Direct identification of the common cheese contaminant Penicillium commune in factory air samples as an aid to factory hygiene

F. LUND. 1996. Creatine sucrose dichloran agar (CREAD) was used as a selective medium for Penicillium commune and related species found in air samples in a cheese factory. Using growth and simple colony characters on CREAD together with detection of indole metabolites with a filter paper method, it was possible to identify all 22 P. commune isolates from a total of 43 Penicillium isolates. Penicillium commune numbers on CREAD were compared with those found on a general isolation medium, dichloran 18% glycerol agar.     

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.     

Predicting noncompliant levels of ochratoxin A in cereal grain from Penicillium verrucosum counts

AIMS: To model the probability of exceeding the European legislative limit of 5 microg ochratoxin A (OTA) per kilogram grain in relation to Penicillium verrucosum levels and storage conditions, and to evaluate the possibilities of using P. verrucosum colony counts for predicting noncompliant OTA levels. METHODS AND RESULTS: Cereal samples were inoculated with P. verrucosum spores and stored for up to 9 months at temperatures and water activities ranging from 10-25 degrees C and aw 0.77-0.95. A logistic regression analysis showed that the probability of exceeding 5 microg OTA kg(-1) grain was related to colony counts of P. verrucosum and water activity. The sensitivity and specificity of various P. verrucosum count thresholds for predicting noncompliant OTA levels were estimated, using data from the storage trial and natural cereal samples. CONCLUSION: The risk of exceeding 5 microg OTA kg(-1) grain increased with increasing levels of P. verrucosum, and with increasing water activities. A threshold of 1000 CFU P. verrucosum per gram grain is suggested to predict whether or not the legislative limit is exceeded. SIGNIFICANCE AND IMPACT OF THE STUDY: This study has provided a tool to evaluate the levels of P. verrucosum in grain in relation to OTA levels. Hence, mycological analyses can be used to identify cereal samples with high risk of containing OTA levels above the legislative limit.     

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.     

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.     

Ochratoxin A producing Penicillium verrucosum isolates from cereals reveal large AFLP fingerprinting variability

AIMS: To examine if molecular amplified fragment length polymorphism (AFLP) fingerprinting of the only ochratoxin A-producing species in European cereals, Penicillium verrucosum, can be used as a method in hazard analysis using critical control points (HACCP). METHODS AND RESULTS: A total of 321 isolates of P. verrucosum were isolated from ochratoxin A-contaminated cereals from Denmark (oats), UK (wheat and barley) and Sweden (wheat). Of these, 236 produced ochratoxin A as determined by thin layer chromatography; 185 ochratoxin A-producing isolates were selected for AFLP fingerprinting. A total of 138 isolates had unique AFLP patterns, whereas 52 isolates could be allocated to small groups containing from two to four isolates with similar AFLP patterns. A total of 155 clones were found among the 185 P. verrucosum isolates, thus 84% of the isolates may represent different genets of P. verrucosum. As the few isolates that were grouped often came from the same farm, and those groups that contained AFLP-identical isolates from different countries were morphotypically different. On single farms up to 35 clones were found. The few groups of ramets from the same genet indicated that a HACCP approach based on clones may require a very large number of AFLP analysis to work in practice, we recommend basing the HACCP approach on the actual species P. verrucosum. A more detailed characterization should rather be based on the profile of species present at different control points, or analysis of the mycotoxins ochratoxin A and citrinin in the isolates. Examination of 86 isolates with HPLC and diode array detection of P. verrucosum showed that 66% produced ochratoxin A, 87% produced citrinin, 92% produced verrucin and 100% produced verrucolone. CONCLUSIONS: Among 184 ochratoxin A-producing Penicillium verrucosum, 155 clonal lineages were indicated by AFLP fingerprinting, indicating a high genetical diversity, yet the species P. verrucosum is phenotypically distinct and valid. SIGNIFICANCE AND IMPACT OF THE STUDY: AFLP fingerprinting of Penicillium verrucosum indicates that genetic recombination takes place in this fungus.     

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.     

A selective and indicative medium for groups of Penicillium viridicatum producing different mycotoxins in cereals

A medium, pentachloronitrobenzene-rose bengal-yeast extract-sucrose agar (PRYES), for the isolation of moulds occurring during storage of cereals has been developed and compared with other selective media. The basal medium is yeast extract agar containing 15% sucrose (w/v). In addition to the sucrose content further selective measures include the addition of antibacterial antibiotics chloram-phenicol and chlortetracycline (50 mg/l), the fungicides rose bengal (25 mg/l each), and pentachloronitrobenzene (1 g/l) and a low incubation temperature (20°C). Members of the Mucorales were completely inhibited, and fast-growing species of other moulds were slightly inhibited, allowing important storage moulds to develop. The important ochratoxin A and citrinin-producing Penicillium viridicatum group II was indicated by a typical violet brown reverse on PRYES. Producers of xanthomegnin and viomellein (P. viridicatum group I and P. aurantiogriseum ) were indicated on PRYES by their yellow reverse and obverse colours. The medium was used for screening 40 samples of barley, and moulds with the characteristic colours were all identified as the species mentioned above.     

A selective and indicative medium for groups of Penicillium viridicatum producing different mycotoxins in cereals

A medium, pentachloronitrobenzene-rose bengal-yeast extract-sucrose agar (PRYES), for the isolation of moulds occurring during storage of cereals has been developed and compared with other selective media. The basal medium is yeast extract agar containing 15% sucrose (w/v). In addition to the sucrose content further selective measures include the addition of antibacterial antibiotics chloramphenicol and chlortetracycline (50 mg/l), the fungicides rose bengal (25 mg/l each), and pentachloronitrobenzene (1 g/l) and a low incubation temperature (20 degrees C). Members of the Mucorales were completely inhibited, and fast-growing species of other moulds were slightly inhibited, allowing important storage moulds to develop. The important ochratoxin A and citrinin-producing Penicillium viridicatum group II was indicated by a typical violet brown reverse on PRYES. Producers of xanthomegnin and viomellein (P. viridicatum group I and P. aurantiogriseum) were indicated on PRYES by their yellow reverse and obverse colours. The medium was used for screening 40 samples of barley, and moulds with the characteristic colours were all identified as the species mentioned above.     

<Emphasis Type="Italic">Penicillium verrucosum</Emphasis> occurrence and Ochratoxin A contents in organically cultivated grain with special reference to ancient wheat types and drying practice

This study addresses the relationship between the ochratoxigenic strains of Penicillium verrucosum and ochratoxin A (OTA) contents in organically cultivated grain. It included 37 combined, non-dried grain samples from farmers with no drying facilities as well as 19 non-dried and 22 dried samples from six farms with on-farm drying facilities (Case studies 1–6). The study focused on the ancient wheat type spelt but also included samples of wheat, rye, barley, oats, triticale, emmer, and einkorn. All 78 samples were analysed for moisture content (MC) and occurrence of P. verrucosum. The latter was assessed by plating non-disinfected kernels on DYSG agar and counting those contaminated by the fungus. Fifty-five samples were analysed for OTA. Most of the combine harvested samples (82%) were contaminated with P. verrucosum prior to drying. This was ascribed to difficult harvest conditions and many samples of spelt, which was significantly more contaminated by P. verrucosum than oats, wheat and barley. Though not statistically significant, the results also indicated that spelt was more contaminated than rye, which is usually regarded the most sensitive small grain cereal. No correlation was found between number of kernels contaminated by P. verrucosum and OTA content. Despite many non-dried samples being contaminated by P. verrucosum, only two exceeded the EU maximum limit for grain (5 ng OTA g^–1), both being spring spelt with 18 and 92 ng g^–1, respectively. The problems were most likely correlated to a late harvest and high MC of the grain. The case studies showed exceedings of the maximum limit in a batch of dried oats and spring wheat, respectively, probably to be explained by insufficient drying of late harvested grain with high MC. Furthermore, our results clearly indicate that OTA is not produced in significant amounts in samples with MCs below 17%. All dried samples with MCs above 18% exceeded the 5 ng OTA g^–1 limit in grain. However, no correlation between MC and the amount of OTA produced was found.     

Determination of volatile metabolites originating from mould growth on wall paper and synthetic media

Microbial volatile organic compounds (MVOCs) emitted from the mould species Penicillium expansum, P. chrysogenum, Aspergillus versicolor, A. fumigatus, A. niger and Cladosporium cladosporoides were analyzed by means of solid phase microextraction (SPME) and GCMS. The mould species were cultivated on the synthetic agar dichloran chloramphenicol (DG 18) and on wet wall paper. The production of MVOCs was monitored over several weeks to detect changes in the emission rates between the initial stage and later periods of growth. The cultivation on the synthetic agar resulted in MVOC patterns with a wide variety of signals. In contrast, the growth on wet wall paper led to changed MVOC patterns with less signals. The emission rates were drastically reduced. Components emitted by all six fungi species on wall paper were 2-pentanol and 2-pentanone. 1-Octen-3-ol was emitted by five fungi species. 2-Pentanol was only detected in considerable amounts during the first days of growth whereas 1-octen-3-ol had a more constant emission rate over the whole period of growth. On the basis of our studies some MVOCs could be proposed as specific for single fungi on wall paper, e.g. 1,3-dimethoxybenzene for A. versicolor and 2,4-pentandione for A. fumigatus.     

Water, temperature and gas composition interactions affect growth and ochratoxin A production by isolates of Penicillium verrucosum on wheat grain

AIMS: To examine the effect of interactions between water, temperature and gas composition on growth and ochratoxin A (OTA) production by isolates of Penicillium verrucosum in vitro and in situ on grain-based media and wheat grain. METHODS AND RESULTS: Three isolates of P. verrucosum were examined in relation to radial growth rate and OTA production, and to interacting conditions of water activity (a(w)), temperature and gas composition on a milled wheat medium. Subsequently, detailed temporal studies were carried out on gamma irradiated wheat grain over the range 0.75-0.995 a(w), 10-25 degrees C and air, 25 or 50% CO(2). This showed that optimum growth of P. verrucosum was at 0.98 a(w) in vitro at 25 degrees C, but at 0.95 a(w) and 25 degrees C on wheat grain. The a(w) minimum for growth was about 0.80 a(w), although no OTA was produced under this condition even after 56 days. Significant inhibition of growth and OTA production occurred with 50% CO(2), and 0.90-0.995 a(w) at 25 degrees C. CONCLUSIONS: The optimum and marginal conditions for growth and OTA production on wheat grain have been identified. At least 50% CO(2) is needed to inhibit growth and OTA production by >75% in moist grain (0.90-0.995 a(w)). SIGNIFICANCE AND IMPACT OF THE STUDY: First detailed identification of optimal and marginal interacting conditions of water/temperature and gas composition on growth and OTA production by P. verrucosum on wheat grain. This is a critical component of the postharvest management strategy for minimizing contamination by this important mycotoxin and predicting risk, based on environmental conditions, during drying and storage.     

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.     

Airborne Fungal Colony-Forming Units in Outdoor and Indoor Environments in Yokohama,Japan

The fungal concentration and flora in indoor and outdoor air in Yokohama, Japan were analyzed with a Reuter centrifugal air sampler and dichloran 18% glycerol agar (DG18), and compared with the levels assessed with potato dextrose agar (PDA). The number of fungal colony-forming units (CFU) in outdoor air was < 13–2750/m³; Cladosporium spp. predominated, followed by Alternaria spp. and Penicillium spp. The fungal concentration in outdoor air peaked in September. The concentrations of fungi in outdoor air (n = 288) were significantly correlated with the maximum temperature of the day, minimum temperature of the day, average temperature of the day, average velocity of wind of the day, average temperature of the month, average relative humidity of the month and precipitation of the month. In indoor air, the fungal CFU was < 13–3750/m³. Cladosporium spp. predominated, followed by the xerophilic fungi such as the Aspergillus restrictus group, Wallemia sebi, the A. glaucus group, and Penicillium spp. The fungal concentration in indoor air peaked in October. The concentrations of fungi in indoor air (n = 288) were significantly correlated with the indoor temperature, indoor relative humidity and the outdoor climatic factors mentioned above, except for the average velocity of wind of the day. This revised version was published online in June 2006 with corrections to the Cover Date.     

Mycoflora and ochratoxin-producing strains of Aspergillus section Nigri in wine grapes in Argentina

AIMS: The aims of this work were to evaluate the mycoflora and to identify the species of Aspergillus with the potential to produce ochratoxin A (OA) from different wine grape varieties from Mendoza, Argentina. Likewise, the capacity to produce OA by Aspergillus section Nigri was studied. METHODS AND RESULTS: Fifty samples of wine grapes were obtained from a winery of Mendoza province, Argentina. The surface-disinfection method was used for mycoflora determination using the medium dichloran 18% glycerol agar (DG18). Alternaria, Aspergillus and Penicillium were identified at species level. OA production was tested in 63 strains belonging to section Nigri. Alternaria genus was the most frequent (80% of the samples) followed by Aspergillus (70%). Alternaria alternata was the only specie identified from the Alternaria genus, followed by A. niger var. niger, A. flavus among others. From Penicillium genus, P. crysogenum was the most frequent specie. From 63 strains of Aspergillus section Nigri, 41.3% were OA producers. The levels of produced toxin ranged from 2 to 24.5 ng ml-1 of culture medium. CONCLUSIONS: The presence of ochratoxigenic strains of Nigri section in this substrate suggests that they may be an important source of OA in grapes from tropical and subtropical zones. Therefore, the industry should work further to diminish the growth of these fungi and mycotoxins formation in grapes, with the aim to reduce OA content in wine products. SIGNIFICANCE AND IMPACT OF THE STUDY: The wine grape contamination with A. alternata and Aspergillus section Nigri was significant.     

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.     

Mycotoxin production from fungi isolated from grapes

AIMS: In order to assess the potential for producing mycotoxins, fungi were isolated from wine producing grapes. METHODS AND RESULTS: The isolates were identified and Penicillium expansum, the most well recognized mycotoxin producer, was analysed for mycotoxin production by TLC. Many of the strains produced patulin and/or citrinin, often depending on whether they were grown on a grape or yeast extract sucrose media. CONCLUSION: Citrinin was produced by all strains grown in the yeast extract sucrose medium, but only one strain (from 51) was able to produce this compound in grape juice medium. Patulin was produced in the yeast extract medium by 20 strains and in grape juice medium by 33 strains. SIGNIFICANCE AND IMPACT OF THE STUDY: The presence of mycotoxins in wine producing grapes is discussed. Grapes contamination with patulin seems not to contribute to wine contamination, and no ochratoxin producing fungi was identified.     

Creatine sucrose agar, a differential medium for mycotoxin producing terverticillate Penicillium species

Creatine sucrose agar, containing creatine as sole N-source and bromocresol purple as pH indicator, was a good differential medium in subgenus Penicillium , subdividing taxa and subgroups based on mycotoxin production in approximately two equal groups.     

Guttation droplets of <Emphasis Type="Italic">Penicillium nordicum</Emphasis> and <Emphasis Type="Italic">Penicillium verrucosum</Emphasis> contain high concentrations of the mycotoxins ochratoxin A and B

Eight of eleven ochratoxigenic isolates of Penicillium nordicum and Penicillium verrucosum produced guttation droplets when grown on Czapek yeast extract (CYA) agar for 10–14 days at 25°C. Parallel cultivation of one strain each of P. nordicum and P. verrucosum on malt extract agar demonstrated that higher volumes of exudate are produced on this agar. However, HPLC analyses revealed higher concentrations of ochratoxin A (OTA) and B (OTB) in droplets originating from cultures on CYA. For quantitative determination of the mycotoxin contents, triplicates of three isolates each of P. nordicum and P. verrucosum were grown as single spot cultures on CYA for up to 14 days at 25°C. Guttation droplets were carefully collected between day 11 and 14 with a microliter syringe from each culture. Extracts from exudates and corresponding mycelia as well as fungal free agar were analyzed by HPLC for the occurrence of ochratoxin A (OTA) and B (OTB). Mean concentrations ranging between 92.7–8667.0 ng OTA and 159.7–2943.3 ng OTB per ml were detected in the guttation fluids. Considerably lower toxin levels were found in corresponding samples of the underlying mycelia (9.0–819.3 ng OTA and 4.5–409.7 ng OTB/g) and fungal free agar (15.3–417.0 ng OTA and 12.7–151.3 ng OTB/g). This is the first report which shows that high amounts of mycotoxins could be excreted from toxigenic Penicillium isolates into guttation droplets.     

Molecular characterization of ochratoxin A producing penicillia

Sixty-six strains classified as P. verrucosum based on morphological criteria were characterized by RAPD analysis. Two groups could be identified which differed in their ability to produce ochratoxin A (OTA). Group I originating from proteinacious foods containing mainly high OTA producing strains, and group II originating from plants containing moderate to non-producing strains. The differences at the RAPD level are consistent with the recent reclassification of ochratoxin A producing penicillia to be either P. verrucosum or P. nordicum.