Complete mitochondrial genome of compactin-producing fungus Penicillium solitum and comparative analysis of Trichocomaceae mitochondrial genomes

We determined the complete mitochondrial genome sequence of the compactin-producing fungus Penicillium solitum strain 20-01. The 28?601-base pair circular-mapping DNA molecule encodes a characteristic set of mitochondrial proteins and RNA genes and is intron-free. All 46 protein- and RNA-encoding genes are located on one strand and apparently transcribed in one direction. Comparative analysis of this mtDNA and previously sequenced but unannotated mitochondrial genomes of several medically and industrially important species of the Aspergillus/Penicillium group revealed their extensive similarity in terms of size, gene content and sequence, which is also reflected in the almost perfect conservation of mitochondrial gene order in Penicillium and Aspergillus. Phylogenetic analysis based on concatenated mitochondrial protein sequences confirmed the monophyletic origin of Eurotiomycetes.     

Mycological survey of selected health foods.

A survey was conducted to compare the total viable fungal content and the number of different mold species encountered in 10 types of health foods labeled organically grown and in the same foods without such a label. The foods were wheat flour, corn meal, brown rice, figs, split peas, pinto beans, soybeans, walnuts, pecans, and peanuts. Results showed no consistent difference in either the total viable fungal content or the number of different mold species encountered between the labeled and unlabeled foods. Two genera of yeasts (Rhodotorula and Saccharomyces) and 22 gener of molds, including more than 65 species, were encountered. The mold flora was dominated by Aspergillus glaucus, Aspergillus niger, Aspergillus flavus, Aspergillus candidus, Penicillium cyclopium, and Penicillium viridicatum. Isolates of the genera Alternaria, Cladosporium, Fusarium, and Helminthosporium also occurred in certain foods. At least 10 toxicogenic species of Aspergillus and Penicillium were encountered. A total of 87 cultures of these species, all isolated from health foods, were screened for laboratory production of their respective toxins. Toxin production potential of these 87 cultures did not differ from that of cultures of the same species isolated from conventional foods.     

Mycological survey of selected health foods.

A survey was conducted to compare the total viable fungal content and the number of different mold species encountered in 10 types of health foods labeled organically grown and in the same foods without such a label. The foods were wheat flour, corn meal, brown rice, figs, split peas, pinto beans, soybeans, walnuts, pecans, and peanuts. Results showed no consistent difference in either the total viable fungal content or the number of different mold species encountered between the labeled and unlabeled foods. Two genera of yeasts (Rhodotorula and Saccharomyces) and 22 gener of molds, including more than 65 species, were encountered. The mold flora was dominated by Aspergillus glaucus, Aspergillus niger, Aspergillus flavus, Aspergillus candidus, Penicillium cyclopium, and Penicillium viridicatum. Isolates of the genera Alternaria, Cladosporium, Fusarium, and Helminthosporium also occurred in certain foods. At least 10 toxicogenic species of Aspergillus and Penicillium were encountered. A total of 87 cultures of these species, all isolated from health foods, were screened for laboratory production of their respective toxins. Toxin production potential of these 87 cultures did not differ from that of cultures of the same species isolated from conventional foods.     

Hypersaline waters - a potential source of foodborne toxigenic aspergilli and penicillia

Previous studies of hypersaline environments have revealed the dominant presence of melanized yeast-like fungi and related Cladosporium spp. In this study, we focused on the genera Aspergillus and Penicillium and their teleomorphic forms. From oligotrophic and eutrophic hypersaline waters around the world, 60 different species were identified, according to their morphological characteristics and extrolite profiles. For the confirmation of five new species, additionally, sequence analysis of the internal transcribed spacer region, the partial large subunit-rDNA and the partial β-tubulin gene was performed. The species Aspergillus niger, Eurotium amstelodami and Penicillium chrysogenum were detected with the highest frequencies at all of the sampled sites; thus, they represent the pan-global stable mycobiota in hypersaline environments. Possible candidates were also Aspergillus sydowii and Eurotium herbariorum, as they were quite evenly distributed among the sampled sites, and Aspergillus candidus, which was abundant, but more locally distributed. These species and their byproducts can accumulate downstream following evaporation of brine, and they can become entrapped in the salt crystals. Consequently, marine salt used for consumption can be a potential source of food-borne fungi and their byproducts. For example, ochratoxin-A-producing species Penicillium nordicum was recovered from brine, salt and salted meat products.     

Host specificity of Eupenicillium ochrosalmoneum, E. cinnamopurpureum and two Penicillium species associated with the conidial heads of Aspergillus

The genus Penicillium comprises species that mostly colonize plant matter. However early reports suggest that several species are capable of parasitizing Aspergillus and sporulating on the conidial heads of the host. More recently Eupenicillium ochrosalmoneum and E. cinnamopurpureum, both with Penicillium anamorphs, have been observed sporulating on the heads of Aspergillus species belonging to section Flavi during the colonization of peanut seeds. Little is known about the host specificity underlying these Aspergillus-Penicillium associations. In this study Aspergillus species representing nine taxonomic sections were paired in culture with E. ochrosalmoneum, E. cinnamopurpureum and two unnamed Penicillium species. Eupenicillium ochrosalmoneum, E. cinnamopurpureum and Penicillium sp. 1 sporulated predominantly on the heads of section Flavi species. In contrast Penicillium sp. 2 was restricted to the heads of section Nigri species. All species spread across Aspergillus colonies by means of aerial hyphae that grew from head to head. Additional studies are required to clarify whether Eupenicillium and Penicillium species are parasitic or simply epibiotic on their hosts.     

Identification of the pathogenic Aspergillus species by nested PCR using a mixture of specific primers to DNA topoisomerase II gene

For PCR-based identification of Aspergillus species, a common primer of the DNA topoisomerase II genes of Candida, Aspergillus and Penicillium, and species-specific primers of the genomic sequences of DNA topoisomerase II of A. fumigatus, A. niger, A. flavus (A. oryzae), A. nidulans and A. terreus were tested for their specificities in PCR amplifications. The method consisted of amplification of the genomic DNA topoisomerase II gene by a common primer set, followed by a second PCR with a primer mix consisting of 5 species-specific primer pairs for each Aspergillus species. By using the common primer pair, a DNA fragment of approximately 1,200 bp was amplified from the Aspergillus and Penicillium genomic DNAs. Using each species-specific primer pair, unique sizes of PCR products were amplified, all of which corresponded to a species of Aspergillus even in the presence of DNAs of several fungal species. The sensitivity of A. fumigatus to the nested PCR was found to be 100 fg of DNA in the reaction mixture. In the nested PCR obtained by using the primer mix (PsIV), the specific DNA fragment of A. fumigatus was amplified from clinical specimens. These results suggest that this nested PCR method is rapid, simple and available as a tool for identification of pathogenic Aspergillus to a species level.     

Selective partitioning of conidia of some Penicillium and Aspergillus species in aqueous two-phase systems.

Conidia of Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, Penicillium brevi-compactum, Penicillium frequentans, Penicillium spinulosum, and Penicillium verrucosum var. cyclopium were subjected to partition at varying pH values in an aqueous two-phase system containing charged polyethylene glycol. In the system, the partition behavior of the conidia of the Penicillium species varied when the pH was raised, while the conidia of the Aspergillus species seemed unaffected. P. brevi-compactum was separated from P. verrucosum var. cyclopium after only 10 transfers when subjected to stepwise partitioning. In the same way, 10 transfers were needed to separate P. verrucosum var. cyclopium from a mixture of conidia of three Aspergillus species. The partition behavior was influenced by the culture media used.     

Comparative analysis of the complete mitochondrial genomes of Aspergillus niger mtDNA type 1a and Aspergillus tubingensis mtDNA type 2b.

To understand the differences in the organization of mitochondrial genomes of the very closely related Aspergillus niger and Aspergillus tubingensis species, we determined the complete genome sequence of the 1a mtDNA type of A. niger and 2b mtDNA type of A. tubingensis and now we provide a comparative analysis of the two mtDNAs. We found that (1) the organization (gene content and order) of the two genomes is almost identical and (2) the size difference between them is principally attributed to the different intron content of their cox1, atp9 and ndh4L genes.     

球孢白僵菌线粒体序列分析及系统进化树构建

线粒体是真核细胞的重要的细胞器,在细胞能量代谢和细胞衰老方面扮演了重要角色,同时也是研究物种进化关系的重要资源。本文应用球孢白僵菌的线粒体序列分析了白僵菌与粪壳菌纲其他11属的13种真菌间的进化关系,为阐明球孢白僵菌的进化地位提供了新的论据。线粒体编码的rnl,rns,25个tRNAs和14个蛋白质基因均由同一条链编码,大部分的tRNA分布为三个tRNA簇,这与粪壳菌纲的其他物种相似。与NCBI上公布的序列比对发现球孢白僵菌线粒体序列与蝇蚧霉线粒体序列最相似,相似率达73%,用14个蛋白编码基因通过邻接法(Neighbor-joint)和最大简约法(Maximum parsimony)构建的系统进化树也得到相同结论,其支持率均为100%。     

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.     

甘草药材上的污染真菌类群及其产毒素特性

对药材市场上霉变甘草样品的污染真菌进行分析,共得到4属7种真菌,包括Penicillium、Aspergillus、Fusarium、Mucor属,其中Penicillium polonicum、Aspergillus parasiticus以及P.crustosum是优势真菌。采用高效液相色谱-质谱联用技术对优势菌菌株产黄曲霉毒素及赭曲霉毒素A的特性进行检测。结果表明A.parasiticus主要产生黄曲霉毒素(AFG2、AFG1、AFB2、AFB1)和赭曲霉毒素A(OTA);而Penicillium polonicum主要产生赭曲霉毒素A(OTA)。     

Molecular biology of mycotoxin biosynthesis

Mycotoxins are secondary metabolites produced by many important phytopathogenic and food spoilage fungi including Aspergillus, Fusarium and Penicillium species. The toxicity of four of the most agriculturally important mycotoxins (the trichothecenes, and the polyketide-derived mycotoxins; aflatoxins, fumonisins and sterigmatocystin) are discussed and their chemical structure described. The steps involved in the biosynthesis of aflatoxin and sterigmatocystin and the experimental techniques used in the cloning and molecular characterisation of the genes involved in the pathway are described in detail. The biosynthetic genes involved in the fumonisin and trichothecene biosynthetic pathways are also outlined. The potential benefits gained from an increased knowledge of the molecular organisation of these pathways together with the mechanisms involved in their regulation are also discussed.     

Comparison of fungal glucose oxidases. Chemical, physicochemical and immunological studies.

Glucose oxidases (beta-D-glucose:oxygen 1-oxidoreductase, EC 1.1.3.4) from two fungal genera (Aspergillus and Penicillium) were studied chemically, physicochemically and immunologically to elucidate the similarities and dissimilarities between these enzymes. Investigation of circular dichroism spectra revealed that these enzymes proteins possess essentially identical conformations. However, differences found in thermal inactivation parameters, catalytic parameters and quantitative immunological reactivities indicate that these enzymes must have some minor but distinct variations in their structures. Interestingly, it was observed that the Penicillium enzyme cross-reacted with the antiserum against the Aspergillus enzyme with an association constant of two orders of magnitude lower than that of the Aspergillus enzyme, and that the precipitin one of the Penicillium enzyme fused together with that of the Aspergillus enzyme in the immunodouble diffusion test. These results lead to the conclusion that these enzymes are closely related but not completely identical, and suggest that they might have evolved from a common ancestral precursor.     

Quantitative PCR analysis of selected Aspergillus, Penicillium and Paecilomyces species

A total of 65 quantitative PCR (QPCR) assays, incorporating fluorigenic 5' nuclease (TaqMan) chemistry and directed at the nuclear ribosomal RNA operon, internal transcribed spacer regions (ITS1 or ITS2) was developed and tested for the detection of selected Aspergillus, Penicillium and Paecilomyces species. The assays varied in specificity from species or subspecies to closely related species groups, subject to the amount of nucleotide sequence variation in the different organisms. A generic assay for all target species of Aspergillus, Penicillium and Paecilomyces was also developed and tested. Using a previously reported DNA extraction method, estimated conidia detection limits for target species ranged from less than one to several hundred per sample for the different assays. Conidia detection limits for non-target species were at least 1,000 fold higher in nearly all instances. The assays were used to analyze ten HVAC dust samples from different sources around the US. Total quantities of Aspergillus, Penicillium and Paecilomyces conidia in the samples, determined by the generic assay and the summed totals from the specific assays, were in general agreement, suggesting that all of the numerically dominant species in the samples were accounted for by the specific assays. QPCR analyses of these samples after spiking them with selected target organisms indicated that the enumeration results were within approximately a one-half log range of the expected values 95% of the time. Evidence is provided that the commonly used practices of enumerating Aspergillus and Penicillium as a single group or only by genus can be misleading in understanding the indoor populations of these organisms and their potential health risks.     

Differentiation of allergenic fungal spores by image analysis, with application to aerobiological counts

The ability of an image analysis routine to differentiate between spores of eleven allergenic fungal genera was tested using analysis based on seven basic and up to 17 more complex features, extracted from digitised images. Fungal spores of Alternaria, Cladosporium, Fusarium, Aspergillus, Penicillium, Botrytis, Epicoccum, Exserohilum, Ustilago, Coprinus and Psilocybe were examined in a series of experiments designed to differentiate between spores at the genus and species level. Linear and Quadratic Discriminant Analysis of feature measurements, recorded for 100 to 1600 spores per taxon, differentiated between genera and species with a high level of accuracy. Genus comparisons using only seven basic features resulted in 98% accuracy for the recognition of conidia belonging to Cladosporium, Fusarium and Epicoccum. Differentiation between conidia of Aspergillus and Penicillium was the least reliable, with 56% of Aspergillus conidia correctly identified and 41% misidentified as Penicillium. At the species level, conidia of Cladosporium macrocarpum, Fusarium moniliforme (microconidia), F. oxysporum (microconidia), F. solani (macroconidia), Alternaria helianthi and A. brassicae were consistently identified with 86--100% accuracy. Reduced levels of accuracy in the identification of spores by image analysis reflected similarities between species in their spore morphology. The application of image analysis to aerobiological counting methods is discussed in relation to the results obtained.     

Detection of aflatoxigenic molds in grains by PCR.

Aflatoxins are carcinogenic metabolites produced by several members of the Aspergillus flavus group in grains and floods. Three genes, ver-1, omt-1, and apa-2, coding for key enzymes and a regulatory factor in aflatoxin biosynthesis, respectively, have been identified, and their DNA sequences have been published. In the present study, three primer pairs, each complementing the coding portion of one of the genes, were generated. DNA extracted from mycelia of five Aspergillus species, four Penicillium species, and two Fusarium species was used as PCR template for each of the primer pairs. DNA extracted from peanut, corn, and three insect species commonly found in stored grains was also tested. Positive results (DNA amplification) were achieved only with DNA of the aflatoxigenic molds Aspergillus parasiticus and A. flavus in all three primer pairs. The detection limit of the PCR was determined by using the primer pairs complementing the omt-1 and ver-1 genes. Sterile corn flour was inoculated separately with six different molds, each at several spore concentrations. Positive results were obtained only after a 24-h incubation in enriched media, with extracts of corn inoculated with A. parasiticus or A. flavus, even at the lowest spore concentration applied (10(2) spores per g). No DNA spores per g). It is concluded that genes involved in the aflatoxin biosynthetic pathway may form the basis for an accurate, sensitive, and specific detection system, using PCR, for aflatoxigenic strains in grains and foods.     

Collagenolytic activity in several species of deuteromycetes under various storage conditions

The ability of deuteromycetes of the genera Penicillium, Aspergillus, and Botrytis to retain collagenolytic activity was studied after both 2 and 10 years of storage on a Czapek medium under a layer of mineral oil at 4 degrees C, as well as in silica gel granules at 20 and -60 degrees C. The enzymatic activity of several species, including Botrytis terrestris, Penicillium janthinellum, Penicillium chrysogenum, and Penicillium citrinum, was retained under both conditions of storage. Aspergillus repens retained enzymatic activity only if stored under a layer of mineral oil. The viability of conidia and the collagenolytic activity of Botrytis terrestris, P. janthinellum, P. chrysogenum, and Penicillium citrinum, maintained on silica gel for 10 years, depended on the storage temperature. The viability of the test strains improved after storage on a silica gel at -60 degrees C. A strain of Aspergillus repens lost its ability to dissolve collagen at various storage temperatures on the silica gel. The index of lysis for three strains of Penicillium deuteromycetes (Penicillium janthinellum, Penicillium chrysogenum, and Penicillium citrinum) increased after a 10-year storage on silica gel at -60 degrees C.     

Effect of the raw extracts of Arthrinium strains (Hyphomycetes, Dematiaceae) on the growth of some deleterious fungi in poultry feed

In previous work the authors have shown that some species of the Arthrinium genus are characterized by being able to produce secondary metabolites with antibiotic activity. The aim of this study was to evaluate the effect of raw extracts of the growth of three different Arthrinium strains against Aspergillus flavus, Aspergillus nidulans, Fusarium moniliforme and Penicillium purpurogenum when they were present in poultry feed. The results showed that the extracts reduced the growth of Aspergillus flavus and Fusarium moniliforme but could not inhibit the development of Aspergillus nidulans. Only the raw extract of A. aureum inhibited the growth of Penicillium purpurogenum.