Chemical and structural similarities in wall polysaccharides of some Penicillium, Eupenicillium and Aspergillus species

Various fractions were extracted from cell-wall material of Eupenicillium crustaceum, Penicillium brevi-compactum, P. decumbens, Aspergillus flavipes and A. ochraceus. The most characteristic fractions, which may have chemotaxonomic relevance, were F1I, an alpha-(1-3) glucan (alkalisoluble, water-insoluble), which amounted to 16.2-32.5% of the cell-wall material, and F1S (alkali and water-soluble) which represented 2.5-6.2% of the cell-wall material and was identified as a beta-(1-5) galactan. 13C-NMR spectra of the F1S fractions showed the same pattern for all the fungal species, characteristic of beta-(1-5) linked galactofuranose.     

Multilocus sequence analysis of Penicillium and Eupenicillium species

Taxonomy of Hyphomycetes has always been a challenging problem, with experts viewing species in different ways and modifying the taxonomy of groups to reflect their best evaluation of species limits and concepts. The advent of phylogenetic analysis, relatively easy DNA sequencing techniques and PCR has provided an opportunity for mycology to move from a strictly morphological analysis of species to phylogenetic analysis of DNA sequences. Phylogenetic theory dictates that data from different loci will produce congruent or at least non-contradictory evolutionary histories of a clonal lineage. Tests of tree congruence such as the index of association can show whether lineages are clonal, and has revealed that some species long thought to be clonal are cryptically recombining. Genealogical concordance phylogenetic species recognition allows unambiguous identification of species boundaries.     

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.     

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.     

New structural features of the antigenic extracellular polysaccharides of Penicillium and Aspergillus species revealed with exo-beta-D-galactofuranosidase.

To study the structures of the epitopes of the extracellular polysaccharides from Penicillium and Aspergillus species, an exo-beta-D-galactofuranosidase was purified from a commercial crude enzyme preparation from Trichoderma harzianum. Analysis of ring size and linkage position of the galactose residues of the extracellular polysaccharide of Penicillium digitatum, before and after enzymatic treatment, was determined by the reductive-cleavage technique. In addition to terminal and beta (1-5)-linked galactofuranosides, beta (1-6)-linked and beta (1,5,6)-linked branched galactofuranose residues could be identified. After degradation with the purified exo-beta-D-galactofuranosidase, all initial linkages of the galactofuranose residues were still present, but the amount of beta (1-5)-linked galactofuranose residues had decreased considerably. Treatment of the extracellular polysaccharides of Penicillium and Aspergillus species with the purified exo-beta-D-galactofuranosidase resulted in complete disappearance of the enzyme-linked immunosorbent assay reactivity of these polysaccharides, using immunoglobulin G antibodies raised against P. digitatum. Therefore, with the use of this enzyme, it was proved that the beta (1-5)-linked galactofuranosyl residues only are responsible for the antigenicity of the extracellular polysaccharides of Penicillium and Aspergillus molds. A new structural model for the antigenic galactofuranose side chains of the galactomannan from P. digitatum is proposed.     

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.     

Purification and characterization of the conidial laccase of Aspergillus nidulans.

Conidial laccase of Aspergillus nidulans was purified by standard protein purification methods. Although the purified material showed a cluster of several protein bands on a nondenaturing gel, each of these protein bands had laccase activity. All bands of activity, however, were absent in a strain carrying a mutation in the structural gene for laccase. Concentrated solutions (greater than 1 mg/ml) were bright blue, suggesting that, like other laccases, this enzyme contains copper. The enzyme contained asparagine-linked carbohydrate (12% by weight) which could be removed by digestion with endo-beta-N-acetylglucosaminidase H. The molecular weight of native enzyme as determined by gel filtration was 110,000, but the largest component in a sodium dodecyl sulfate gel was 80,000. Several smaller components (55,000 and 36,000 molecular weight) were also visible. We present evidence which suggests that the smaller components are in vivo cleavage products tightly associated with enzymatically active molecules. Comparison of the laccase from a white-spore (wA) and a green-spore (wA+) strain showed, surprisingly, that the enzymes differed in electrophoretic pattern, in vitro heat stability, and in vivo metabolic stability. The difference was manifested for enzymes isolated from cultures after conidial pigmentation of the wA+ strain had occurred. If examined earlier, before pigmentation, the enzymes were indistinguishable. Since wA strains lack the precursor of the wild-type green pigment, i.e., the laccase substrate, we suggest that the transformation of the enzyme of the wA strain is due to its failure to interact with its normal substrate.     

Survey of Aspergillus species associated with the human respiratory tract

A comprehensive survey has been carried out on the occurrence ofAspergillus species in the respiratory tract of patients of bronchopulmonary diseases in Delhi. In all, 1238 clinical specimens, which included 1082 sputa, 143 bronchial aspirates and 13 throat swabs obtained from 812 patients, were examined. Of these 61.7 per cent patients were culturally positive yielding 29 different species ofAspergillus. The prevalence of aspergilli in sputa was significantly higher than in the bronchial aspirates.Aspergillus niger was the commonest species isolated showing a prevalence of 36.7 per cent. It was followed byA. flavus, A. nidulans, A. terreus, A. versicolor, A. sydowi, A. japonicus andA. oryzae. None of theAspergillus species showed a significant correlation with any of the diseases, or the type of treatment the patients had received. Of the 8 broad occupational groups investigated farmers and labourers showed higher prevalence ofA. niger andA. flavus. The prevalence ofAspergillus species in the throats of healthy persons was 16 per cent withA. versicolor being the commonest species followed byA. flavus, A. amstelodami, A. sydowi andA. terreus. A comparison of the prevalence ofAspergillus species in the patients, healthy individuals and atmosphere of Delhi appears to support the view that the aspergilli are transient residents in the human respiratory tract following their inhalation from the environment.     

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.     

Production of xanthomegnin and viomellein by isolates of Aspergillus ochraceus, Penicillium cyclopium, and Penicillium viridicatum.

Fungal isolates from legumes were cultured on rice and examined for production of the toxic mold metabolites xanthomegnin and viomellein. Six of 14 Aspergillus ochraceus isolates produced from 0.3 to 1.3 mg of xanthomegnin per g and 0.1 to 1.0 mg of viomellein per g. One of nine isolates of Penicillium cyclopium produced 0.1 mg of xanthomegnin per g and 0.06 mg of viomellein per g. Three of nine P. viridicatum isolates produced from 0.4 to 1.6 mg of xanthomegnin per g and 0.2 to 0.4 mg of viomellein per g. This is the first report of xanthomegnin and viomellein production by A. ochraeus and P. cyclopium.     

Relationship between conidial enzymes and germination of the apple blue mold,Penicillium expansum

The relationship between conidial enzymes of Penicillium expansum and spore germination was examined. The activities of xylanase and pectinase, but not of cellulase and amylase, were detected in the conidia. The levels of xylanase and pectinase were greatly enhanced by xylan and pectin as respective carbon sources in the basal medium. No conidia germinated in the basal medium without a carbon source. The type of carbon source and the enzyme levels of the conidia did not affect the rate of germination. However, a relationship was found between the enzyme levels and the elongation of the germ tubes.     

Host specificity of two species of Gnathia (Isopoda) determined by DNA sequencing blood meals

Host specificity data for gnathiid isopods are scarce because the parasitic stages are difficult to identify and host-parasite contact is often brief. We examined two common nocturnal species, Gnathia falcipenis and Gnathia sp. C, collected in light traps from two locations at Lizard Island on the Great Barrier Reef, Australia. Engorged third stage gnathiids were photographed and permitted to moult into adults to allow identification. We compared approximately 580 bp sequences of 16S mtDNA from blood meals with host sequences available on GenBank using BLASTn. Where homology was <98%, familial identity was investigated with neighbour-joining trees. All blood meal sequences (n=60) and homologous fish sequences (n=87) from GenBank were used in a Bayesian analysis, which identified all but three sequences to family. The host frequency distributions used by each species were significantly different; only four host families were shared. No gnathiids fed on elasmobranchs, blennies or apogonids, and most fed on host families whose representatives are typically large. Gnathia sp. C showed a distinct predilection for nemipterids. Gnathia falcipenis often parasitised sand-dwelling families, and unlike sympatric diurnal gnathiid species, it also frequently parasitised pomacentrids. We conclude that G. falcipenis and Gnathia sp. C operate as generalist micropredators with preferences.     

Proteome of conidial surface associated proteins of Aspergillus fumigatus reflecting potential vaccine candidates and allergens

Aspergillus fumigatus is a mold causing most of the invasive fungal lung infections in the immunocompromised host. In addition, the species is the causative agent of certain allergic diseases. Both in invasive and in allergic diseases, the conidial surface mediates the first contact with the human immune system. Thus, conidial surface proteins may be reasonable vaccine candidates as well as important allergens. To broaden the list of those antigens, intact viable Aspergillus conidia were extracted with mild alkaline buffer at pH 8.5 in the presence of a 1,3-beta-glucanase. The proteome of this fraction was separated by two- dimensional gel electrophoresis (2-DE) and analyzed by liquid chromatography coupled with tandem mass spectrometry. Altogether 26 different A. fumigatus proteins were identified, twelve of which contain a signal for secretion. Among these were the known major conidial surface protein rodlet A, one acid protease PEP2, one lipase, a putative disulfide isomerase and a putative fructose-1,6-biphosphatase. The known allergen Aspf 3 was identified among the proteins without a signal for secretion. On the basis of the recently annotated A. fumigatus genome (Nature 2005, 438, 1151-1156), proteome analysis is now a powerful tool to confirm expression of hypothetical proteins and, thereby to identify additional vaccine candidates and possible new allergens of this important fungal pathogen.     

Penicillium brocae, a new species associated with the coffee berry borer in Chiapas, Mexico

Penicillium brocae is a new monoverticillate species isolated from coffee berry borers collected at coffee plantations in Mexico near Cacahoatán, Chiapas, and from borers reared on artificial diets at ECOSUR laboratory facilities in Tapachula, Chiapas. Phenotypically, it is in Penicillium series Implicatum, but because it does not conform to known species we have described it as new. ITS and large subunit rDNA were sequenced and compared to determine the phylogenetic position of this species. It is most closely related to Penicillium adametzii. Penicillium brocae has only been found in association with the coffee berry borer and is one of several fungi that grow in coffee berry borer galleries. Penicillium brocae may provide the exogenous sterols necessary for the coffee berry borer's development and thus be mutualistically associated with the insect.