for 20 min at 115°C), was used for the preparation of serial dilutions of FP. Eleven twofold dilutions (ranging from 50 to 0.05 µg/ml) were prepared; 180 µl of each dilution and medium control was then transferred in duplicate into wells of 96-well microtitration plates. The inoculum of the fungi was obtained from fresh (7-day old) cultures on potato dextrose agar. Each microwell was inoculated with 20 µl of a conidial suspension of the test isolates at the concentration of 105 conidia/ml in sterile Czapek Dox broth, pH 7.0. Plates were sealed with Parafilm (Pechiney, Chicago, IL) in order to prevent evaporation and incubated at 26 ± 1°C for 7 days. Growth was observed 1, 2, 3, and 7 days after inoculation under a reverse microscope. Conidia were assumed to not be germinated if the germ tube was shorter than the conidium diameter. The MIC was identified as the lowest concentration that resulted in complete inhibition of germination of the test fungus. Antifungal tests were performed three times.
The in vitro inhibitory activity of FP to OTA-producing aspergilli is summarized in Table 1. The MIC method is subject to inherent variability, and therefore, the procedure is generally considered accurate within ±1 twofold dilution (39). The data are presented in Table 1 in the form of a MIC range for each isolate tested. In addition, average MICs of different species were calculated to allow for comparisons of susceptibility. Different susceptibilities to FP of the species tested were found. A. carbonarius was the most sensitive, whereas A. niger and A. tubingensis were less susceptible. Inhibitory effects (reduction of percentage of germinated conidia and germ tube length) were also found at concentrations lower than MICs. Strong inhibition of growth and morphological changes were still observed at half the MIC (sub-MIC) after 7 days (Fig. 2). Under these conditions, germ tubes of A. niger and A. tubingensis exhibited severe thickening and irregular growth. The phenotype of these conidia resembled the phenotype described previously by Ram et al. (31) for A. niger transformants lacking the glutamine-fructose-6-phosphate amidotransferase gene (gfaA) encoding the enzyme responsible for the first step in chitin synthesis. Abnormal swelling of cells and conidia was observed in all of the A. tubingensis isolates, but not in isolates of the other two species. The swollen cells were up to 10 times bigger than normal cells (Fig. 2). Katoh et al. (23) reported an abnormal swelling of A. niger conidia that germinated in the presence of the antibiotic tunicamycin, similar to that observed in A. tubingensis. Tunicamycin is a nonspecific inhibitor of chitin synthesis that acts by blocking the synthesis of a lipid-linked saccharide intermediate involved in protein-chitin complexes (21). Based on the amphiphilic nature of FP, it was hypothesized that FP has an effect on plasma membrane function and integrity, similar to that of antifungal biosurfactants (4). The results of the present work suggest that FP also interferes with chitin synthesis and cell wall structural integrity. This mechanistic hypothesis is supported by the relative insensitivity of yeasts to FP (3). Chitin is a major component in filamentous fungi cell walls, accounting for up to 10 to 30% of the cell wall dry weight (15), while it is only 1 or 2% of the weight in yeast cell walls (25). Should this multiple mechanism of action be confirmed by further studies, the probability that resistance to FP arises in populations of black aspergilli can be predicted to be low. In fact, in this case, the development of resistance might require major structural change of the plasma membrane and cell wall. Variation in sensitivity exhibited by different species in the Aspergillus section Nigri is likely due to diversity in cell wall structure, composition, and permeability to FP as well as to a variable fatty acid composition of plasma membranes (20). Differences in the cell wall composition of species in the Aspergillus section Nigri, besides being a useful character for taxonomy, systematics, and phylogeny studies (2, 8) of this group, might also have practical implications. Some fungicides (e.g., organophosphorus and carboxylic acid amides) act as inhibitors of phospholipids and cell wall component biosynthesis and deposition. Therefore, it is conceivable that variations in plasma membrane and cell wall composition may result in different efficacies of these fungicides in controlling of different ochratoxigenic species.
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FIG. 2. Effects of sublethal concentrations (sub-MIC) of fusapyrone on the growth of germinated conidia of A. carbonarius, A. niger, and A. tubingensis. Germinated conidia of A. niger and A. tubingensis showed severe alterations of morphology that consisted of thickening and irregular growth of hyphae that formed pronounced bulges. Abnormal swelling of cells was observed in the A. tubingensis isolates. (Top) Controls at 48 h. (Middle) Half MIC, 48 h of exposure. (Bottom) Half MIC, 7 days of exposure. Bar = 100 µm.
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Bioassay on artificially inoculated grape berries.
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Bunches of red wine grapes of the Negroamaro variety were collected from vineyards located in the Salento area (southern Apulia, Italy) 7 to 10 days before the regular harvest date. In the laboratory, berries were excised by cutting pedicels 0.5 cm above their points of insertion on the berries and washed thoroughly in tap water to remove dust and pesticide residue. Berries were surface sterilized with 2% sodium hypochlorite for 15 min, repeatedly rinsed in sterile distilled water, and let dry on blotting paper under a fume hood for 2 h. Well-formed and undamaged berries were selected for use in the bioassay. The inoculum of the OTA-producing strain A. carbonarius ITEM 4167 was prepared by flooding 7- to 10-day-old cultures on potato dextrose agar with sterile distilled water, and the suspension was adjusted with sterile distilled water to 5 x 104 conidia/ml by using a hemocytometer. Berries were wounded with a sterile needle (2 mm deep) in two symmetrical abaxial points and sprayed with sterile distilled water (negative control) or the conidial suspension of A. carbonarius ITEM 4167 using an EcoSpray sprayer (Labo Chimie, Aix-en-Provence, France). Enough suspension was sprayed to cover the surface of the berries without dripping. Grapes were allowed to dry under a sterile hood, and then A. carbonarius-inoculated grapes were sprayed with a solution of FP in sterile distilled water at a concentration of either 100 or 50 µg/ml. Grapes sprayed with only the A. carbonarius suspension were used as a positive control. Grapes were then transferred into moist chambers (10 berries per chamber), consisting of plastic food containers (159 by 114 by 54 mm) with moist filter paper to ensure high humidity, which were individually placed in polypropylene bags and sealed. The chambers were incubated at 26 ± 1°C for 10 days. Experiments were arranged in a fully randomized block design, with at least three replicates per treatment. Samples from these experiments were used for assessment of the treatment efficacy in inhibiting A. carbonarius growth and for OTA analyses.
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Inhibition of A. carbonarius by FP on artificially inoculated grape berries.
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Growth of the OTA-producing strain A. carbonarius ITEM 4167 in artificially inoculated grapes was evaluated by determination of CFU. Grapes from each replicate (20 to 30 g) were transferred into centrifuge tubes and weighted. After an equal weight of sterile distilled water was added, grapes were homogenized in a blender (Sterilmixer II; International PBI, Milan, Italy) at high speed for 1 min, transferred into the tubes again, and shaken on an orbitary shaker at 180 rpm for 1 h. Subsequently, three subsamples of 1 g were drawn from each tube and used to prepare serial 1/10 dilutions in sterile distilled water. From each dilution, three 100-µl samples were plated on an Aspergillus-selective medium (24) containing (per liter) 10 g glucose, 5 g peptone, 1 g K2HPO4, 15 g agar, 25 mg rose bengal, 2 mg dicloran, and 100 mg chloramphenicol. Plates were incubated at 26 ± 1°C, and CFU were counted in two adjacent dilutions with less than 100 CFU per plate after 2 and 5 days. Counts were corrected by the dilution factor and averaged to give CFU/g of grape pulp.
Three independent trials were carried out in which FP was applied at both 100-µg/ml and 50-µg/ml rates. Interestingly, in these experiments, A. carbonarius was isolated from the surface-sterilized berries of negative controls. This result suggests endophytic behavior of the pathogen, which may be of importance for optimizing an effective field control strategy. The variability among different experiments in the development of A. carbonarius infections and in the level of natural contamination of the negative control prompted us to evaluate the results of these experiments separately (Table 2). In spite of the above variability, a consistent trend was found among different experiments. FP applied at either a 100- or a 50-µg/ml rate resulted in a significant (P < 0.05) reduction of A. carbonarius infections. The treatment of berries with a solution of FP at 100 µg/ml resulted in a reduction of A. carbonarius biomass from 2 to 6 orders of magnitude. In two out of three experiments, the reduction of A. carbonarius growth achieved with FP at 50 µg/ml was smaller but not statistically different (P < 0.05) from that achieved with FP at 100 µg/ml.
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Effect of FP on OTA content.
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Grape samples (20 to 30 g) were homogenized by blending at high speed for 3 min with a Sorvall Omnimixer (Sorvall, Inc., Newtown, CT). The homogenized slurry (20 g) was added to 50 ml of a solution containing 1% polyethylene glycol 8000 and 5% sodium bicarbonate, cleaned up through an OchraTest immunoaffinity column (Vicam, Watertown, MA), and analyzed for OTA content by high-pressure liquid chromatography/fluorescence detector as described previously and extensively by Cozzi et al. (14). Average recoveries of OTA from grapes spiked at levels from 1.0 to 10 ng/g ranged from 80% to 85%, with relative standard deviations of <2.5% (triplicate experiments). The detection limit was 0.02 ng/g, based on a signal-to-noise ratio of 3:1.
The data of OTA content in artificially inoculated grapes treated with FP are shown in Table 2. In all three experiments, berries treated with either 100 or 50 µg/ml of FP showed a significant (P < 0.05) reduction of OTA content compared to the level for the positive control. The treatment with 100 µg/ml of FP prevented the accumulation of OTA in the berries to a level comparable to that for the negative control (0.01 to 1% of the positive control). The reduction of OTA content obtained with FP at 50 µg/ml was apparently smaller (0.3 to 20% of the positive control), although not statistically different, than the OTA content found in grapes treated with FP at 100 µg/ml.
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Statistical analyses.
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All statistical analyses were performed with the InStat program, version 3.0 (GraphPad Software, San Diego, CA).
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Conclusions.
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FP proved to possess a strong inhibitory activity toward three ochratoxigenic Aspergillus species belonging to the section Nigri that are the major source of OTA in grapes and grape-derived foods and beverages.
We tested the effectiveness of FP on artificially inoculated grapes in conditions that were highly conducive to mold development, viz., skin injuries, a high level of inoculum, absence of competitors, and high relative humidity. FP applied at a rate of 100 µg/ml almost completely controlled A. carbonarius. In grapes treated with a half dosage (50 µg/ml) of FP, the average level of control was lower but not statistically different from the level with the full dosage. Therefore the application rate of 50 µg/ml of FP appears to be enough to achieve satisfactory control of A. carbonarius under the most adverse conditions. Dramatic reductions of the OTA content, compared to the level for the positive control (the average amount of OTA in three independent experiments was 112.5 ng/g of grape), were obtained with application of either 100 or 50 µg/ml of FP (0.6 or 5.1 ng/g of grape, respectively).
In conclusion, our results show that FP is highly effective in inhibiting the growth of black aspergilli, particularly A. carbonarius, and preventing OTA occurrence in infected grape berries. These findings warrant further studies to assess whether the use of FP is a feasible strategy for the prevention of OTA occurrence in grapes and grape-derived products under field conditions.
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ACKNOWLEDGMENTS
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This work was supported by the Italian Ministry of University and Research, projects MAIA (1.1 Microorganisms and Microbial Metabolites in Plant Protection [law 488/92, cluster C06+07]) and SIVINA (Individuazione di metodologie innovative prontamente trasferibili per migliorare la sicurezza dei vini rossi di qualità del Salento [project no. 12818]).
We thank M. Marzano and G. Panzarini for their valuable assistance.
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FOOTNOTES
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* Corresponding author. Mailing address: Istituto di Scienze delle Produzioni Alimentari, CNR, Via Giovanni Amendola 122/O, 70125 Bari, Italy. Phone: 39 80 592 9318. Fax: 39 80 592 9374. E-mail: claudio.altomare{at}ispa.cnr.it 
Published ahead of print on 8 February 2008.
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Applied and Environmental Microbiology, April 2008, p. 2248-2253, Vol. 74, No. 7
0099-2240/08/$08.00+0 doi:10.1128/AEM.01998-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.
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ABSTRACT
INTRODUCTION
