Identification of Volatile Metabolites from Five Fungal Species Cultivated on Two Media

Five fungal species, Aspergillus versicolor, Penicillium commune, Cladosporium cladosporioides, Paecilomyces variotii, and Phialophora fastigiata, were cultivated on two media, malt extract agar and dichloran glycerol agar. Culture flasks provided with inlet and outlet tubes were used and purified, and humidified air was constantly led through the flasks. Air samples from the cultures were sorbed on Tenax GR and analyzed by thermal desorption-gas chromatography. The produced volatile metabolites were analyzed by mass spectrometry. Various hydrocarbons, alcohols, ketones, ethers, esters, sulfur-containing compounds, and terpenes were identified. The most commonly produced substances were 2-methyl-1-propanol, 2-methyl-1-butanol, 3-methyl-1-butanol, 3-methylfuran, and dimethyl disulfide. The production was highly dependent on both medium and species.     

Selective medium for isolation of Fusarium species and dematiaceous hyphomycetes from cereals

A selective medium containing 2 micrograms of dichloran per ml, 200 micrograms of chloramphenicol per ml, and 1.5% bacteriological peptone was developed for the isolation of Fusarium species and dematiaceous hyphomycetes from cereals. The medium, designated DCPA, was shown to select against species of Aspergillus, Penicillium, Cladosporium, and mucoraceous fungi. DCPA was evaluated for use as an enumeration medium and compared satisfactorily with dichloran-rose bengal-chloramphenicol agar when both media were tested with a range of cereal samples. Fusarium species and dematiaceous hyphomycetes produced well-formed colonies with good conidial production on DCPA, permitting rapid identification of such isolates on this medium.     

Selective medium for isolation of Fusarium species and dematiaceous hyphomycetes from cereals.

A selective medium containing 2 micrograms of dichloran per ml, 200 micrograms of chloramphenicol per ml, and 1.5% bacteriological peptone was developed for the isolation of Fusarium species and dematiaceous hyphomycetes from cereals. The medium, designated DCPA, was shown to select against species of Aspergillus, Penicillium, Cladosporium, and mucoraceous fungi. DCPA was evaluated for use as an enumeration medium and compared satisfactorily with dichloran-rose bengal-chloramphenicol agar when both media were tested with a range of cereal samples. Fusarium species and dematiaceous hyphomycetes produced well-formed colonies with good conidial production on DCPA, permitting rapid identification of such isolates on this medium.     

Diagnostic characterization of Penicillium palitans, P. commune and P. solitum

A total of 98 isolates of Penicillium commune and P. solitum were analysed and it was shown that these isolates produced three unique combinations of secondary metabolites. Penicillium commune produced cyclopiazonic acid, cyclopaldic acid and rugulovasine A and B; P. solitum produced compactin and a new group including the ex-type culture of P. palitans produced cyclopiazonic acid and fumigaclavine A. Isolates in all three groups were able to produce cyclopenin, cyclopenol and viridicatin. An optimal thin layer chromatography system to detect the secondary metabolites from P. commune, P. palitans and P. solitum was developed using an agar plug method. The results were confirmed by high performance liquid chromatography with diode array detection. The chemotaxonomic allocations were backed up by differences in conidial colour on Czapek yeast autolysate agar, reverse colour on yeast extract sucrose agar and origin of the isolates. Even though P. palitans previously has been considered synonymous with P. commune or P. solitum it was concluded that P. palitans is a distinct species.     

Penicillium verrucosum in wheat and barley indicates presence of ochratoxin A

AIMS: The aims of this study were to isolate and identify ochratoxin A (OTA) producing fungi in cereals containing OTA and to determine the best selective and indicative medium for recovery of OTA producing fungi. METHODS AND RESULTS: Seventy-six wheat, barley and rye samples from Europe containing OTA and 17 samples without OTA were investigated using three different media, dichloran yeast sucrose agar (DYSG), dichloran rose bengal yeast extract sucrose agar (DRYES) and dichloran 18% glycerol agar (DG18). Hundred kernels were plated on each medium and the kind and number of fungal OTA producers were recorded as percentage of infestation. Penicillium verrucosum was the sole OTA producer found in cereals. The average percentage of infestation of P. verrucosum counts was recorded as 28.3% on DYSG, 10.3% on DRYES and 9.9% on DG18 on the OTA containing samples and 0.8% on DYSG, 0.4% on DRYES and 0.6% on DG18 for the samples without OTA. CONCLUSIONS: Penicillium verrucosum was the sole OTA producer in European cereals. Determination of P. verrucosum infestation and infection was best detected on DYSG after 7 days at 20 degrees C. The percentage of infestation of P. verrucosum found on DYSG and OTA content in cereals were correlated. More than 7% infestation of P. verrucosum indicated OTA contamination. SIGNIFICANCE AND IMPACT OF THE STUDY: The developed method could be used as a cereal quality control.     

Development of selective media for the isolation and enumeration of Alternaria species from soil and plant debris

A new semi-selective medium, acidified weak potato-dextrose agar (AWPDA) with Mertect (active ingredient: thiabendazole), was developed for the isolation and enumeration of Alternaria species from samples of soil and plant debris. The medium was selected based on growth inhibition tests against Alternaria and several other commonly encountered saprobic fungi utilizing three antifungal agents, Botran (active ingredient: dichloran), Bayleton (active ingredient: triadimefon), and Mertect, and two basal media, acidified potato-dextrose agar (APDA) and AWPDA. Botran inhibited growth of Rhizopus stolonifer moderately, but had little effect on Cladosporium cladosporoides, Fusarium oxysporum, Penicillium chrysogenum, or Trichoderma harzianum. Bayleton inhibited growth of R. stolonifer and C. cladosporoides severely, and inhibited growth of F. oxysporum, P. chrysogenum, and T. harzianum moderately. Mertect inhibited growth of C. cladosporoides, F. oxysporum, P. chrysogenum, and T. harzianum completely, but had little or moderate effect on R. stolonifer. All three antifungal agents inhibited growth of Alternaria species slightly or moderately. The combination of Bayleton and Mertect inhibited growth of all fungi severely. A comparison of recovery rates of Alternaria from soil and plant debris samples on AWPDA with Mertect and weak potato-dextrose agar (WPDA) revealed that Alternaria spp. accounted for 63.6%-81.0% of recovered fungal isolates on AWPDA with Mertect as compared to 0.6%-2.7% of recovered isolates on WPDA. The AWPDA medium with Mertect exhibited superior selective growth of Alternaria species from samples of soil and plant debris, and will be useful in studies where the recovery and enumeration of Alternaria species is necessary.     

Broad and complex antifungal activity among environmental isolates of lactic acid bacteria

More than 1200 isolates of lactic acid bacteria isolated from different environments were screened for antifungal activity in a dual-culture agar plate assay. Approximately 10% of the isolates showed inhibitory activity and 4% showed strong activity against the indicator mould Aspergillus fumigatus. The antifungal spectra for 37 isolates with strong activity and five isolates with low or no activity were determined. Several of the strains showed strong inhibitory activity against the moulds A. fumigatus, Aspergillus nidulans, Penicillium commune and Fusarium sporotrichioides, and also against the yeast Rhodotorula mucilaginosa. Penicillium roqueforti and the yeasts Pichia anomala and Kluyveromyces marxianus were not inhibited. Several isolates showed reduced antifungal activity after storage and handling. The majority of the fungal inhibitory isolates were identified by 16S rDNA sequencing as Lactobacillus coryniformis. Lactobacillus plantarum and Pediococcus pentosaceus were also frequently identified among the active isolates. The degree of fungal inhibition was not only related to production of lactic or acetic acid. In addition, antifungal cyclic dipeptides were identified after HPLC separation and several other active fractions were found suggesting a highly complex nature of the antifungal activity.     

Visual clone identification of Penicillium commune isolates

A method for visual clone identification of Penicillium commune isolates was developed. The method is based on images of fungal colonies acquired after growth on a standard medium and involves a high degree of objectivity, which in future studies will make it possible for non-experts to perform a qualified identification of different species as well as clones within a species. A total of 77 P. commune isolates from a cheese dairy were 3-point inoculated on Yeast Extract Sucrose (YES) agar and incubated for 7 days at 25 degrees C. After incubation, the isolates were classified into groups containing the same genotype determined by DNA fingerprinting (AFLP). Each genotype also has a specific phenotype such as different colony colours. By careful image acquisition, colours were measured in a reproducible way. Prior to image analysis, each image was corrected with respect to colour, geometry and self-illumination, thereby gaining a set of directly comparable images. A method for automatic extraction of a given number of concentric regions was used. Using the positions of the regions, a number of relevant features--capturing colour and colour-texture from the surface of the fungal colonies--was extracted for further analysis. We introduced the Jeffreys-Matusitas (JM) distance between the feature distributions to express the similarity between regions in two colonies, and to evaluate the overall (weighted) similarity. The nearest neighbour (NN) classification rule was used. On a dataset from 137 isolates, we obtained a "leave-one-out" cross-validation identification rate of approximately 93-98% compared with the result of DNA fingerprinting.     

Comparison of Biomass Dry Weights and Radial Growth Rates of Fungal Colonies on Media Solidified with Different Gelling Compounds

Penicillium commune, Aureobasidium pullulans, and Paecilomyces farinosus were grown on two different media solidified with agar, Pluronic F-127, Carrageenan X-4910, or Carrageenan X-4910 overlaid with cellophane. Growth on Carrageenan X-4910 was generally the same as that on agar, as was the visual appearance of the colonies, e.g., the pigmentation. The Carrageenan X-4910 gels had a melting point, depending on the medium, of 41 to 46(deg)C, and the dry weights of the colonies were readily determined at 60(deg)C. To determine the dry weights of the colonies grown on agar plates, the gels were boiled for 10 min to melt the agar. Comparison of these two procedures showed that the boiling procedure resulted in a 22% reduction of the biomass dry weight. Cellophane membranes did not affect the radial growth rate profoundly. The biomass density was almost halved for P. commune and P. farinosus grown with membranes, whereas the presence of the membrane did not affect the biomass density of A. pullulans. The biomass densities of the colonies grown on Pluronic F-127 were significantly reduced, while in most cases, the radial growth rates of colonies grown on Pluronic F-127 were significantly higher than those obtained on agar or Carrageenan X-4910. Furthermore, the morphology of the leading hyphae was altered, and the hyphal growth unit length was more than twice that obtained on agar and Carrageenan X-4910. Carrageenan X-4910 is a valuable gelling compound for the study of the growth of fungi, as the biomass dry weight is readily determined and growth is similar to that obtained on agar gels.     

Differentiating Penicillium species by detection of indole metabolites using a filter paper method

The indole secondary metabolites chaetoglobosin C, cyclopiazonic acid, isofumigaclavine A and rugulovasine A and B produced by several Penicillium species growing on Czapek yeast autolysate agar were detected directly in the culture using filter paper wetted with Ehrlich reagent dissolved in ethanol. The filter paper was placed on the mycelial side of an agar plug and the metabolites were visualized as a violet zone on the paper within 10 min. It was shown that the combined characters of the violet reaction on filter paper and the ability to grow on creatine sucrose agar occurred in 5 out of 16 species of Penicillium examined. A few additional simple morphological and physiological criteria were then sufficient for identification of P. camemberti, P. commune, P. discolor, P. expansum and P. roqueforti var. roqueforti.     

Antigenic characterization of Penicillium camemberti and related common cheese contaminants

Twenty-four isolates of Penicillium (including a green-spored mutant from a French Brie cheese, Penicillium camemberti) with a proposed relationship to the white cheese mold P. camemberti were investigated by immunological procedures. These penicillia, which are representative of species that have caused considerable taxonomic confusion, had common micromorphology (terverticillate penicilli with rough and smooth stipes and smooth ellipsoidal to subglobose [(3 to 5) X 2 1/2 to 4 1/2 microns] conidia); growth rates; good growth on creatine sucrose agar, cheese, and other products with a high amount of protein and lipid as a primary habitat; production (with the exception of Penicillium solitum) of cyclopiazonic acid; and the ability to grow at low temperatures and water activities. The isolates that were investigated proved to be strictly antigenically related. Absorbed antiserum of the green-spored mutant of P. camemberti showed a specific precipitin band when tested by immunodiffusion either with its homologous reference antigen or with the exoantigens obtained from different isolates. The precipitin band was not present in any P. camemberti starter culture but in many unwanted cheese contaminants. The precipitin band can be used in the purity control of P. camemberti starter culture spore preparations. Analysis of the exoantigens of all the cultures by reversed phase high-performance liquid chromatography allowed us to subdivide these penicillia into nine groups below the species level. The results indicate that P. commune Thom is the wild-type ancestor of P. camemberti.     

Antigenic characterization of Penicillium camemberti and related common cheese contaminants.

Twenty-four isolates of Penicillium (including a green-spored mutant from a French Brie cheese, Penicillium camemberti) with a proposed relationship to the white cheese mold P. camemberti were investigated by immunological procedures. These penicillia, which are representative of species that have caused considerable taxonomic confusion, had common micromorphology (terverticillate penicilli with rough and smooth stipes and smooth ellipsoidal to subglobose [(3 to 5) X 2 1/2 to 4 1/2 microns] conidia); growth rates; good growth on creatine sucrose agar, cheese, and other products with a high amount of protein and lipid as a primary habitat; production (with the exception of Penicillium solitum) of cyclopiazonic acid; and the ability to grow at low temperatures and water activities. The isolates that were investigated proved to be strictly antigenically related. Absorbed antiserum of the green-spored mutant of P. camemberti showed a specific precipitin band when tested by immunodiffusion either with its homologous reference antigen or with the exoantigens obtained from different isolates. The precipitin band was not present in any P. camemberti starter culture but in many unwanted cheese contaminants. The precipitin band can be used in the purity control of P. camemberti starter culture spore preparations. Analysis of the exoantigens of all the cultures by reversed phase high-performance liquid chromatography allowed us to subdivide these penicillia into nine groups below the species level. The results indicate that P. commune Thom is the wild-type ancestor of P. camemberti.     

Toxigenic Aspergillus and Penicillium Isolates from Weevil-Damaged Chestnuts

Aspergillus and Penicillium were among the most common genera of fungi isolated on malt-salt agar from weevil-damaged Chinese chestnut kernels (16.8 and 40.7% occurrence, respectively). Chloroform extracts of 21 of 50 Aspergillus isolates and 18 of 50 representative Penicillium isolates, grown for 4 weeks at 21.1 C on artificial medium, were toxic to day-old cockerels. Tweleve of the toxic Aspergillus isolates were identified as A. wentii, eight as A. flavus, and one as A. flavus var. columnaris. Nine of the toxic Penicillium isolates were identified as P. terrestre, three as P. steckii, two each as P. citrinum and P. funiculosum, and one each as P. herquei (Series) and P. roqueforti (Series). Acute diarrhea was associated with the toxicity of A. wentii and muscular tremors with the toxicity of P. terrestre, one isolate of P. steckii, and one of P. funiculosum.     

Organization of the gene cluster for biosynthesis of penicillin in Penicillium nalgiovense and antibiotic production in cured dry sausages

Several fungal isolates obtained from two cured meat products from Spain were identified as Penicillium nalgiovense by their morphological features and by DNA fingerprinting. All P. nalgiovense isolates showed antibiotic activity in agar diffusion assays, and their penicillin production in liquid complex medium ranged from 6 to 38 microgram. ml-1. We constructed a restriction map of the penicillin gene cluster of P. nalgiovense and found that the organization of the penicillin biosynthetic genes (pcbAB, pcbC, and penDE) is the same as in Penicillium chrysogenum and Aspergillus nidulans. The pcbAB gene is located in an orientation opposite that of the pcbC and penDE genes in all three species. Significant amounts of penicillin were found in situ in the casing and the outer layer of salami meat during early stages of the curing process, coinciding with fungal colonization, but no penicillin was detected in the cured salami. The antibiotic produced in situ was sensitive to penicillinase.     

Dichloran as an Inhibitor of Mold Spreading in Fungal Plating Media: Effects on Colony Diameter and Enumeration

Problems associated with overgrowth by spreading molds are not addressed by currently recommended fungal enumeration media. Twenty-two fungi, including 12 mold and 3 yeast genera, were evaluated for the effects of dichloran (2,6-dichloro-4-nitroaniline), previously identified as a mold-spreading inhibitor, on colony diameter and enumeration. On malt agar or antibiotic-potato-dextrose agar (APDA), colony diameters were effectively reduced when dichloran was added. Colony diameters decreased as the dichloran concentration increased. Counts obtained with mixed mold spore suspensions were lower on APDA supplemented with 25 μg of dichloran per ml than on APDA and were higher than APDA with the addition of 5 μg of dichloran per ml (APDA-D-5). Overall counts of mixed and individual mold spore and yeast suspensions were higher in APDA-D-5 than in APDA. The additional advantages of APDA-D-5 may be useful in routine enumeration of fungi.     

Dichloran-glycerol medium for enumeration of xerophilic fungi from low-moisture foods.

A low water activity (alpha omega) medium (0.95 alpha omega) containing 18% (wt/wt) glycerol and 2 micrograms of dichloran per ml was developed for enumerating the fungal flora of dried and semidried foods. The medium, designated DG18, was shown to be significantly better than Christensen malt salt agar when both media were tested with foodstuffs and with pure culture inocula. The need for a medium of reduced alpha omega for enumerating xerophilic fungi from low-moisture foods was demonstrated by comparing fungal counts obtained on both high-alpha omega and low-alpha omega media.     

Identification of cheese-associated fungi using selected ion monitoring of volatile terpenes

The cheese-associated fungi Penicillium commune , P. roqueforti , P. solitum , P. discolor and Aspergillus versicolor have been investigated for production of volatile terpenes for chemical identification, when grown on yeast extract agar. Volatiles were collected by headspace solid-phase microextraction. Selected ion monitoring of four to seven of the most characteristic ions of mainly sesquiterpenes made it possible to identify the fungi to species level within 2 d. In a mixed culture of P. roqueforti and P. commune , inoculated in a ratio of 1000 : 1, volatiles from both fungi could be detected within 3 d, making identification possible.     

Survey of mycoflora and ochratoxin A in dried vine fruits from Argentina markets

AIMS: The aims of this work were to identify the mycoflora and to evaluate the natural occurrence of OA in dried vine fruits. Likewise, the capacity to produce OA by Aspergillus section Nigri was studied. MATERIALS AND METHODS: Fifty samples of dried vine fruits were obtained from Mendoza and San Juan provinces. The surface disinfection method was used for mycoflora determination using the medium dichloran 18% glycerol agar (DG18) and dichloran Rose Bengal chloramphenicol agar (DRBC). RESULTS: Statistical analysis demonstrated that the species A. niger var. niger and Aspergillus niger var. awamori were isolated in higher frequency from black dried vine fruits from DRBC and DG18 media (P < 0.01). OA was found in 74% of the dried vine fruits samples. Sixty-two strains (28%) of Aspergillus section Nigri, were OA producers. In the species A. carbonarius the highest percentages of ochratoxigenic strains were detected (82.6%). CONCLUSIONS: The presence of ochratoxigenic strains of Nigri section in dried vine fruits suggests that they may be an important source of OA in this substrate. Dried vine fruits can also be an important source of OA people who consume large amounts. SIGNIFICANCE AND IMPACT OF THE STUDY: The dried vine fruits contamination with Aspergillus section Nigri and OA was significant.     

Biochemical characterization of ochratoxin A-producing strains of the genus Penicillium

In order to explore the biochemical scope of ochratoxin A-producing penicillia, we screened 48 Penicillium verrucosum isolates for the production of secondary metabolites. Fungal metabolites were analyzed by high-pressure liquid or gas chromatography coupled to diode array detection or mass spectrometry. The following metabolites were identified: ochratoxins A and B, citrinin, verrucolones, verrucines, anacines, sclerotigenin, lumpidin, fumiquinazolines, alantrypinones, daldinin D, dipodazine, penigequinolines A and B, 2-pentanone, and 2-methyl-isoborneol. By use of average linking clustering based on binary (nonvolatile) metabolite data, the 48 isolates could be grouped into two large and clearly separated groups and a small outlying group of four non-ochratoxin-producing isolates. The largest group, containing 24 isolates, mainly originating from plant sources, included the type culture of P. verrucosum. These isolates produced ochratoxin A, verrucolones, citrinin, and verrucines and had a characteristic dark brown reverse color on yeast extract-sucrose agar medium. Almost all of a group of 20 isolates mainly originating from cheese and meat products had a pale cream reverse color on yeast extract-sucrose agar medium and produced ochratoxin A, verrucolones, anacines, and sclerotigenin. This group included the former type culture of P. nordicum. We also found that P. verrucosum isolates and three P. nordicum isolates incorporated phenylalanine into verrucine and lumpidin metabolites, a finding which could explain why those isolates produced relatively lower levels of ochratoxins than did most isolates of P. nordicum.     

Secondary metabolites produced by solid fermentation of the marine-derived fungus Penicillium commune QSD-17

The marine sediment-derived fungus Penicillium commune QSD-17 was re-investigated and cultured on rice solid medium. Two new compounds, isophomenone (1) and 3-deacetylcitreohybridonol (2), together with seven known derivatives (3-9), were identified. Their structures were determined by spectroscopic analysis.