Molecular Identification of Species from the Penicillium roqueforti Group Associated with Spoiled Animal Feed

The Penicillium roqueforti group has recently been split into three species, P. roqueforti, Penicillium carneum, and Penicillium paneum, on the basis of differences in ribosomal DNA sequences and secondary metabolite profiles. We reevaluated the taxonomic identity of 52 livestock feed isolates from Sweden, previously identified by morphology as P. roqueforti, by comparing the sequences of the ribosomal internal transcribed spacer region. Identities were confirmed with random amplified polymorphic DNA analysis and secondary metabolite profiles. Of these isolates, 48 were P. roqueforti, 2 were P. paneum, and 2 were Penicillium expansum. No P. carneum isolates were found. The three species produce different mycotoxins, but no obvious relationship between mold and animal disease was detected, based on medical records. P. roqueforti appears to dominate in silage, but the ecological and toxicological importance of P. carneum and P. paneum as feed spoilage fungi is not clear. This is the first report of P. expansum in silage.     

Fungi isolated from contaminated baled grass silage on farms in the Irish Midlands

The incidence of fungal growth on baled grass silage was recorded on 35 farms in the Irish Midlands in 2003. Fungal colonies were visible on 58 of 64 bales examined and the number of colonies per bale ranged from 1 to 12. On average, 5% of bale surface areas were affected. The fungus most prevalent on bales was Penicillium roqueforti, present on 86% of bales and representing 52% of all isolates. Other moulds isolated were Penicillium paneum, Geotrichum, Fusarium and mucoraceous species. Schizophyllum commune was observed protruding through the plastic film on bales on 17 of the 35 farms.     

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.     

Headspace sorptive extraction and GC-TOFMS for the identification of volatile fungal metabolites

Volatiles from fungi cultivated in Petri dishes were collected by a simple headspace polydimethylsiloxane (PDMS) sorptive extraction technique (HSSE), thermally desorbed into a gas chromatographic capillary column and detected and identified by gas chromatography-time-of-flight mass spectrometry (GC-TOFMS). The method was used to compare metabolite profiles of seven species of fungi grown on two types of sterile agars - potato dextrose and Sabouraud dextrose. Three species from the genus Penicillium (P. italicum, P. camemberti, and P. roqueforti) and four outgroups, each from a different phylum (Saprolegnia sp.; Sordaria fimicola, wild-type; Coprinus cinereus; and Rhizopus stolonifer) were grown on the two types of agars and analyzed. Multivariate analysis (PCA) was used to determine whether separate classes of fungi can be distinguished from one another based on their metabolite profiles. PCA showed clear class separation between the three Penicillium samples and the outgroups. Slight differences were observed in metabolite profiles as a function of growth medium. HSSE/GC-TOFMS appears to be a relatively simple and accurate technique for classification of fungi based on their volatile metabolite profiles. The volatiles sampling technique reported here is non-destructive, so it can be applied with traditional methods for studying fungal growth and metabolism.     

Mass spectrometry-based chemotaxonomic classification of Penicillium species (P. echinulatum, P. expansum, P. solitum, and P. oxalicum) and its correlation with antioxidant activity

In this study, 4 Penicillium species (17 strains) were classified on the basis of metabolite profile (chemotaxonomy) by using liquid chromatography-electrospray ionization ion trap-mass spectrometry (LC-ESI-MS), gas chromatography-ion trap-mass spectrometry (GC-IT-MS) and multivariate statistical analysis. The LC-ESI-MS-based dendrogram was similar to the internal transcribed spacer (ITS)-based dendrogram, in that Penicillium oxalicum was separated from the other 3 species. Moreover, vermiculidiol, meleagrin, oxaline, glandicolin A and B, and secalonic acid D were identified as metabolites that enable discrimination of Penicillium species by partial least squares discriminant analysis (PLS-DA). Evaluation of the species-specific metabolites produced by P. expansum, P. echinulatum, and P. solitum revealed that the 3 species differed from each other. On the other hand, GC-IT-MS-based dendrogram revealed that P. expansum was clearly classified separately from the other 3 species, and this result correlated with the antioxidant activity of the 4 species: P. expansum had a higher radical scavenging activity than the other 3 species. The metabolites produced in higher amounts in P. expansum were gluconic acid (12, 29, 33); andrastin A (16), B (15), and C (17); chaetoglobosin C (14), a class of sugar (31, 32); and salicylic acid (28). The results of this study demonstrated that metabolite-based chemotaxonomy could be used not only as a classification method but also as a tool for evaluation of species-specific activities.     

Microbiological and molecular determination of mycobiota in fresh and ensiled maize silage

The mycobiota of fresh and ensiled maize was studied with culturing techniques and a DNA sequence-based approach. Freshly chopped and ensiled maize were collected for 2 y from 12 farms in Pennsylvania. Samples were plated on selective media and isolates identified by morphology and sequences of the internal transcribed spacer regions of rDNA, 800-900 bp of the 5' end of the translation elongation factor 1-alpha gene and a portion of the rodA gene (Aspergillus fumigatus only). ITS regions were amplified from total silage DNA, cloned, sequenced and compared to fungal ITS sequences in GenBank with the BLAST-N algorithm. For samples analyzed by both methods, the molecular technique detected a greater number of species than selective plating. Plating recovered several Penicillium and Fusarium species and Aspergillus fumigatus, while molecular analysis detected Alternaria, Penicillium and Fusarium species. Data from both methods found that Fusarium and Penicillium were the dominant mycotoxigenic fungi in silage, while yeast made up the majority all fungi recovered or detected. Known mycotoxigenic species often accounted for 50% or more of the total number of species isolated or detected at each site. Viable Fusaria were not isolated from or detected in ensiled maize, suggesting that Fusarium species do not survive the ensiling process. Results from this study suggest that given the numerous species of fungi present in silage with mycotoxin producing ability, there is a strong possibility that silage may be contaminated with multiple toxins simultaneously.     

Mycophenolic acid in silage

We examined 233 silage samples and found that molds were present in 206 samples with counts between 1 x 10(3) and 8.9 x 10(7) (mean, 4.7 x 10(6)) CFU/g. Mycophenolic acid, a metabolite of Penicillium roqueforti, was detected by liquid chromatography-mass spectrometry in 74 (32%) of these samples at levels ranging from 20 to 35,000 (mean, 1,400) microg/kg. This compound has well-known immunosuppressive properties, so feeding with contaminated silage may promote the development of infectious diseases in livestock.     

Antagonistic activity of the food-related filamentous fungus Penicillium nalgiovense by the production of penicillin.

Defined strains of the genus Penicillium used as starter cultures for food and strains isolated from mold-fermented foods were analyzed for their ability to inhibit the growth of Micrococcus luteus DSM 348 used as an indicator organism. Most of the strains belonging to the species Penicillium nalgiovense showed antagonistic activity in agar diffusion assays. Penicillium camemberti and Penicillium roqueforti strains proved to be inactive in these tests. The inhibitory substance excreted by P. nalgiovense strains was totally inactivated when treated with beta-lactamase (penicillinase), indicating that a beta-lactam antibiotic is produced by these strains. This observation was verified by PCRs with primer sets specific to the [delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine] synthetase gene (pcbAB), the isopenicillin-N-synthase gene (pcbC), and the acyl coenzyme A:6-aminopenicillanic acid acyltransferase gene (penDE) from Penicillium chrysogenum using chromosomal DNA of the fungal strains as a template. These results indicate that penicillin biosynthesis is a characteristic often found in strains of P. nalgiovense. No specific PCR signal could be identified with DNA from P. camemberti and P. roqueforti.     

New Penicillium species associated with bulbs and root vegetables

Taxa of the Penicillium series Corymbifera are known for their strongly fasciculate growth and association with the rhizosphere of vegetables and flower bulbs. Using micromorphology, colony characteristics on various media and chemotaxonomic profiling, P. albocoremium sensu stricto and two new species, P. radicicola and P. tulipae, are redescribed during a taxonomic survey of P. albocoremium isolates contained within the IBT culture collection. Although these novel taxa are micromorphologically quite similar, their unique secondary metabolite profiles individually distinguish them from isolates of P. albocoremium. Moreover, the following metabolites produced by these species are known mycotoxins: citrinin, penicillic acid and terrestric acid by P. radicicola and terrestric acid and penitrem A by P. tulipae.     

Physiological criteria and mycotoxin production as AIDS in identification of common asymmetric penicillia

The taxonomy of the asymmetric (predominantly terverticillate) penicillia is based on morphological differences that leave identification difficult. The application of physiological criteria facilitated the identification of the common asymmetric penicillia investigated. Changes in the placement of some strains of these penicillia made the connection to mycotoxin-producing ability clearer. The classical criterion of conidium color was deemphasized and replaced by the following criteria: (i) growth on nitrite-sucrose agar and (ii) growth and acid (and subsequent base) production on creatine-sucrose agar (containing bromocresol purple). Other criteria used or developed were: (iii) growth on sorbic acid plus benzoic acid agar (50 + 50 ppm, pH 3.8), (iv) growth on an agar containing 1,000 ppm propionic acid (pH 3.8), (v) growth on an agar containing 0.5% acetic acid, (vi) growth at 37 degrees C, (vii) growth rate on an agar containing 0.1% pentachloronitrobenzene, (viii) production of extracellular tricaproinase, and (ix) fasciculation on a medium containing 10 ppm botran (2,6-dichloro-4-nitroanilin). The pattern of extracellular metabolites after thin-layer chromatography was used as a chemotaxonomic criterion. The species investigated, the number of isolates investigated, and the toxins which some of these isolates produce were: Penicillium roqueforti (18) (patulin), P. citrinum (11) (citrinin), P. patulum (9) (patulin and griseofulvin), P. expansum (patulin and citrinin), P. hirsutum (13), P. brevicompactum (19), and P. chrysogenum (12). Widespread species of the P. cyclopium, P. viridicatum, and P. expansum series of Raper and Thom (A Manual of the Penicillia, 1949) were subdivided into four new groups: "P. crustosum pA" (29) (penitrem A), "P. melanochlorum" (29), "P. cyclopium p" (119) (penicillic acid and infrequently penitrem A), and "P. viridicatum o-c" (43) (ochratoxin A and citrinin). "P. viridicatum o-c" was separated from "P. cyclopium p" due to its ability to grow on nitrite as sole nitrogen source. The species and groups investigated were related to the new taxonomic classification of the genus Penicillium according to Pitt.     

Patulin Production in Apples Decayed by Penicillium expansum

Sixty isolates of Penicillium expansum were tested for patulin production in decaying apples. All the isolates were found to produce the mycotoxin patulin as determined by thin-layer chromatography. Since patulin is known to be stable in many apple products, the results indicate that apple products made partially from apples decayed by P. expansum will contain patulin which may present a health hazard. The results also suggest that patulin may be important in the decay of apples by P. expansum.     

<Emphasis Type="Italic">Penicillium svalbardense</Emphasis>, a new species from Arctic glacial ice

During investigations of mycobiota in the coastal Arctic polythermal glaciers, different species of the ubiquitous genus Penicillium were isolated from the extreme subglacial environment. A group of Penicillium strains was obtained that did not belong to any known Penicillium species. This species was isolated in high numbers from the Kongsvegen subglacial ice and was not detected in the surrounding environment. A detailed analysis of secondary metabolite profiles, physiological and morphological characteristics, and partial β-tubulin gene sequences showed that the proposed new species Penicillium svalbardense is closely related but not identical to Penicillium piscarium and Penicillium simplicissimum. It differs in the production of secondary metabolites and in the morphological features of conidia and penicilli, and it is therefore described as a new species.     

Identification of NephrotoxicPenicillium Species from Cereal Grains

A system is described for identifying grain-inhabiting nephrotoxicPenicillium spp. based on their colony characters on Czapek yeast extract agar, yeast extract sucrose agar, and malt extract agar media, and their secondary metabolite profiles on thin layer chromatography plates. Using this system, the identity of 11Penicillium species, or their chemotypes, producing nephrotoxic metabolites could be confirmed. The species areP. verrucosum chemotype I, P.verrucosum chemotype II,P. expansum, P. citrinum, P. aurantiogriseum, P. freii, P. tricolor, P. polonicum, P. viridicatum, P. cyclopium, and P. melanoconldlum. Other non-nephrotoxicPenicillium species present on stored grains were separated from nephrotoxic species by their colony characters and metabolite profiles.     

Toxigenic species of Penicillium, Fusarium, and Aspergillus from weevil-damaged pecans.

Of a total of 2392 fungi isolated from weevil-damaged pecans, 46.4% were Alternaria and Epicoccum, 23.9% were Penicillium, 12.4% were Pestalotia and Monochaeta, 6.5% were Cladosporium, 6.4% were Fusarium, and less than 2% each were Phoma, Aspergillus, Rhizopus, Trichothecium, and miscellaneous. Chloroform extracts of 34 of 105 representative Penicillium isolates, 3 of 28 Fusarium isolates, and 3 of 23 Aspergillus isolates were toxic to day-old cockerels during three bioassays. Eight of the toxic extracts from Penicillium spp. were tremorgenic. One tremorgenic isolate was identified as P. paxilli, four were identified as P. lanoso-coeruleum, and three as P. cyclopium. Nine of the non-tremorgenic isolates were identified as P. citrinum, five as P. aurantio-virens, three as P. oxalicum, and two as P. meleagrinum. Others were identified as one each of P. brevi-compactum (Series), P. nigricans (Series), P. roqueforti, P. rugulosum (Series), P. terrestre, and P. stoloniferium. One was unidentified. Toxigenic Aspergillus isolates were all A. flavus. Two of the toxic Fusarium isolates were F. moniliforme, and one was unidentified.     

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.     

Isolation and metabolite production by Penicillium roqueforti,P. paneum and P. crustosum isolated in Canada

Penicillium roqueforti, P. crustosum and P. paneum grow on ensiled grain and recycled feed unless properly treated. The former two species occur also on cut lumber in Canada. These are known to produce a number of secondary metabolites including roquefortine. In cooler dairy production areas, including Scandinavia and North America, cattle toxicosis has been associated with silage contaminated by these fungi. We collected strains associated with cow or cattle toxicoses. The principal metabolites were determined making use of a new extraction method and analysis combining HPLC, LC/MS/MS, and LC/NMR. Penicillium roqueforti and P. crustosum required amino acid nitrogen for metabolite formation and their toxins were formed under conditions of low oxygen (20–30% saturation). Production of roquefortine C occurred on depletion of the available nitrogen and penitrem A on depletion of carbon source. Yield was reduced by excess carbon. Medium osmotic tension (a_w) affected metabolite production by the two species differently. Penicillium paneum was associated with ill-thrift of dairy cows and P. roqueforti was associated with more serious symptoms. Our data suggest a physiological basis for the common occurrence of roquefortine C in silage without serious consequences and the alternative, the presence of roquefortine C and toxicoses. The strain isolated from lumber was the best producer of the toxins studied. This is the first report of the toxigenic potential of P. roqueforti and P. paneum from Canada.     

Measurement of mercury methylation in lake water and sediment samples

The production of various eremophilane-type sesquiterpenes by Penicillium roqueforti strains has allowed us to propose a biochemical pathway for PR toxin synthesis. A time-course study of P. roqueforti metabolite production by high-performance liquid chromatography was performed to check this hypothetical pathway. The results obtained suggested that eremofortin C was the direct precursor of PR toxin in the P. roqueforti cell. Attempts to determine the amount of PR toxin in the mycelium failed. It was shown that the absence of PR toxin in mycelium was due to its instability during the extraction procedure.     

M13-microsatellite PCR and rDNA sequence markers for identification of Trichoderma (Hypocreaceae) species in Saudi Arabian soil

Seven fungal isolates were identified as pan-global Hypocrea/Trichoderma species, from section Trichoderma, on the basis of their morphology. These species were H. lixii/T. harzianum and H. orientalis/T. longibrachiatum. PCR-based markers with primer M13 (core sequence of phage M13) and internal-transcribed spacer sequences of ribosomal DNA were used to confirm the identity of the two Trichoderma species. Sequence identification was performed using the TrichOKEY version 2.0 barcode program and the multilocus similarity search database TrichoBLAST. Sequences from the ribosomal DNA internal-transcribed spacer regions showed limited variation among the Trichoderma species. This analysis divided the isolates into two main groups. Grouping the isolates based on cluster analysis of their DNA profiles matched the grouping based on morphological taxonomy. Molecular data obtained from analyses of gene sequences are essential to distinguish phonetically cryptic species in this group and to establish phylogenetic relationships.     

Molecular characterization of Penicillium expansum MUCL mutants

The modern biomolecular analysis of DNA was carried out to determine the identity of Penicillium expansum MUCL V1-V9 Variants with parental strain of Penicillium expansum MUCL 29412 and to compare the results with Penicillium verrucosum, a related species. The extracted DNAs were fragmented by digestion with restriction endonuclease Hind III and the fragments were separated by agarose gel electrophoresis. The DNAs were then denaturated in the gel after partial depurination with dilute acid and were transferred to a nylon membrane. The membrane was incubated with 32P-labeled probe, which was a DNA having a base sequence complementary to the DNA that was to be detected on the filter. The hybridization of the restriction fragments was performed for a highly qualitative comparison of the digested fragments. The analysis of the DNA profile, the most important stage in DNA identity testing, confirms the identity of the DNA for all strains of Penicillium expansum MUCL, except for V5 Variant.     

Antibacterial activity of marine-derived fungi

A total of 227 marine isolates of ubiqituous fungi were cultivated on different media and the secondary metabolite content of the extracts (ethyl acetate/chloroform/methanol 3 : 2 : 1) characterized by HPLC. The fungi were secured from animals, plants and sediments of Venezuelan waters (0–10 m) including mangroves and lagoonal areas. The extracts were tested for antibacterial activity. A total of 7 were active towards Vibrio parahaemolyticus and 55 towards Staphylococcus aureus, representing 18 different fungal species from 8 ascomycetous genera. For 61 strains of Penicillium citrinum antibacterial activity correlated well with content of secondary metabolites as measured by HPLC. Thirteen isolates of Penicillium steckii produced very similar profiles of secondary metabolites and 6 of these had activity against either V. parahaemolyticus or S. aureus or both. This revised version was published online in June 2006 with corrections to the Cover Date.