Antioxidant and antibacterial activities of the chlorine pigment sclerotiorin from Penicillium mallochii and its chemotaxonomic significance

Penicillium mallochii was isolated as an endophyte from Himatanthus sp. and inoculated in liquid potato-dextrose culture medium, with adjusted to pH 3.6, and incubated for 10 days at 24 °C. Successive column chromatography of the hexane extract afforded the chlorine pigment sclerotiorin. Its structure was determined by NMR and by comparison with the literature. Sclerotiorin showed moderate antioxidant activity and moderate antibacterial against Bacillus subtilis, Staphylococcus aureus, and Micrococcus luteus. The isolation of sclerotiorin from P. mallochii support the taxonomic classification within the Penicillium genus, demonstrating a closer evolutionary relationship among the Penicillium species. Therefore, we suggest that sclerotiorin and the sclerotiorin group of metabolites may be used as chemotaxonomic markers for proper identification of Penicillium species.     

Isolation of Oxalic acid tolerating fungi and decipherization of its potential to control Sclerotinia sclerotiorum through oxalate oxidase like protein

Oxalic acid plays major role in the pathogenesis by Sclerotinia sclerotiorum; it lowers the pH of nearby environment and creates the favorable condition for the infection. In this study we examined the degradation of oxalic acid through oxalate oxidase and biocontrol of Sclerotinia sclerotiorum. A survey was conducted to collect the rhizospheric soil samples from Indo-Gangetic Plains of India to isolate the efficient fungal strains able to tolerate oxalic acid. A total of 120 fungal strains were isolated from root adhering soils of different vegetable crops. Out of 120 strains a total of 80 isolates were able to grow at 10?mM of oxalic acid whereas only 15 isolates were grow at 50?mM of oxalic acid concentration. Then we examined the antagonistic activity of the 15 isolates against Sclerotinia sclerotiorum. These strains potentially inhibit the growth of the test pathogen. A total of three potential strains and two standard cultures of fungi were tested for the oxalate oxidase activity. Strains S7 showed the maximum degradation of oxalic acid (23?%) after 60?min of incubation with fungal extract having oxalate oxidase activity. Microscopic observation and ITS (internally transcribed spacers) sequencing categorized the potential fungal strains into the Aspergillus, Fusarium and Trichoderma. Trichoderma sp. are well studied biocontrol agent and interestingly we also found the oxalate oxidase type activity in these strains which further strengthens the potentiality of these biocontrol agents.     

Reconstruction of the Carotenoid Biosynthetic Pathway of Cronobacter sakazakii BAA894 in Escherichia coli

Cronobacter sakazakii could form yellow-pigmented colonies. However, the chemical structure and the biosynthetic pathway of the yellow pigments have not been identified. In this study, the yellow pigments of C. sakazakii BAA894 were purified and analyzed. The major components of the yellow pigments were confirmed as zeaxanthin-monoglycoside and zeaxanthin-diglycoside. A gene cluster containing seven genes responsible for the yellow pigmentation in C. sakazakii BAA894 was identified. The seven genes of C. sakazakii BAA894 or parts of them were reconstructed in a heterologous host Escherichia coli DH5α. The pigments formed in these E. coli strains were isolated and analyzed by thin layer chromatography, UV-visible spectroscopy, high performance liquid chromatography, and electron spray ionization-mass spectrometry. These redesigned E. coli strains could produce different carotenoids. E. coli strain expressing all the seven genes could produce zeaxanthin-monoglycoside and zeaxanthin-diglycoside; E. coli strains expressing parts of the seven genes could produce lycopene, β-carotene, cryptoxanthin or zeaxanthin. This study identified the gene cluster responsible for the yellow pigmentation in C. sakazakii BAA894.     

Quorum sensing involvement in response surface methodology for optimisation of sclerotiorin production by Penicillium sclerotiorum in shaken flasks and bioreactors

Sclerotiorin, an azaphilone produced by some filamentous fungi including Penicillium sclerotiorum, is a pigment with variety of biological activities including lipoxygenase inhibition, reduction of cholesterol levels, and anti-cancer properties. Sclerotiorin has potential use in pharmaceutical as well as food industries. In this context, the purpose of this study was to provide a simple and robust procedure for optimised production of sclerotiorin by P. sclerotiorum using a central composite design developed through response surface methodology (RSM) and to identify the molecule(s) involved in the signalling mechanism in P. sclerotiorum. The optimisation of sclerotiorin production was carried out using RSM in shaken flasks and the obtained results were then replicated using a 2-L stirred tank bioreactor. Penicillium sclerotiorum ethyl acetate culture extract was analysed using thin layer chromatography (TLC) and potential signalling molecules were identified using Gas chromatography-mass spectrometry (GC-MS). The experimental studies suggested an increase in the sclerotiorin production by 2.1-fold and 2.2-fold in shaken flasks and stirred tank bioreactors respectively. Further analysis of P. sclerotiorum ethyl acetate culture extract reported the presence of ricinoleic acid, an oxylipin, belonging to a family of signalling molecules tentatively involved in the enhancement of sclerotiorin production. This paper has highlighted the positive effect of the optimal supplementation of P. sclerotiorum culture extracts for enhanced production of sclerotiorin. It has also examined potential molecules involved in the signalling mechanism in P. sclerotiorum culture extract for the overproduction of sclerotiorin.     

Production of Penicillin by Fungi Growing on Food Products: Identification of a Complete Penicillin Gene Cluster in Penicillium griseofulvum and a Truncated Cluster in Penicillium verrucosum

Mycobiota growing on food is often beneficial for the ripening and development of the specific flavor characteristics of the product, but it can also be harmful due to the production of undesirable compounds such as mycotoxins or antibiotics. Some of the fungi most frequently isolated from fermented and cured meat products such as Penicillium chrysogenum and Penicillium nalgiovense are known penicillin producers; the latter has been shown to be able to produce penicillin when growing on the surface of meat products and secrete it to the medium. The presence of penicillin in food must be avoided, since it can lead to allergic reactions and the arising of penicillin resistance in human-pathogenic bacteria. In this article we describe a study of the penicillin production ability among fungi of the genus Penicillium that are used as starters for cheese and meat products or that are frequently isolated from food products. Penicillium griseofulvum was found to be a new penicillin producer and to have a penicillin gene cluster similar to that of Penicillium chrysogenum. No other species among the studied fungi were found to produce penicillin or to possess the penicillin biosynthetic genes, except P. verrucosum, which contains the pcbAB gene (as shown by hybridization and PCR cloning of fragments of the gene) but lacks pcbC and penDE. Antibacterial activities due to the production of secondary metabolites other than penicillin were observed in some fungi.     

Diversity,ecological role and potential biotechnological applications of marine fungi associated to the seagrass Posidonia oceanica

The marine environment is characterized by high salinity and exerts a strong selective pressure on the biota, favouring the development of halo-tolerant microorganisms. Part of this microbial diversity is made up of fungi, important organisms from ecological and biotechnological points of view. In this study, for the first time, the qualitative and quantitative composition of the mycoflora associated to leaves, rhizomes, roots and matte of the seagrass Posidonia oceanica was estimated. A total of 88 fungal taxa, mainly belonging to Ascomycota, were identified by morphological and molecular methods. The most represented genera were Penicillium, Cladosporium and Acremonium. Most of the species (70) were selectively associated with one district; only two species (Penicillium chrysogenum var. chrysogenum and P. janczewskii) were isolated from all the districts. Moreover the capability to produce laccases, peroxidases and tannases by 107 fungal isolated by the different districts of P. oceanica was carried out. These results show that the mycoflora associated to P. oceanica is very rich and characterized by fungi able to produce ligninolytic enzymes and tannases useful to degrade and detoxify lignocellulose residues in presence of high salt concentrations. These fungi, hence, may play important ecological roles in marine environments but can also be very useful in different biotechnological areas.     

Aerobiological investigations inside repositories of the National Archive of the Republic of Cuba

The objectives of this study were to evaluate the microbial prevalence inside six repositories of the National Archive of the Republic of Cuba in 2?months of the year and to examine some of the physiological features of fungi isolated in order to evaluate their potential for biodeterioration. The microbiological sampling was conducted in February and September using a slit impactor as air sampler. Appropriate selective culture media were used to isolate fungi and bacteria. Temperature and relative humidity were measured during the samplings. The cellulolytic activity and the production of acids and pigments of the fungi isolated were qualitatively determined. Total viable microbiota and bacteria concentrations were greater in February while the fungal concentration was higher in September. Aspergillus, Cladosporium, Penicillium, Curvularia and Alternaria were the predominant fungal genera in February while Cladosporium prevailed in September, although Fusarium, Mucor and Neurospora genera were also isolated in this month. The fungi isolated were capable of degrading cellulose and excreting pigments and acids. The Gram-positive bacteria group prevailed in the air and Corynebacterium, Streptomyces, Bacillus, Streptococcus, Staphylococcus, Enterobacter and Serratia were some of the genera identified.     

The effect of three basidiomycetous fungal species on soil-borne,foliage and fruit-damaging phytopathogens in laboratory experiments

The effect of three basidiomycetous fungi, Meira geulakonigii, M. argovae and Acaromyces ingoldii on strains of seven phytopathogenic fungal species was assayed in the laboratory. The phytopathogens were placed on dialysis membranes covering media in which the basidiomycetous fungi were formerly cultured. A. ingoldii inhibited the growth of all phytopathogens tested, whereas Meira spp. suppressed their growth to a lesser extent. Upon being returned to extract-free media, all phytopathogens resumed their growth, suggesting a fungistatic effect of the basidiomycetous fungi. These fungi inhibited the growth of Sclerotina sclerotiorum on tomato leaves for a few days and Meira spp. hindered the infection of oranges by Penicillium digitatum, whereas A. ingoldii was less effective. Results of preliminary biochemical assays suggest that neither competition for hydrocarbons nor secreted proteases are responsible for the inhibitory activity of the basidiomycetous fungi, which was probably due to the secretion of unidentified substances by these fungi.     

Biological transformation of thiourea

Summary Six strains of fungi: two Aspergilli of the A. glaucus group, Penicillium sp. Trichoderma viride, and two unidentified dark fungi, were found able to use thiourea as a source of nitrogen. One of the Aspergilli and the Penicillium showed the best utilization, but the growth was always slow, and no more than 50% (usually much less) of the thiourea-nitrogen was recovered in the mycelium.Assimilation of thiourea-nitrogen by Aspergillus and Penicillium was accompanied by oxidation of the sulphur to sulphate in roughly approximate stoichiometric proportion. The same fungi were able to produce sulphate from cystine as combined source of nitrogen and sulphur.     

Isolation and characterization of rhizosphere bacteria for the biocontrol of the damping-off disease of tomatoes in Tunisia

Fluorescent Pseudomonas spp., isolated from tomato and pepper plants rhizosphere soil, was evaluated in vitro as a potential antagonist of fungal pathogens. Pseudomonas strains were tested against the causal agents of tomatoes damping-off (Sclerotinia sclerotiorum), root rot (Fusarium solani), and causal agents of stem canker and leaf blight (Alternaria alternata). For this purpose, dual culture antagonism assays were carried out on 25% tryptic soy agar, King B medium and potato dextrose agar to determine the effect of the strains on mycelial growth of the pathogens. In addition, strains were screened for their ability to produce exoenzymes and siderophores. All the strains significantly inhibited Alternaria alternata, particularly in 25% TSA medium. Antagonistic effect on Sclerotinia sclerotiorum and Fusarium solani was greater on King B medium. Protease was produced by 30% of the strains, but no strain produced cellulase or chitinase. Finally, the selected Pseudomonas strain, Psf5, was evaluated on tomato seedling development and as a potential candidate for controlling tomato damping-off caused by Sclerotinia sclerotiorum, under growth chamber conditions. In vivo studies resulted in significant increases in plant stand as well as in root dry weight. Psf5 was able to establish and survive in tomato plants rhizosphere after 40 days following the planting of bacterized seeds.     

Isolation and identification of endophytic fungus SX01, a red pigment producer from <Emphasis Type="Italic">Ginkgo biloba</Emphasis> L

Fungal pigments are a potential resource as natural food colorant. Endophytic fungus SX01, which is able to produce abundant soluble red pigments, was isolated from the twigs of Ginkgo biloba L. For further research and utilization of this strain and its secondary metabolites, morphological and molecular characteristics of SX01 was identified. Morphological identification which employed light microscope and scanning electron microscope showed that SX01 was a species of genera Penicillium. ITS1-5.8S-ITS2 region and 18S rRNA gene were then cloned, and the molecular phylogeny of these two sequences proved that strain SX01 was Penicillium purpurogenum.     

Identification of Ochratoxin A Producing Fungi Associated with Fresh and Dry Liquorice

The presence of fungi on liquorice could contaminate the crop and result in elevated levels of mycotoxin. In this study, the mycobiota associated with fresh and dry liquorice was investigated in 3 producing regions of China. Potential toxigenic fungi were tested for ochratoxin A (OTA) and aflatoxin B1 (AFB1) production using liquid chromatography/mass spectrometry/mass spectrometry. Based on a polyphasic approach using morphological characters, β-tubulin and RNA polymerase II second largest subunit gene phylogeny, a total of 9 genera consisting of 22 fungal species were identified, including two new Penicillium species (Penicillium glycyrrhizacola sp. nov. and Penicillium xingjiangense sp. nov.). The similarity of fungal communities associated with fresh and dry liquorice was low. Nineteen species belonging to 8 genera were detected from fresh liquorice with populations affiliated with P. glycyrrhizacola, P. chrysogenum and Aspergillus insuetus comprising the majority (78.74%, 33.33% and 47.06% of total) of the community from Gansu, Ningxia and Xinjiang samples, respectively. In contrast, ten species belonging to 4 genera were detected from dry liquorice with populations affiliated with P. chrysogenum, P. crustosum and Aspergillus terreus comprising the majority (64.00%, 52.38% and 90.91% of total) of the community from Gansu, Ningxia and Xinjiang samples, respectively. Subsequent LC/MS/MS analysis indicated that 5 fungal species were able to synthesize OTA in vitro including P. chrysogenum, P. glycyrrhizacola, P. polonicum, Aspergillus ochraceus and A. westerdijkiae, the OTA concentration varied from 12.99 to 39.03 µg/kg. AFB1 was absent in all tested strains. These results demonstrate the presence of OTA producing fungi on fresh liquorice and suggest that these fungi could survive on dry liquorice after traditional sun drying. Penicillium chrysogenum derived from surrounding environments is likely to be a stable contributor to high OTA level in liquorice. The harvesting and processing procedure needs to be monitored in order to keep liquorice free of toxigenic fungi.     

Endophytic Fungi Isolated from Oil-Seed Crop Jatropha curcas Produces Oil and Exhibit Antifungal Activity

Jatropha curcas L., a perennial plant grown in tropics and subtropics is popularly known for its potential as biofuel. The plant is reported to survive under varying environmental conditions having tolerance to stress and an ability to manage pest and diseases. The plant was explored for its endophytic fungi for use in crop protection. Endophytic fungi were isolated from leaf of Jatropha curcas, collected from New Delhi, India. Four isolates were identified as Colletotrichum truncatum, and other isolates were identified as Nigrospora oryzae, Fusarium proliferatum, Guignardia cammillae, Alternaria destruens, and Chaetomium sp. Dual plate culture bioassays and bioactivity assays of solvent extracts of fungal mycelia showed that isolates of Colletotrichum truncatum were effective against plant pathogenic fungi Fusarium oxysporum and Sclerotinia sclerotiorum. Isolate EF13 had highest activity against S. sclerotiorum. Extracts of active endophytic fungi were prepared and tested against S. sclerotiorum. Ethyl acetate and methanol extract of C. truncatum EF10 showed 71.7% and 70% growth inhibition, respectively. Hexane extracts of C. truncatum isolates EF9, EF10, and EF13 yielded oil and the oil from EF10 was similar to oil of the host plant, i.e., J. curcas.     

Diversity and biocontrol potential of endophytic fungi in Brassica napus

This study was conducted to isolate endophytic fungi from oilseed rape (Brassica napus), to identify the fungal endophytes based on morphology and ITS (ITS1-5.8S rDNA-ITS2) sequences, and to evaluate their efficacy in suppression of the plant pathogenic fungi Sclerotinia sclerotiorum and Botrytis cinerea. Selected endophytic fungal isolates were further tested for promoting growth of oilseed rape in potting experiments. A total of 97 endophytic fungal isolates were obtained from roots (35), stems (49) and leaves (13) of B. napus. Forty fungal species were identified and most species (80%) belong to Ascomycota. The species composition is highly diversified with Simpson’s diversity index reaching 0.959. Alternaria alternata is the dominant species accounting for 12.4% of the isolates. Twenty-four isolates exhibited antifungal activity against S. sclerotiorum in dual cultures on potato dextrose agar forming inhibition zones of 3–17 mm in width. The culture filtrates of Aspergillus flavipes CanS-34A, Chaetomium globosum CanS-73, Clonostachys rosea CanS-43 and Leptosphaeria biglobosa CanS-51 in potato dextrose broth exhibited consistent and effective suppression of oilseed rape leaf blight caused by S. sclerotiorum. Fusarium oxysporum CanR-46 was detected capable of production of volatile organic compounds highly inhibitory to S. sclerotiorum and B. cinerea. Moreover, A. alternata CanL-18, Fusarium tricinctum CanR-70 and CanR-71r, and L. biglobosa CanS-51 exhibited growth-promoting effects on oilseed rape. These results suggest that B. napus harbors diversified endophytic fungi, from which potential biocontrol agents against S. sclerotiorum and B. cinerea, and for promoting growth of B. napus can be screened.     

Chemical characterization and toxicity evaluation of fungal pigments for potential application in food,phamarceutical and agricultural industries

Concerns associated with the use of synthetic colourants backs the demand for natural colourants. Thus, the current study aimed at characterizing crude fungal pigments produced by Penicillium multicolour, P. canescens, Talaromyces verruculosus, Fusarium solani and P. herquie. This included their antioxidant and antimicrobial properties together with acute toxicity evaluation on zebrafish embryos. The identification of pigment compounds was achieved through MS and IR data. The study demonstrated a substantial radical scavenging activity of extracts ranging from 65.49 to 74.46%, close to that of ascorbic acid (89.21%). Penicillium canescens and F. solani exhibited a strong antimicrobial activity against Escherichia coli and Enterococcus aerogenes and Salmonella typhi, Staphylococcus aureus and Bacillus cereus at MIC values ranging from 1.5 to 2.5 mg/mL. However, some levels of toxicity were observed for all extracts at a concentration range of 3–5 mg/mL. Pigment by P. multicolour, T. verruculosus and F. solani were tentatively identified through IR and MS data as sclerotiorin (yellow), rubropunctamine (red) and bostrycoidin (red). In conclusion, the study demonstrates a market potential of filamentous fungi pigments due to their antioxidant, antimicrobial activities, and prominent colours. Although there are some toxicity concerns, further tests must be done using molecular docking, albino mice and cell linings.     

Antifungal effects of volatile organic compounds from the endophytic fungus Cryptosporiopsis ericae Cc-HG-7 isolated from Coptis chinensis Franch

In this study, 136 strains of endophytic fungi were isolated from the Chinese traditional medicinal plant Coptis chinensis Franch. Of these, 129 strains were classified into 12 different genera according to morphological traits and internal transcribed spacer (ITS) gene sequence analyses. Their antifungal activities were assessed against the following fungi: Magnaporthe oryzae, Pythium graminicola, Cylindrocarpon destructans, Fusarium oxysporum, Cercospora zeae-maydis, Sclerotinia sclerotiorum, Setosphaeria turcica and Botrytis cinerea. Fourteen endophytic strains were active against at least one of the selected fungi. The most active strain Cc-HG-7 identified as Cryptosporiopsis ericae displayed inhibition rates of 81.42% and 72.00%, respectively, against S. sclerotiorum and S. turcica in dual culture technique. The volatile antifungal compounds were identified using headspace solid-phase microextraction, followed by gas chromatography mass spectrometry to investigate the potential biocontrol mechanisms of strain Cc-HG-7. The results suggested that the strain Cc-HG-7 could be a potential agent for the biological control of S. sclerotiorum and S. turcica.     

Alteration of protein patterns in black rock inhabiting fungi as a response to different temperatures

Rock inhabiting fungi are among the most stress tolerant organisms on Earth. They are able to cope with different stressors determined by the typical conditions of bare rocks in hot and cold extreme environments. In this study first results of a system biological approach based on two-dimensional protein profiles are presented. Protein patterns of extremotolerant black fungi – Coniosporium perforans, Exophiala jeanselmei – and of the extremophilic fungus – Friedmanniomyces endolithicus – were compared with the cosmopolitan and mesophilic hyphomycete Penicillium chrysogenum in order to follow and determine changes in the expression pattern under different temperatures. The 2D protein gels indicated a temperature dependent qualitative change in all the tested strains. Whereas the reference strain P. chrysogenum expressed the highest number of proteins at 40 °C, thus exhibiting real signs of temperature induced reaction, black fungi, when exposed to temperatures far above their growth optimum, decreased the number of proteins indicating a down-regulation of their metabolism. Temperature of 1 °C led to an increased number of proteins in all of the analysed strains, with the exception of P. chrysogenum. These first results on temperature dependent reactions in rock inhabiting black fungi indicate a rather different strategy to cope with non-optimal temperature than in the mesophilic hyphomycete P. chrysogenum.     

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.     

Halotolerant laccases from Chaetomium sp., Xylogone sphaerospora, and Coprinopsis sp. isolated from a Mediterranean coastal area

Laccases (EC 1.10.3.2) are phenoloxidases involved in the transformation of the recalcitrant fraction of organic matter in soil. These enzymes are also able to transform certain aromatic pollutants such as polycyclic aromatic hydrocarbons (PAHs) and are known to be inhibited by chloride ions. This study aims to test the potential of some fungal strains newly isolated from natural environments subjected to high osmotic pressure such as coastal ecosystems, to produce chloride tolerant laccases. Three strains were identified as Chaetomium sp., Xylogone sphaerospora (two Ascomycota), and Coprinopsis sp. (a Basidiomycota) and the laccases produced by these fungi were weakly inhibited by chloride ions compared with previous data from literature. Moreover, we tested their reactivity towards various PAHs which are widespread anthropic pollutants. They were able to transform anthracene to 9,10-anthraquinone and we determine 7.5 eV as the threshold of ionization potential for PAH oxidation by these laccases.