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.     

A monoclonal antibody specific for conidia and mycelium wall layer of Penicillium and Aspergillus.

A monoclonal antibody was obtained from BALB/c mice immunized with Penicillium frequentans mycelium. The specificity of the antibody was evaluated by enzyme-linked immunosorbent and indirect immunofluorescence assays against the same mycelium. This IgM antibody cross-reacted with various strains of the Penicillium and Aspergillus genera. By indirect immunofluorescence assays, the antibody was able to stain about 10% of Penicillium and Aspergillus conidia, but major part of conidia did not absorb the fluorescence-labeled antibody before swelling. During germination of P. frequentans conidia, the germ tube wall which constitutes a continuation of an inner wall layer was also stained. During germination of P. griseofulvum, the protrusion of the germ tube wall was not always recognized by the antibody because the germ tube wall was constituted by a continuation of an outer spore wall layer. The study of the staining patterns of the spores and the protrusions suggests that the antibody specifically recognizes an antigen of the inner spore wall layer. The monoclonal antibody reacts with extracellular galactomannans produced by genera Aspergillus and Penicillium but is not directed against beta-(1,5)-linked galactofuranose units.     

Production and Some Properties of a New Type of Acid Carboxypeptidase of Penicillium Molds

Among some 38 strains of the genus Penicillium we investigated seven wild-type strains (P. daleae IFO-6087, P. frequentans AHU-8328, P. funiculosum IAM-7013, P. janthinellum IFO-8070, IAM-7026, P. lividum IAM-7200, and P. oxalicum AHU-8336) that were found to be excellent strains for a new type of acid carboxypeptidase production in a surface koji culture at 25 C. The production of acid carboxypeptidase was determined in various culture conditions in a koji culture. The maximum yields of acid carboxypeptidase were obtained by P. janthinellum IFO-8070. Partial purification and isolation of the acid carboxypeptidase from strains of Penicillium were performed with gel filtration on Bio-Gel P-100. Characterization studies indicate that the acid carboxypeptidases from P. daleae IFO-6087, P. funiculosum IAM-7013, P. janthinellum IFO-8070, and P. oxalicum AHU-8336 have some properties similar to those of the enzyme of Aspergillus saitoi with regard to the hydrolysis of several peptides at acidic pH range but have other slightly different properties with regard to stability, pH optima, inhibitors, and molecular weights.     

Control of microbiological quality and shelf-life of catfish (Clarias gariepinus) by chemical preservatives and smoking

Fresh catfish ( Clarias gariepinus ) were subjected to different concentrations of sodium benzoate or potassium sorbate and smoked traditionally before evaluation for microbiological, chemical and organoleptic characteristics during ambient tropical storage. Unsmoked fish samples showed diverse microflora ( Enterobacter, Escherichia, Serratia, Bacillus, Staphylococcus, Streptococcus, Penicillium, Aspergillus and Achlya genera) while smoked samples were dominated by Gram-positive bacterial flora ( Bacillus, Staphylococcus and Streptococcus ) and spoilage moulds ( Penicillium verrucosum, Aspergillus flavus and Achlya spp.). Significant reduction in microbial population occurred in most samples following smoking with samples subjected to 0.4% (w/v) potassium sorbate showing the lowest microbial load and maximum shelf-stability. However, marked microbial increase occurred after day 4 of storage in control and benzoate-treated samples. Changes in pH were marginal but decreased after day 12 of storage. Moisture content decreased sharply after smoking and remained low after day 4 of storage. Overall, potassium sorbate treatment (0.4% w/v) was most effective in controlling microbial quality and extended the shelf-life of the samples by 8 d.     

Penicillium viridicatum, Penicillium verrucosum, and production of ochratoxin A

The taxonomy of the important mycotoxigenic species Penicillium viridicatum and P. verrucosum was reviewed to clarify disagreements relating to the three P. viridicatum groups erected by Ciegler and coworkers (A. Ciegler, D. I. Fennell, G. A. Sansing, R. W. Detroy, and G. A. Bennett, Appl. Microbiol. 26:271-278, 1973) and the mycotoxins produced by them. Cultures derived from the types of these two species and authentic cultures from each group and from many other sources were examined culturally, microscopically, and for mycotoxin production. It was concluded that P. viridicatum group II has affinities with P. verrucosum and not with P. viridicatum, as indicated by J. I. Pitt in the 1979 monograph (The Genus Penicillium and Its Teleomorphic States Eupenicillium and Talaromyces). As a result of this study it can now be unequivocally stated that the mycotoxins ochratoxin A and citrinin are not produced by P. viridicatum. Of species in subgenus Penicillium, only P. verrucosum is known to produce ochratoxin A.     

Penicillium viridicatum, Penicillium verrucosum, and production of ochratoxin A.

The taxonomy of the important mycotoxigenic species Penicillium viridicatum and P. verrucosum was reviewed to clarify disagreements relating to the three P. viridicatum groups erected by Ciegler and coworkers (A. Ciegler, D. I. Fennell, G. A. Sansing, R. W. Detroy, and G. A. Bennett, Appl. Microbiol. 26:271-278, 1973) and the mycotoxins produced by them. Cultures derived from the types of these two species and authentic cultures from each group and from many other sources were examined culturally, microscopically, and for mycotoxin production. It was concluded that P. viridicatum group II has affinities with P. verrucosum and not with P. viridicatum, as indicated by J. I. Pitt in the 1979 monograph (The Genus Penicillium and Its Teleomorphic States Eupenicillium and Talaromyces). As a result of this study it can now be unequivocally stated that the mycotoxins ochratoxin A and citrinin are not produced by P. viridicatum. Of species in subgenus Penicillium, only P. verrucosum is known to produce ochratoxin A.     

Cloning a part of the ochratoxin A biosynthetic gene cluster of Penicillium nordicum and characterization of the ochratoxin polyketide synthase gene

Penicillium nordicum is a fungal species able to produce high amounts of ochratoxin A. A 10kb genomic DNA fragment of P. nordicumn has been cloned which carries three long open reading frames. One open reading frame (otapksPN) has homology to fungal polyketide synthases. The second open reading frame (npsPN) has homology to non-ribosomal peptide synthetases and the third open reading frame (aspPN) has homology to fungal alkaline serine proteinases. The non-ribosomal peptide synthetase and the polyketide synthase are convergently transcribed. Interestingly, the polyketide synthase can be identified by PCR only in P. nordicum strains and not in the related species Penicillium verrucosum or in ochratoxigenic Aspergillus species, indicating that the ochratoxin polyketide synthases are different in the important ochratoxigenic species. In contrast, the non-ribosomal peptide synthetase can be identified in P. nordicum and P. verrucosum, but not in other species. An inactivation of the polyketide synthase resulted in strains with abolished capacity to produce ochratoxin A. Expression of the polyketide synthase correlates with ochratoxin A biosynthesis.     

Antifungal activity of lactobacilli isolated from salami

Sixty-five strains of lactobacilli isolated from salami were tested for their antifungal activity in early and late phases of growth. Ten strains showed inhibitory activity in the early phase of growth towards moulds such as Aspergillus and Penicillium. The active compounds identified were phenyl-lactate and hydroxy-phenyl-lactate. All strains tested had activity in the late phase, after autolysis. The compounds released were peptidic and showed antifungal activity.     

Etest for assessing the susceptibility of filamentous fungi

The purpose of this study was to evaluate the Etest as an in vitro antifungal susceptibility test method for different moulds originating from human samples and from the environment. A total of 50 isolates (1 Acremonium, 18 Aspergillus, 2 Cladosporium, 1 Epicoccum, 15 Penicillium, 2 Scopulariopsis and 11 Trichoderma strains) were tested by the Etest. Forty-six of the tested moulds (92%) were resistant to fluconazole with minimal inhibitory concentrations (MICs) > or = 256 microg ml(-1). There were strains resistant to ketoconazole among Aspergillus niger, A. ochraceus and Cladosporium spp. with MICs > 32 microg ml(-1). For fluconazole, no differences were observed using two different inocula, while for itraconazole, ketoconazole and amphotericin B, a 1 or less step 2-fold dilution difference in MIC was seen for the most of 10 selected strains. The MICs of fluconazole and amphotericin B obtained for Trichoderma strains by the Etest and the agar dilution method were also compared. MICs for fluconazole were in agreement, while MICs for amphotericin B were higher with 1 or 2 steps of 2-fold dilutions for most of Trichoderma strains in the case of the agar dilution method.     

Antifungal activity of phenyllactic acid against molds isolated from bakery products

Phenyllactic acid (PLA) has recently been found in cultures of Lactobacillus plantarum that show antifungal activity in sourdough breads. The fungicidal activity of PLA and growth inhibition by PLA were evaluated by using a microdilution test and 23 fungal strains belonging to 14 species of Aspergillus, Penicillium, and Fusarium that were isolated from bakery products, flours, or cereals. Less than 7.5 mg of PLA ml(-1) was required to obtain 90% growth inhibition for all strains, while fungicidal activity against 19 strains was shown by PLA at levels of < or = 10 mg ml(-1). Levels of growth inhibition of 50 to 92.4% were observed for all fungal strains after incubation for 3 days in the presence of 7.5 mg of PLA ml(-1) in buffered medium at pH 4, which is a condition more similar to those in real food systems. Under these experimental conditions PLA caused an unpredictable delaying effect that was more than 2 days long for 12 strains, including some mycotoxigenic strains of Penicillium verrucosum and Penicillium citrinum and a strain of Penicillium roqueforti (the most widespread contaminant of bakery products); a growth delay of about 2 days was observed for seven other strains. The effect of pH on the inhibitory activity of PLA and the combined effects of the major organic acids produced by lactic acid bacteria isolated from sourdough bread (PLA, lactic acid, and acetic acid) were also investigated. The ability of PLA to act as a fungicide and delay the growth of a variety of fungal contaminants provides new perspectives for possibly using this natural antimicrobial compound to control fungal contaminants and extend the shelf lives of foods and/or feedstuffs.     

Influence of selected soil saprotrophes on gas exchange,growth and yield of <Emphasis Type="Italic">Solanum tuberosum</Emphasis>

The influence of saprotrophic soil fungi: Aspergillus versicolor, Penicillium frequentans, Penicillium verrucosum var. cyclopium, and Trichocladium asperum on development, wholesomeness, gas exchange and yield of potato cv. Mila was studied in pot experiments during 1998–2000. The presence of each of the mentioned fungi species in soil accelerated potato germination and stimulated growth of overground plant parts in comparison with control plants. Additionally, the presence of the tested saprotrophes in soil prologed the potato growing period by inhibiting chloroses of necroses. The tested saprotrophic fungi also modified plant physiological processes, such as transpiration and assimilation. The contact of plant root system with soil saprotrophes diminished significantly assimilation and transpiration intensity of overground parts in comparison with the control plants, in all the years of the experiment. However, this response did not reduce the yield of tubers.     

A Selective Method for Cultivating Heat Resistant Moulds, particularly those of the Genus Byssochlamys, and their Presence in Dutch Soil

S ummary : Heat resistant moulds of the genera Byssochlamys, Aspergillus, Penicillium and Paecilomyces can be cultivated selectively in the presence of Bacillus spp. when at least 10 p/m of chloramphenicol or chlortetracycline is added to the growth medium. The inhibitory agents sulphafurazole, penicillin, streptomycin, oxytetracycline and furazolidone, do not give completely selective and optimal growth of these moulds. Selective cultivation of heat resistant moulds from 200 samples of Dutch soil showed that Byssochlamys nivea occurs more frequently than was expected, whereas Byss. fulva occurs by way of exception. Other heat resistant moulds observed in soil are, in order of frequency, species of the genera Aspergillus, Paecilomyces and Penicillium .     

南极乔治王岛的丝状真菌

本文报道从南极乔治王岛26个样品中分离到60株丝状真菌,其中6个样品未分离到任何真菌,经初步鉴定有8属10个种。根据本次温度实验和该地寒冷的气候,只有桔灰青霉(Penicillium aurantiogriseum Dierckx),大毛霉Mucor mucedo(L.)Fres.)和毡状金孢霉(chrysosporium pannorum(Link)Hughes)在本地能生长繁殖。其中毡状金孢霉占明显优势。     

MYCOLOGICAL SYNTHESIS OF FAT FROM WHEY. I. PRELIMINARY STUDIES WITH STATIONARY CULTURES

SUMMARY: Forty mould species, 30 of which were from the genera Aspergillus, Penicillium, Fusarium and Mucor , were examined for their ability to utilize the lactose in whey and to produce fat in the mycelium in stationary cultures.
In terms of lactose consumed and weight of mycelial felt produced the most promising species were A. ustus, P. frequentans, P. oxalicum and P. notatum. The first three of these warrant further study as fat producers.     

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.     

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.     

Study of the lipolytic system of Penicillium verrucosum var cyclopium (Westling). II. Conditions for the production of the lipase system

The specie Penicillium verrucosum var cyclopium (Westling) has showed that Media containing peptones, especially, trypticase, as a source of nitrogen and maltose or glucose as a source of carbon are most efficient for a good stimulation of the lipolytic activity. Salt, especially Mg++ ion and some oligo- elements exhibit a marked effect on enzyme production. On the other hand, addition of lipids to the growth medium inhibited the lipase production. Shaking of the medium decreases the amount of lipase production but allows an early growth of P. verrucosum var cyclopium (Westling).     

MYCOLOGICAL SYNTHESIS OF FAT FROM WHEY. II. COMPARATIVE STUDIES WITH SHAKEN AND STATIONARY CULTURES USING SELECTED MOULDS

SUMMARY: In detailed studies of the growth of Aspergillus ustus, Penicillium oxalicum, P. frequentans and P. notatum in whey with fat production in view, the first two showed the highest lactose utilization and felt weights in shaken cultures while the last two gave as good or better felt yields in stationary cultures.
When A. ustus was grown in shaken culture in whey with and without the addition of 1·14 g/1. of ammonium nitrate, the extra nitrogen led to the production of larger amounts of fat, but P. frequentans did not form additional fat in these circumstances. A. ustus was the best mould; in whey plus ammonium nitrate it utilized up to 96% of the lactose and formed, per litre of whey, about 17 g of mycelial felt containing 13% of protein and 28% of fat.     

Morphological changes of conidiophores and mycelium of some hyphomycetes

The formation of enlarged cells and other abnormalities of mycelium and conidiophores occurring inAspergillus flavus Link,Penicillium chrysogenum Thom,Penicillium notatum Westling,Penicillium variabile Sopp.,Scopularipsis brevicaulis Bainier,Fusarium moniliforme Sheld.,Botrytis cinera Pers. ex Fr.,Botrytis allii Rud.,Humicola brunnea var.africana Fass. andTritirachium heimii var.grisea Fass. under the influence of uranium acetate, radioactive sediment from P?íbram mines (uranium mines in P?íbram, Bohemia), fungicidal compounds (fungizon, fungistatin, nitrofungin), antibiotic nystatin and antifungal bacteriumBacillus subtilis Cohn emend. Prazmowski were investigated. Enlarged phialides or phialides with extended orifices and collarettes and occasionally a conversion of phialides to spherical enlarged cells were observed inAspergillus flavus grown on a Sabouraud agar supplemented with uranium acetate. InPenicillium chrysogenum some conidiophores were completely reduced to enlarged cells. Less abnormalities were observed on the Czapek-Dox agar. The radioactive sediment as a component of malt agar caused either complete reduction of conidiophores inAspergillus flavus to dichotomously branched hyphae or sometimes only an enlargement of phialides. The antifungal effect ofBacillus subtilis was demonstrated in all studied strains. Enlarged cells instead of whole conidiophores were formed under the influence of the bacterium. This effect was observed inBotrytis allii, Botrytis cinerea and to a lesser extent inPenicillium notatum, Aspergillus flavus andFusarium moniliforme. The enlarged cells could still germinate and can be considered as reduced conidiophores. The used fungicidal compounds and the antibiotic nystatin inhibited to a certain degree the studied strains, however, their application did not bring about any abnormalities of mycelium and conidiophores.     

Effect of preharvest fungicides and interacting fungi on Aspergillus carbonarius growth and ochratoxin A synthesis in dehydrating grapes

AIMS: To evaluate the effect of preharvest grape pesticides in Aspergillus section Nigri infection in dehydrating grapes and the final ochratoxin A (OTA) content. Additionally, the effect of coinoculation of moulds frequently isolated from grapes and raisins on Aspergillus section Nigri infection was studied. METHODS AND RESULTS: Fungicide-treated grapes were inoculated with Aspergillus carbonarius, Aspergillus niger aggregate, Eurotium amstelodami and Penicillium janthinellum in different combinations, then dehydrated by reducing a(w) for 20 days. The percentages of colonized grapes treated with fungicides were, in general, lower, but no differences were observed among fungicides. The untreated grapes always showed higher concentrations of OTA, regardless of the inoculum applied. In general, Chorus was the most effective antifungal treatment in reducing OTA accumulation in grapes during dehydration. Penicillium janthinellum reduced Aspergillus section Nigri colonization and OTA accumulation in grapes during dehydration. CONCLUSIONS: The four preharvest fungicides studied reduced the Aspergillus section Nigri growth and OTA production by A. carbonarius during dehydration of grapes. SIGNIFICANCE AND IMPACT OF THE STUDY: The success of these chemical treatments might depend on the mycobiota composition of grapes.