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.     

Molecular characterization of fungal endophytes from Calotropis procera plants in Taif region (Saudi Arabia) and their antifungal activities

Fungal endophytes were isolated from the leaves of Calotropis procera (Apocynaceae) collected from Taif region (Saudi Arabia). Thirty-three different taxa were recovered. The overall foliar colonization rate was 35.1%. A total of 161 isolates were obtained and identified into 33 distinct operational taxonomic units based on the sequencing of the internal transcribed spacer regions of the rRNA gene. The most prevalent fungi were Aspergillus flavus, Chaetomium globosum, Cochliobolus lunatus, Fusarium dimerum, F. oxysporum, and Penicillium chrysogenum. A total of 161 isolates were tested for antifungal activities against four plant pathogenic fungi (Alternaria alternata, Fusarium oxysporum, Botrytis cinerea, and Pythium ultimum), of which 33 isolates showed antifungal activity against at least one plant pathogenic fungi. Four isolates of Chaetomium globosum and three isolates of Myrothecium verrucaria showed the strongest antifungal activity. This study reported the occurrence of a much wider spectrum of fungi, when compared with previous work. Also, it confirmed the variation of different isolates from the same species in terms of antifungal activity.     

Evaluation of aerial microbial pollutants in Al-Haram Al-Nabawi during pilgrimage of 2013

Al-Madinah Al-Munawwarah is the second holiest site in Islam. The possibility of new emerging microbes is valid due to the increased number of pilgrims. The objectives of the current study were to estimate the numbers of fungi and bacteria inside and outside Al-Haram Al-Nabawi and to find whether new bacterial and fungal species have emerged compared to previous studies. Air samples were collected twice a day from 12 spots and four directions during the pilgrim year of 2013 for four consecutive weeks by using the sedimentation method. Thirty five genera and fifty eight species were identified. The most recovered bacterial genera were Staphylococcus, Micrococcus, Bacillus, and Dermacoccus with 32.47%, 18.18%, 12.85%, and 11.23%, respectively. Fifty nine isolates of fungi were molecularly identified. Aspergillus species had the highest percentage (78%). The other fungal genera identified (Alternaria triticina, Emericella nidulans, Emericella striata, Mucor circinelloides, Penicillium chrysogenum, Penicillium minioluteum, Rhizopus arrhizus, Rhizopus oryzae, and Syncephalastrum racemosum) had less than 5% frequency. In places such as Al-Haram Al-Nabawi, a large and crowded public (millions) exist especially during pilgrimages and Ramadan, thus, exposure to microorganisms is high. On the other hand, microorganism infectivity depends on many factors including their virulence, landing site, and person’s immunity. For those reasons, many aspects should be considered to avoid aerosol contaminants.     

Desert soil fungi isolated from Saudi Arabia: cultivable fungal community and biochemical production

Desert soils harbor fungi that have survived under highly stressed conditions of high temperature and little available moisture. This study was designed to survey the communities of cultivable fungi in the desert soils of the Arabian Peninsula and to screen the fungi for the potentially valuable antioxidants (flavonoids, phenols, saponins, steroids, tannins, terpenoids, and alkaloids) and enzymes (cellulase, laccase, lipase, protease, amylase, and chitinase). Desert soil was sampled at 30 localities representing different areas of Saudi Arabia and studied for physico-chemical soil properties. Five types of soil texture (sand, loamy sand, sandy loam, silty loam, and sandy clay loam) were observed. A total of 25 saprotrophic species was identified molecularly from 68 isolates. Our survey revealed 13 culturable fungal species that have not been reported previously from Arabian desert soils and six more species not reported from Saudi Arabian desert soils. The most commonly recorded genera were Aspergillus (isolated from 20 localities) and Penicillium (6 localities). The measurements of biochemicals revealed that antioxidants were produced by 49 and enzymes by 52 isolates; only six isolates did not produce any biochemicals. The highest biochemical activity was observed for the isolates Fusarium brachygibbosum and A. phoenicis. Other active isolates were A. proliferans and P. chrysogenum. The same species, for instance, A. niger had isolates of both high and low biochemical activities. Principal component analysis gave a tentative indication of a relationship between the biochemical activity of fungi isolated from soil and soil texture variables namely the content of silt, clay and sand. However, any generalizable relation between soil properties and fungal biochemical activities cannot be suggested. Each fungal isolate is probable to produce several antioxidants and enzymes, as shown by the correlation within the compound groups. Desert soil warrants further research as a promising source of biochemicals.     

Natural shifts in communities of rhizosphere fungi of common oak after felling

Fungal communities in the rhizospheres of oak roots were shown to change after the trees were felled. The ecological significance of this is considered. Fungal communities were isolated from rhizospheres of thin roots (0.5–1 mm diam.) of living trees and their stumps two years after felling in 30- and 50-year-old stands of Quercus robur, with samples taken at two locations in each stand. In total, 128 species of fungi were isolated. Six species, Absidia cylindrospora, Penicillium adametzii, P. citrinum, P. daleae, P. janczewskii and Umbelopsis vinacea were detected in the rhizospheres of living oaks and stumps at all locations. The abundance of Geomyces spp.?+?Pseudogymnoascus roseus, Gymnoascus reessii and Zygomycetes was greater, and of Penicillium spp. was smaller, usually significantly, in rhizospheres of stump roots than of living tree roots. Among Zygomycetes, the populations of A. cylindrospora and Mucor hiemalis decreased, while those of Mortierella spp., particularly M. macrocystis, increased after felling. Species of Penicillium favoured by roots are P. adametzii, P. citrinum, P. daleae, P. herquei, P. janczewskii, P. raistrickii. On stump roots they are replaced by P. chrysogenum, P. islandicum and P. spinulosum. The combination of Chalara spp. with oak roots indicates the occurrence of specific relationships, suggesting that the organic food base provided by the plant is a primary factor determining the composition of the fungal community. Edaphic and other environmental factors may be less important in this effect.     

Species-specific PCR to describe local-scale distributions of four cryptic species in the Penicillium chrysogenum complex

Penicillium chrysogenum is a ubiquitous airborne fungus detected in every sampled region of the Earth. Owing to its role in Alexander Fleming's serendipitous discovery of Penicillin in 1928, the fungus has generated widespread scientific interest; however its natural history is not well understood. Research has demonstrated speciation within P. chrysogenum, describing the existence of four cryptic species. To discriminate the four species, we developed protocols for species-specific diagnostic PCR directly from fungal conidia. 430 Penicillium isolates were collected to apply our rapid diagnostic tool and explore the distribution of these fungi across the London Underground rail transport system revealing significant differences between Underground lines. Phylogenetic analysis of multiple type isolates confirms that the ‘Fleming species’ should be named Penicillium rubens and that divergence of the four ‘Chrysogenum complex’ fungi occurred about 0.75 million yr ago. Finally, the formal naming of two new species, Penicillium floreyi and Penicillium chainii, is performed.     

A comparative study of siderophore production by fungi from marine and terrestrial habitats

Siderophore producing potential of 20 fungal isolates (same 10 species from each marine and terrestrial habitat) were examined and compared. Except marine Aspergillus flavus, all isolates produced siderophores as evidenced by positive reaction in FeCl₃ test, CAS assay and CAS agar plate test. The results indicated widespread occurrence of siderophores in both the habitats. Examination of the chemical nature of siderophores revealed that mucoraceous fungi produced carboxylate, while others produced hydroxamate siderophores. Thus, the nature of siderophore was found to be independent of habitat. Among all the isolates, Cunninghamella elegans (marine form) was maximum siderophore producer (1987.5 μg/ml) followed by terrestrial form of C. elegans (1248.75 μg/ml). There was no marked variation in siderophore concentration of Penicillium funiculosum strains. Comparison of quantification of siderophore production between marine and terrestrial revealed that four terrestrial isolates (Aspergillus niger, Aspergillus ochraceous, Penicillium chrysogenum, Penicillium citrinum) were ahead in siderophore production, while, the other four marine isolates (Aspergillus versicolor, C. elegans, Rhizopus sp., Syncephalastrum racemosum) were found to be more potent siderophore producers, indicating that they were equally competent.     

Environmental factors and preservatives affect carbon utilization patterns and niche overlap of food spoilage fungi

Nutrient assimilation by two mycotoxigenic spoilage fungi (Penicillium verrucosum, Aspergillus ochraceus) and four other food spoilage fungi (Penicillium coryolophilum, P. roqueforti, Cladosporium herbarum, Eurotium repens), of 32 key C-sources in wheat bread were examined in relation to abiotic factors of water availability, pH, temperature and the presence/absence of a preservative, potassium sorbate. These studies were to understand the relative potential co-existence, nutritional partitioning and niche exclusion in bread-based matrices. The niche size decreased significantly with decrease in water availability, temperature and pH. There were also significant interactive effects between pH and the preservative. The data were used to determine the niche overlap indices (NOI) of competing fungi relative to the two ochratoxigenic species. These showed that P. verrucosum and A. ochraceus were nutritionally dominant over the other species. The NOIs showed that potential co-existence and/or niche exclusion was influenced by environmental factors. Overall, the addition of the preservative increased the interspecific competition for nutrients. The level of co-existence and nutritional partitioning was significantly modified by abiotic factors, and this can help explain the dominance of specific fungal species in food matrices. This approach could also be useful for understanding competitiveness and dominance of fungal species in other ecosystems, especially in relation to impacts of abiotic and biotic factors.     

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.     

Purification and characterization of a novel antifungal protein secreted by Penicillium chrysogenum from an Arctic sediment

A fungal strain, Penicillium chrysogenum A096, was isolated from an Arctic sediment sample. Its culture supernatant inhibited mycelial growth of some plant pathogenic fungi. After saturation of P. chrysogenum A096 culture supernatant with ammonium sulfate and ion exchange chromatography, a novel antifungal protein (Pc-Arctin) was purified and identified by matrix assisted laser desorption ionization-time of flight-time of flight-mass spectrometry (MALDI-TOF-TOF-MS). The gene encoding for Pc-Arctin consisting of 195 nucleotides was cloned from P. chrysogenum A096 to confirm the mass spectrometry result. Pc-Arctin displays antifungal activity against Paecilomyces variotii, Alternaria longipes, and Trichoderma viride at minimum inhibitory concentrations (MIC) of 24, 48, and 192 ng/disc, respectively. Pc-Arctin was most sensitive to proteinase K and then to trypsin but insensitive to papain. Pc-Arctin possesses high thermostability and cannot be antagonized by common surfactants, except for sodium dodecyl sulfate (SDS). Divalent ions, such as Mn²⁺, Mg²⁺, and Zn²⁺, inhibited the antifungal activity of Pc-Arctin. Hemagglutination assays showed that Pc-Arctin had no hemagglutinating or hemolytic activity against red blood cells (RBC) from rabbits, rats, and guinea pigs. Therefore, Pc-Arctin from Arctic P. chrysogenum may represent a novel antifungal protein with potential for application in controlling plant pathogenic fungal infection.     

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.     

Enrichment of biologically active 18-β glycyrrhetinic acid in Glycyrrhiza glabra root by solid state fermentation

This paper describes the in situ bioconversion of glycyrrhizin of Glycyrrhiza glabra root to 18-β glycyrrhetinic acid by solid state fermentation. Fermentation was carried out with two different fungal strains, Penicillium chrysogenum and Rhizopus oryzae. The solid state fermentation was carried out under stationary state and under rotating state. Penicillium chrysogenum is a better producer of 18-β glycyrrhetinic acid than Rhizopus oryzae. The induced P. chrysogenum seed culture produces higher 18-β glycyrrhetinic acid with 2.955 mg g^?1 and maximum β-glucuronidase activity of 3,583.8 U ml^?1 under stationary solid state fermentation. The mycelium growth and bioconversion rate is highest at pH of 5.5 and 4.5, respectively. G. glabra root supplemented with a solution of dextrose 9 g l^?1, MnSO₄?·?H₂O 3 g l^?1 and (NH₄)₂SO₄ 0.540 g l^?1 produces 48.580 mg of 18-β glycyrrhetinic acid per gram of G. glabra root, i.e. 86.74 % bioconversion by P. chrysogenum in 96 h under stationary state solid state fermentation.     

Diversity of mucoralean fungi in soils of papaya (Carica papaya L.) producing regions in Mexico

Mexico is the fifth largest producer of papaya worldwide and has recently reported problems with mucoralean fungi in this crop. These fungi are considered saprophytes in the soil and are ubiquitous in nature. In this work, they were isolated from soil in regions of intensive papaya cultivation in Mexico. Collections were made in the states of Colima, Oaxaca and Veracruz in Apr 2016. A total of 72 mucorales fungal isolates was obtained and morphologically characterized and then molecular characterization (28S ribosomal region) of 25 representative isolates was carried out. Phylogenetic analysis of the sequences confirmed the presence of the species Gilbertella persicaria, Rhizopus oryzae, Rhizopus stolonifer, Mucor circinelloides and Mucor hiemalis, which cause soft rot in papaya fruits, therefore, spores of these fungi found in the orchard soils can be considered as a constant source of contamination that affects healthy fruits. Additionally, Choanephora cucurbitarum, Mucor ellipsoideus, Rhizopus homothallicus, Rhizopus microsporus, Rhizopus schipperae, Lichteimia ramosa, Gongronella butleri, Cunninghamella bertholletiae and Cunninghamella blakesleeana were identified which are considered to have agricultural, biotechnological and medical importance.     

Dermatophyte and non dermatophyte fungi in Riyadh City,Saudi Arabia

BackgroundDermatophytes are a scientific label for a group of three genera (Microsporum, Epidermophyton and Trichophyton) of fungus that causes skin disease in animals and humans. Conventional methods for identification of these fungi are rapid and simple but are not accurate comparing to molecular methods.ObjectiveThis study aimed to isolate human pathogenic dermatophytes which cause dermatophytosis in Riyadh City, Saudi Arabia and to identify these fungi by using conventional and molecular methods.MethodsThe study was conducted in Medical Complex, Riyadh and King Saud University. Samples of infected skin, hairs and nails were collected from 112 patients. Diagnosis of skin infections, direct microscopic test, isolation and identification of dermatophytes by conventional and molecular methods were carried out.ResultsThe results indicated that the tinea capitis infection had the highest prevalence among the patients (22.3%) while Tinea barbae had the lowest. In this study the identified dermatophyte isolates belong to nine species as Trichophyton violaceum, Trichophyton verrucosum, Trichophyton rubrum, Trichophyton mentagrophytes, Trichophyton schoenleinii, Trichophyton concentricum, Microsporum canis, Microsporum audouinii and Epidermophyton floccosum which cause skin infections were isolated during this study. Non dermatophyte isolates included 5 isolates from Aspergillus spp. 4 isolates from Acremonium potronii and 15 isolates from Candida spp. M. canis were the most common species (25% of isolated dermatophytes). Out of the 52 dermatophyte isolates identified by conventional methods, there were 45 isolates identified by the molecular method.ConclusionsThe results concluded that approximately M. canis caused a quarter of dermatophyte cases, tinea capitis infection was prevalent and the molecular method was more accurate than conventional methods.     

Mycobiota and molecular detection of Aspergillus flavus and A. parasiticus aflatoxin contamination of Strawberry (Fragaria ananassa Duch.) fruits

Strawberry fungi were isolated from fresh fruits and juice on the two types of media (Sabouraud dextrose agar, SDA and potato-dextrose agar, PDA) at 28 °C. Nineteen fungal species belong to 12 genera were isolated from fruits and juice on both isolation media. The most common fungal genera and species were Aspergillus flavus, A. niger, Mucor racemosus, Neurospora crassa, Penicillium chrysogenum, Rhizopus stolonifer and Trichoderma harzianum. Twenty A. flavus and A. parasitics isolates were assayed for their abilities to produce aflatoxins. The concentration of aflatoxins ranged between 25.8–75.2 and 23.6–71.1 ng/ml at 350 and 365 nm, respectively. Among A. flavus and A. parasiticus strains tested, aflatoxin B contributed 30–60% of total isolates. However, G type contributed 85–90%. The R_f values of B₁, B₂, G₁ and G₂ were 0.79, 0.61, 0.44 and 0.32, respectively. High-performance liquid chromatography analysis of extracts revealed the presence of aflatoxins with variable levels.     

PcMtr, an aromatic and neutral aliphatic amino acid permease of Penicillium chrysogenum

The gene encoding an aromatic and neutral aliphatic amino acid permease of Penicillium chrysogenum was cloned, functionally expressed and characterized in Saccharomyces cerevisiae M4276. The permease, designated PcMtr, is structurally and functionally homologous to Mtr of Neurospora crassa, and unrelated to the Amino Acid Permease (AAP) family which includes most amino acid permeases in fungi. Database searches of completed fungal genome sequences reveal that Mtr type permeases are not widely distributed among fungi, suggesting a specialized function.     

Characterization of a 52 kDa Exoantigen of <Emphasis Type="Italic">Penicillium chrysogenum</Emphasis> and Monoclonal Antibodies Suitable for its Detection

The indoor clade of Penicillium chrysogenum, the so-called Fleming clade, is the most common species of Penicillium on moldy building materials. In a previous study, we identified a 52 kDa human antigen characteristic of the indoor clade of P. chrysogenum not present in a taxonomically diverse selection of fungi. Further investigations revealed that it is a modestly glycosylated mature protein with a pI 5.3. The protein is apparently identical to a glucoamylase previously reported from an aluminum-tolerant P. chrysogenum mutant. Based on sequence similarity, molecular weight, and pI, it is distinct from a number of other glucoamylases from domesticated strains of Aspergillus oryzae and A. niger used to produce industrial enzymes. Surprisingly, it had not been reported as an allergen. The monoclonal antibodies developed have the potential for use in assays of P. chrysogenum antigens in spores and spore/mycelial fragments in dust.     

Microbiological and molecular identification of bacterial species isolated from nasal and oropharyngeal mucosa of fuel workers in Riyadh,Saudi Arabia

This study aimed to determine the bacterial species colonizing the nasal and oropharyngeal mucosa of fuel workers in Central Riyadh, Saudi Arabia on a microbiological and molecular level. Throat and nasal swab samples were obtained from 29 fuel station attendants in the period of time extending from March to May 2014 in Riyadh, Saudi Arabia. Microbiological identification techniques were utilized to identify the bacterial species isolated. Antibiotic sensitivity was assessed for each of the bacterial isolates. Molecular identification techniques based on PCR analysis of specific genomic sequences was conducted and was the basis on which phylogeny representation was done for 10 randomly selected samples of the isolates. Blood was drawn and a complete blood count was conducted to note the hematological indices for each of the study participants. Nineteen bacterial species were isolated from both the nasal cavity and the oropharynx including Streptococcus thoraltensis, alpha-hemolytic streptococci, Staphylococcus hominis, coagulase-negative staphylococci, Leuconostoc mesenteroides, Erysipelothrix rhusiopathiae and several others. We found 100% sensitivity of the isolates to ciprofloxacin, cefuroxime and gentamicin. Whereas cefotaxime and azithromycin posted sensitivities of 85.7% and 91.4%, respectively. Low sensitivities (<60% sensitivity) to the antibiotics ampicillin, erythromycin, clarithromycin and norfloxacin were observed. Ninety-seven percent similarity to the microbial bank species was noted when the isolates were compared to it. Most hematological indices recorded were within the normal range. In conclusion, exposure to toxic fumes and compounds within fuel products may be a contributing factor to bacterial colonization of the respiratory tract in fuel workers.