Dichloran Rose Bengal Chloramphenicol Agar Research Articles

P362 Seasonal trend of fungal flora in water of tertiary care hospital in North India

Poster session 3, September 23, 2022, 12:30 PM - 1:30 PMObjectivesThe study was conducted to assess the seasonal variation of fungal flora in hospital water of a tertiary care hospital in North India.MethodsA total of 200 water samples from the main reservoir, overhead and underground tanks, and taps of critical care units of the hospital were collected. The water samples were filtered by membrane filtration technique (0.22 micron) and cultured on dichloran rose-Bengal chloramphenicol agar with and without benomyl. The plates were incubated for upto 15 days and fungal colonies recovered were sub-cultured on Sabouraud Dextrose Agar and identified by phenotypic methods. Yeasts were identified by Matrix Assisted Laser Desorption Ionization Time of Flight (MALDI-TOF-MS).ResultsMycelial fungi were isolated from 100% of the water samples which included Alternaria, Curvularia, Nigrospora, Penicillium, Aspergillus, Paecilomyces, Scytillidium, and Mycelia sterilia as depicted in Figure 1. Different fungi were prevailing in different water storage units like: Advance eye center— A. fumigatus, A. flavus, Fusarium solani, Alternaria spp. Rhizopus arrhizus, Ustilago spp., mycelia sterilia, Trichosporon spp.; Advance trauma Center—Cladophaialophora spp., Alternaria alternata, Penicillium, spp. A. flavus, A. nidulans, A. fumigatus, F. solani, Rhodotorula spp.: Bone marrow transplant unit— Alterneria alternata, A. niger, A. flavus, A. versicolor, Cladosporium spp., Fonsecaea pedrosoi, Fusarium spp., Nigrospora spherica, Penicillium spp., Rhodotorula spp., Trichosporon ashaii. The seasonal variation of fungal isolation is depicted in Figure 2. Isolation rate of Aspergillus species was 35% in winters, 31% in post-monsoon, 25% in summers. Isolation rate of Penicillium species was 19% in post-monsoon, 16% in winter and 15% in summers. Maximum number of dematiaceous fungi were isolated in summer season with isolation rate of 30% in summers as compared with 21.5% in post-monsoon and 19% in winters. Few yeasts isolated were Rhodotorula, Trichosporon, and Ustilago. Mucorales isolated rarely included Rhizopus, Absidia, Syncephalastrum, and Mucor species. Fungal colony forming units in the water samples ranged from 50 to 450 colony forming units/liter of water.ConclusionThe distribution of fungi in hospital water showed diversity and seasonal variability. Aspergillus species were isolated in maximum number in the winter season, Penicillium species in post-monsoon season and dematiaceous fungi in the winter season. Water as a source of fungal infection in critical care units, remains a relatively neglected area. Water supply could be a source of nosocomial fungal infections. Improving the quality of water by regular testing for fungal contamination and appropriate action to reduce its burden may reduce the hospital-acquired fungal infections.

Evaluation of environmental Mucorales contamination in and around the residence of COVID-19-associated mucormycosis patients.

IntroductionRecently, India witnessed an unprecedented surge of coronavirus disease 2019 (COVID-19)-associated mucormycosis (CAM) cases. In addition to patient management issues, environmental Mucorales contamination possibly contributed to the outbreak. A recent study evaluated environment contamination by Mucorales in the hospital setting. However, a considerable number of CAM patients were never admitted to a hospital before the development of the disease. The present study, therefore, planned to evaluate Mucorales contamination of patients’ residences.MethodsThe residential environment of 25 patients with CAM living in north India was surveyed. Air samples were collected from indoor and immediate outdoor vicinity of the patients’ residence and cultured on Dichloran Rose–Bengal Chloramphenicol (DRBC) agar with benomyl for selective isolation of Mucorales. Surface swab samples were also collected from the air coolers fitted in those residences and cultured on DRBC agar. The isolates were identified by phenotypic and genotypic methods. Amplified fragment length polymorphism (AFLP) was employed to evaluate the genetic relatedness of the environmental and patients’ clinical isolates.ResultsThe median spore count (mean ± SD, cfu/m3) of Mucorales in the air of patients’ bedrooms was significantly higher than in the air in other rooms in those residences (3.55 versus 1.5, p = 0.003) or the air collected directly from the front of the air cooler (p < 0.0001). The Mucorales spore count in the environment did not correlate with either ventilation of the room or hygiene level of the patients’ residences. Rhizopus arrhizus was isolated from the environment of all patients’ residences (n = 25); other Mucorales species isolated were Cunninghamella bertholletiae (n = 14), Rhizopus microsporus (n = 6), Rhizopus delemar (n = 6), Syncephalastrum racemosum (n = 1), Lichtheimia corymbifera (n = 1), and Mucor racemosus (n = 1). Genetic relatedness was observed between 11 environmental isolates from the patients’ bedrooms and respective clinical isolates from patients.DiscussionThe study supported the view that the patients might have acquired Mucorales from the home environment during the post-COVID-19 convalescence period. Universal masking at home during patients’ convalescence period and environmental decontamination could minimize exposure in those susceptible patients.

Matrix Extension of the CompactDry™ "Nissui" YMR for Enumeration of Yeasts and Molds in Dried Cannabis Flower: AOAC Performance Tested MethodSM 092002.

CompactDry™ Yeast/Mold Rapid (YMR) is a ready-to-use dry media sheet using a chromogenic medium with selective agents for the enumeration of yeasts and molds in a variety of food products after incubation at 25 ± 1°C for 3 days. The method is certified as AOAC Performance Tested MethodSM 092002. The CompactDry YMR method was validated for a matrix extension to cannabis flower through the AOAC Emergency Response Validation process. The performance of the CompactDry YMR was compared to Dichloran Rose Bengal Chloramphenicol (DRBC) agar for the enumeration of yeasts and molds in cannabis flower. Matrix data were normalized by log10 transformation, and performance indicators included repeatability, difference of means (DOM), and inclusivity/exclusivity. The results demonstrated that the CompactDry YMR method is equivalent to the DRBC agar method at 72 h of incubation. In the independent laboratory validation study, there was no significant difference in detection, enumeration, or repeatability between the CompactDry YMR method and DRBC agar at 72 h. Eighteen inclusivity and 16 exclusivity strains specific to cannabis plant materials that were not evaluated in the original CompactDry YMR method validation were tested in this study. All inclusivity organisms produced typical colonies on the CompactDry YMR. The two exclusivity bacterial strains that showed growth on CompactDry YMR at 72 h were inoculated at a high concentration. CompactDry YMR is equivalent in performance to traditional culture media detection methods of yeasts and molds. CompactDry YMR will streamline dried cannabis flower testing.

Is there an association between zinc and COVID-19-associated mucormycosis? Results of an experimental and clinical study.

BackgroundThe enormous increase in COVID‐19–associated mucormycosis (CAM) in India lacks an explanation. Zinc supplementation during COVID‐19 management is speculated as a contributor to mucormycosis. We conducted an experimental and clinical study to explore the association of zinc and mucormycosis.MethodsWe inoculated pure isolates of Rhizopus arrhizus obtained from subjects with CAM on dichloran rose Bengal chloramphenicol (DRBC) agar enriched with (three different concentrations) and without zinc. At 24 h, we counted the viable colonies and measured the dry weight of colonies at 24, 48 and 72 h. We also compared the clinical features and serum zinc levels in 29 CAM cases and 28 COVID‐19 subjects without mucormycosis (controls).ResultsWe tested eight isolates of R arrhizus and noted a visible increase in growth in zinc‐enriched media. A viable count percentage showed a significantly increased growth in four of the eight isolates in zinc‐augmented DRBC agar. A time‐ and concentration‐dependent increase in the mean fungal biomass with zinc was observed in all three isolates tested. We enrolled 29 cases of CAM and 28 controls. The mean serum zinc concentration was below the reference range in all the subjects and was not significantly different between the cases and controls.ConclusionsHalf of the R arrhizus isolates grew better with zinc enrichment in vitro. However, our study does not conclusively support the hypothesis that zinc supplementation contributed to the pathogenesis of mucormycosis. More data, both in vitro and in vivo, may resolve the role of zinc in the pathogenesis of CAM.

Exploring potentials of indigenous yeasts in fermentation of Chambourcin Grapes

Background: Indigenous yeasts present on grape berries have been shown to impact the winemaking process and final wine quality. In this study, we used Chambourcin, a hybrid grape cultivar, to isolate indigenous yeasts for potential use in winemaking. Hybrid grapes are of particularly interest due to higher resistance to cold temperature and fungal diseases. Methods: Yeasts were isolated from spontaneous fermentation of crushed grapes by plating on Dichloran Rose Bengal Chloramphenicol agar and identified by Sanger sequencing. Yeast candidates were selected for their ability to grow in Yeast Extract-Peptone-Dextrose broth supplemented with varying ethanol concentration. Candidate yeasts were chosen for mock fermentation using sterile Chambourcin juice. Volatile and non-volatile compounds that predict wine quality (flavor and aroma) were measured by UPLC and GC-MS. Results: Hanseniaspora uvarum, Starmerella bacillaris, Candida californica, and Zygoascus meyerae were predominantly isolated from Chambourcin. S. bacillaris isolate 180002 and C. californica isolate 180004 were able to tolerate up to 10% ethanol compared to S. cerevisiae (ethanol tolerance up to 12%). Our results demonstrate that isolate 180002 grown in ethanol supplemented medium is comparable to the commercial S. cerevisiae. Various aroma profiles of three main chemical families – esters, higher alcohol and volatile acids in products fermented by S. bacillaris and other isolates were observed. Conclusion: Starmerella bacillaris isolate 180002 demonstrates potential for use in winemaking with hybrid grapes based on ethanol tolerance and production of wine related chemical compounds. This study further supports the application of indigenous grape-associated yeasts in creating flavor and aroma diversity of wine.

Characterization of Saccharomyces cerevisiae Strains from Palm Wine for Baking Leaven Production in Republic of Congo

This study was carried out to select the autochthon yeast strains from the palm wine. The work aims to wildly isolate, characterize and identify Saccharomyces cerevisiae in order to locally and safely produce a bio-leaven and to assess their ability to ferment wheat flour dough used for the bread making and other products. Palm wine samples have been bought in the village around Brazzaville. Samples have been collected in sterile glass bottles then stored under refrigeration condition at 4°C until they were processed. Yeasts were enumerated and isolated on Dichloran Rose Bengal Chloramphenicol agar medium at 37°C. Molecular identification was carried out by a PCR-based method using species-specific primer pairs to Saccharomyces genus without sequencing stage. The results showed that palm wine contains about 3.3106 CFUmL-1. Fifty-one isolates were obtained with a percentage of 13% corresponding to Saccharomyces cerevisiae. This identity was confirmed by the molecular characterization. Five yeast strains showed high ability to ferment wheat flour dough analogous of the control. The better growth of isolates was between 28 and 37°C. However, the near neutral pH gives the higher growth. High concentration of NaCl and glucose in medium has a strong negative impact on the growth of isolates.

Short communication: Pseudomonas azotoformans causes gray discoloration in HTST fluid milk

Pseudomonas species are well recognized as dairy product spoilage organisms, particularly due to their ability to grow at refrigeration temperatures. Although Pseudomonas-related spoilage usually manifests itself in flavor, odor, and texture defects, which are typically due to production of bacterial enzymes, Pseudomonas is also reported to cause color defects. Because of consumer complaints, a commercial dairy company shipped 4 samples of high temperature, short time (HTST)-pasteurized milk with distinctly gray colors to our laboratory. Bacterial isolates from all 4 samples were identified as Pseudomonas azotoformans. All isolates shared the same partial 16S rDNA sequence and showed black pigmentation on Dichloran Rose Bengal Chloramphenicol agar. Inoculation of one pigment-producing P. azotoformans isolate into HTST-pasteurized fluid milk led to development of gray milk after 14 d of storage at 6°C, but only in containers that had half of the total volume filled with milk (∼500 mL of milk in ∼1,000-mL bottles). We conclusively demonstrate that Pseudomonas can cause a color defect in fluid milk that manifests in gray discoloration, adding to the palette of color defects known to be caused by Pseudomonas. This information is of considerable interest to the dairy industry, because dairy processors and others may not typically associate black or gray colors in fluid milk with the presence of microbial contaminants but rather with product tampering (e.g., addition of ink) or other inadvertent chemical contamination.

Mycobiota of Slovak wine grapes with emphasis on aspergillus and penicillium species in the south Slovak wine region

The Southern Slovak wine growing region is warmest part of Slovakia and is suitable for cultivating the grapes for production of wines at high quality. From the eight vineyards were collected 8 samples of wine grapes (white 7, blue 1) during harvesting 2011, 2012 and 2013. The aim of this work was to gain more knowledge about mycobiota on grapes originating from Slovakia, to identify Aspergillus and Penicillium species according to their morphopogy and evaluate the presence of secondary metabolites (also including intracellular and extracellular mycotoxins) produced in in vitro conditions by thin layer chromatography method from fresh grape berries. Fifty wine grapes per bunch (approximately 7 - 8 berries per plate) that showed no symptoms were randomly selected on Dichloran Rose Bengal Chloramphenicol agar medium. The plates were then incubated aerobically at 25 ±1 °C for 5 to 7 days in the dark. Of these samples were identified 17 genera. One hundred percent of samples were colonies by the genus Penicillium and 75% by the genus Aspergillus. During the survey, 135 isolates belonging to 9 Penicillium species (P. aurantiogriseum, P. canescens, P. citrinum, P. crustosum, P. decumbens, P. expansum, P. funiculosum, P. chrysogenum and P. purpurogenum) and 26 isolates belonging to 3 Aspergillus species (A. clavatus, A. flavus and A. section Nigri) were isolated and identified from exogenous contamination. The main occurring penicillium species of the samples were P. expansum (37.5% Fr), followed P. citrinum, P. chrysogenum and P. crustosum (25% Fr). The main occurring aspergillus species of the samples were A. section Nigri (62.5%). Eight potentially toxigenic species were tested for their toxigenic ability. It was confirmed the production of various mycotoxins such as aflatoxin B1, citrinin, patulin, cyclopiazonic acid, penitrem A and roquefortin C. Out of 34 strains, 56% produced at least one mycotoxin.

Species of genera Botrytis, Fusarium and Rhizopus on grapes of the Slovak origin

Our research was focused to identify the Botrytis, Fusarium and Rhizopus species from grapes of the Slovak origin. A further goal of the project was to characterized toxinogenic potential of chosen strains of species Fusarium. 50 samples of grapes, harvested in years 2011, 2012 and 2013 from various wine-growing regions were analyzed in this study. For the isolation of species the of direct plating method was used: a) surface-sterilized berries (using 1% freshly pre-pared chlorine) b) berries and c) damaged berries on DRBC (Dichloran Rose Bengal Chloramphenicol agar). For each analysis were used 50 berries (or all damaged berries from sample). The cultivation was carried at 25 ±1°C, for 5 to 7 days in dark. After incubation, the colonies of Botrytis, Fusarium and Rhizopus were transferred to identification media and after incubation strains were identified to species level. Thirteen species of fusaria (F. acuminatum, F. avenaceum, F. culmorum, F. equiseti, F. graminearum, F. oxysporum, F. proliferatum, F. semitectum, F. solani, F. sporotrichioides, F. subglutinans F. tricinctum and F. verticilioides) were identified. Frequency of fusaria isolation was 92 %. Botrytis cinerea was determined from 86% samples and Rhizopus from 94%. Chosen strains of species of genus Fusarium were able to produce following mycotoxins: deoxynivalenol, T-2 toxin, HT-2 toxin and diacetoxyscirpenol in in vitro conditions as determinated by thin-layer chromatography. Thirty-two (68%) of tested isolates of Fusarium species were able to produce at least one mycotoxin.

Role of Fungal Biomass in N-Hexane Biofiltration

The biofiltration of n-hexane is studied to optimize determinants factors of hydrophobic VOC filtration efficiency. Four trickle-bed air biofilters (TBABs) were employed; two of which were supplied with nutrients buffered at a neutral pH, while another two at an acidic pH of 4 to induce and enhance fungal growth. The loading rate of n-hexane was kept constant in all TBABs at 13 g/m3/h. At each pH levels studied, the biomass of the TBABs was pre-acclimated using different ratios of n-hexane and methanol. The fungal biomass responsible for the degradation of n-hexane was then examined and quantified. Dichloran Rose Bengal Chloramphenicol agar was used for fungi quantification, and optical microscopy for classification. Effluent biomass was validated by measuring volatile suspended solids. Fungal counts resulting from n-hexane biodegradation were related to nitrate and carbon consumption. It was found that n-hexane elimination capacity closely followed biomass growth, and reached a steady-state at an optimum biomass density of roughly 3000 cfu/ml. Major shifts in fungal species were observed in all TBABs. Dominant fungal species grew slowly to become the most numerous, and were found to provide maximum elimination capacity, although TBABs pre-acclimated to higher methanol concentrations took less time to reach this steady-state. It was concluded, therefore, that steady and monitored growth of TBAB biomass is an essential factor in maximizing fungi’s ability to metabolize VOCs and that a new ecological biofiltration model may be the most effective at VOC purification. Keywords

Prelimanary Observation On Microbial Population in The Soaking Water During Bedak Sejuk Production

Background/Objectives: During the production of Bedak sejuk (local face powder cosmetic made from rice), the soaking water were changed intermittently because of its pungent smell, believe to be caused by microbial reactions. In this initial investigation, we monitored the microbial population in the series of batch fermentation of Bedak sejuk. Methods: Local polished rice grains (Indica) were soaked in tap water (w/v) at ambient temperature mimicking the usual preparation of Bedak sejuk. The overall soaking period was 28 days (two times of soaking, 14 days for each interval). The soaking water was serially diluted and then spread over several types of agar. To count the Lactic Acid Bacteria (LAB), the diluted samples were spread on the MRS media, yeast and mould were spread on Dichloran Rose Bengal Chloramphenicol agar (DRBC) while coliforms were spread on Petri-Film. The results were indicated as Colony-Forming Units per milligram (CFU/mL) of soaking water. Findings: For the first batch fermentation, the microbial count in soaking water was 11.44 log CFU/mL. LAB counts increased from 3.02 to 6.14 log CFU/mL at the end of fermentation time. Yeasts count increased from 1.68 to 2.78 log CFU/mL, mould count also increased from 0.73 to 1.31 log CFU/mL while coliform count decreased from 3.88 to 1.21 log CFU/mL. While for the second batch fermentation, microbial count in soaking water was 13.15 log CFU/mL. LAB counts increased from 5.65 to 6.81 log CFU/mL at the end of fermentation time. Yeasts count increased from 2.34 to 3.37 log CFU/mL, mould count also increased from 1.33 to 2.08 log CFU/mL while coliform count decreased from 2.38 to 0.89 log CFU/ml. Application: The results of the study have given the initial background information on the varieties of microbial populations that presence during the natural fermentation of Bedak sejuk.

Colonization of grapes berries by Alternaria sp. and their ability to produce mycotoxins

Our research focused on identify the Alternaria species from grapes (surface sterilized berries and non-surface sterilized berries) of Slovak origin and characterize their toxinogenic potential in in vitro conditions. We analyzed 47 samples of grapes, harvested in years 2011, 2012 and 2013 from various wine-growing regions. For the isolation of species, the method of direct plating berries and surface-sterilized berries (using 1 % freshly pre-pared chlorine) on DRBC (Dichloran Rose Bengal Chloramphenicol agar) was used. For each analysis was used 50 berries. Only undamaged berries have been used for analysis. The cultivation was carried at 25 ±1°C, for 5 to 7 days in dark. After incubation, the colonies of Alternaria were transferred on PCA - potato-carrot agar and CYA - Czapek-yeast extract agar and cultured for 7 days at room temperature and natural light. A total 4 species-groups of the genus Alternaria were isolated from grapes berries: Alternaria alternata (1369 isolates), Alternaria arborescens (734 isolates), Alternaria infectoria (143 isolates), and Alternaria tenuissima (3579 isolates). According to European Union legislation mycotoxins produced by species genus Alternaria are not monitored in foods and food commodities. Mycotoxins such as alternariol and alternariol monomethylether are mutagenic and genotoxic in various in vitro systems. Selected strains were tested for production of altenuene, alternariol monomethylether and alternariol. In neither case of A. infectoria species-group isolates was confirmed the production of tested mycotoxins in in vitro conditions by TLC method. The ability to produce altenuene, alternariol monomethylether and alternariol in in vitro conditions was detected in isolates of Alternaria alternata, Alternaria arborescens and Alternaria tenuissima species-groups. Isolates of Alternaria alternata species-group (44 tested isolates) were able to produce altenuene (24 isolates), alternariol monomethyleter (42 isolates) and alternariol (43 isolates). Only one isolate did not produce any mycotoxins. Isolates of Alternaria arborescens species-group (38 tested isolates) were able to produce altenuene (24 isolates), alternariol monomethyleter (33 isolates) and alternariol (36 isolates). Only two isolates did not produce any mycotoxins. Isolates of Alternaria tenuissima species-group (87 tested isolates) were able to produce altenuene (42 isolates), alternariol monomethyleter (41 isolates) and alternariol (73 isolates). Thirteen isolates did not produce any mycotoxins.

Mycobiota of Slovak wine grapes with emphasis on Aspergillus and Penicillium species in the small carpathian area

The Slovak wine-growing region is divided into six viticulture areas. The largest in size and the most important over the centuries has been the Small Carpathian area (around 5800 ha of vineyards) spreads in the western of Slovakia. The objectives of this study were: to gain more knowledge about mycobiota on grapes originating from Slovakia, with a focus on genera Aspergillus and Penicillium and their ability to produce mycotoxins in in vitro conditions by thin layer chromatography method. From the twelve vineyards were collected 14 samples of wine grapes (white 6, blue 8) during harvesting 2011, 2012 and 2013. Fifty wine grapes per bunch (approximately 7-8 berries per plate) that showed no symptoms were randomly selected on Dichloran Rose Bengal Chloramphenicol agar medium. The plates were then incubated aerobically at 25 ±1 °C for 5 to 7 days in the dark. Of these samples were identified 22 genera. Ninety-three percent of samples were colonies by the genus Penicillium and 79% by the genus Aspergillus. During the survey, 251 isolates belonging to 14 Penicillium species (P. aurantiogriseum, P. citrinum, P. coprophylum, P. crustosum, P. expansum, P. funiculosum, P. glabrum, P. griseofulvum, P. chrysogenum, P. oxalicum, P. polonicum, P. purpurogenum, P. roqueforti and P. thomii) and 37 isolates belonging to 7 Aspergillus species (A. clavatus, A. flavus, A. section Nigri, A. ostianus, A. parasiticus, A. versicolor and A. westerdijkiae) were isolated and identified from exogenous contamination. The main occurring penicillium species of the samples were P. chrysogenum (36% Fr), followed P. crustosum (29% Fr), P. griseofulvum (21% Fr) and P. expansum (21% Fr). The main occurring aspergillus species of the samples were A. section Nigri (64%). Thirteen potentially toxigenic species were tested for their toxigenic ability. It was confirmed the production of various mycotoxins such as aflatoxin B1, G1, citrinin, griseofulvin, patulin, cyclopiazonic acid, penitrem A, roquefortin C and sterigmatocystin. Out of 124 strains, 84% produced at least one mycotoxin.

Colonization of grapes berries and cider by potential producers of patulin

The aim of this study was to detect potential producers of mycotoxin patulin from grapes (berries, surface sterilized berries - endogenous mycobiota and grape juice) of Slovak origin. We analyzed 47 samples of grapes, harvested in 2011, 2012 and 2013 from various wine-growing regions. For the isolation of species we used the method of direct plating berries and surface-sterilized berries (using 1% freshly pre-pared chlorine) berries on DRBC (Dichloran Rose Bengal Chloramphenicol agar). For the determination of fungal contamination of grape juice we used plate-dilution method and DRBC and DG18 (Dichloran 18% Glycerol agar) as media. The cultivation in all modes of inoculation was carried at 25 ±1 °C, for 5 to 7 days. After incubation Aspergillus and Pencillium isolates were inoculated on the identification media. The potential producers of patulin were isolated from 23 samples berries, 19 samples of surface-sterilized berries and 6 samples of grape juice. Overall, the representatives of producers of patulin were detected in 32 (68.1%) samples (75 isolates). In this work we focused on the detection of potential producers of patulin, Penicillium expansum (the most important producer of patulin in fruits), Penicillium griseofulvum and Aspergillus clavatus were isolated. Chosen isolates of potential patulin producers were tested for the ability to produce relevant mycotoxins in in vitro conditions using thin layer chromatography method. The ability to produce patulin in in vitro condition was detected in 82% of isolates of Penicillium expansum, 65% of Penicillium griseofuvum and 100% of Aspergillus clavatus. Some isolates of Penicillium expansum were able to produce citrinin and roquefortine C, Penicillium griseofulvum cyclopiazonic acid, griseofulvin and roquefortin C, also.

ENDOGENAL COLONIZATION OF GRAPES BERRIES

The aim of study was to detect the microscopic filamentous fungi from wine surface of sterilized grapes berries of Slovak origin. We analyzed 21 samples of grapes, harvested in the year 2012 of various wine-growing regions. For the isolation of species we used the method of direct plating surface-sterilized berries (using 0.4% freshly pre-pared chlorine) on DRBC (Dichloran Rose Bengal Chloramphenicol agar). The cultivation was carried at 25±1°C, for 5 to 7 days. A total number of 2541 fungal isolates pertaining to 18 genera including Mycelia sterilia were recovered. Isolates of genus Alternaria were found in all of tested samples with the highest relative density 56.4%. The second highest isolation frequency we detected for genus Fusarium (90.48% positive samples), but with low relative density (31 isolates and 2.99% RD). Another genera with higher isolation frequency were Cladosporium (Fr 85.71%, RD 14.6%), Mycelia sterilia (Fr 85.71%, RD 4.25%), Penicillium (Fr 80.95%, RD 13.42%), Botrytis (Fr 71.43%, RD 2.95%) Rhizopus (Fr 66.66%, RD 1.34%), Aspergillus (Fr 57.14%, RD 0.87%), Epicoccum (Fr 47.62%, RD 1.22%), Trichoderma (Fr 42.86%, RD 1.26%). Isolation frequency of another eight genera (Arthrinium, Dichotomophtora, Geotrichum, Harzia, Chaetomium, Mucor, Nigrospora and Phoma) was less than 10% and relative density less than 0.5%. Chosen isolates of potential producers of mycotoxin (species of Alternaria, Aspergillus, Fusarium and Penicillium) were tested for the ability to produce relevant mycotoxins in in vitro conditions using TLC method. None isolate of Aspergillus niger aggregate (13 tested) did not produce ochratoxin A – mycotoxin monitored in wine and another products from grapes berries. Isolates of potentially toxigenic species recovered from the samples were found to produce another mycotoxins: aflatoxin B1, altenuene, alternariol, alternariol monomethylether, citrinin, diacetoxyscirpenol, deoxynivalenol, HT-2 patulin, penitrem A and T-2 toxin in in vitro conditions. In conclusion, another research should be performed to detect the occurrence of these mycotoxins in grapes, must, wine and another products from grape.

MICROFUNGI AND MYCOTOXINS OF GRAPES FROM EASTERN SLOVAK WINE REGION

The current study investigated an endogenous mycobiota of grapes in Eastern wine region, Slovakia and detection a potentially pathogenic isolates to produce selected mycotoxins. Intact berries from four wine grape cultivars were tested. Seven/eight berries superficially sterilized from each samples were placed on a Dichloran Rose Bengal Chloramphenicol agar in a total of 50 and incubated at 25 °C, 5 - 7 days. A total of 582 isolates were obtained that belonged to ten genera: Alternaria, Aspergillus, Botrytis, Cladosporium, Epicoccum, Fusarium, Mucor, Penicillium, Rhizopus, Trichoderma and one unidentified genus Mycelium sterillium without creation fruiting bodies. The most frequent were genera Alternaria, Aspergillus, Botrytis and Penicillium with 100 % frequention. The most frequent Aspergillus species was Aspergillus section Nigri (100 %) and Penicillium species was Penicillium chrysogenum (50 %). The largest number of isolates belonged to Alternaria (275 isolates), Cladosporium (114 isolates) and Penicillium (92 isolates). For that reason the relative density of both genera were the highest 42 %, 19.6 % and 15.8 %, respectively. The selected isolates – Aspergillus flavus, Aspergillus section Nigri, Penicillium citrinum, Penicillium expansum and Penicillium chrysogenum were tested for patulin, citrinin, penitrem A, roquefortin C, ochratoxin A, aflatoxin B1, G1 and cyclopiazonic acid producing ability. Out of 11 strains 54 % produced at least one mycotoxin. In our research ochratoxigenic microfungi (some species of Aspergillus section Nigri) were found in grape samples but without production of OTA.

MICROFUNGI AND MYCOTOXINS OF GRAPES FROM TOKAJ WINE REGION

The aim of this study was to investigate a surface mycobiota of grapes and detection a potentially pathogenic isolates to produce selected mycotoxins. Three samples of wine grapes Furmint, Lipovina and Yellow muscat were collected in the Tokaj wine region in Viničky during the November harvest in 2012. Seven/eight berries from each sample were placed on a Dichloran Rose Bengal Chloramphenicol agar in a total of 50 and incubated at 25 °C for 5 - 7 days. After incubation from the samples were isolated following filamentous fungi: Alternaria, Aspergillus, Botrytis, Cladosporium, Mucor, Penicillium, Phoma, Rhizopus, Trichoderma and Mycelium sterillium. The most frequent were genera Aspergillus, Botrytis and Penicillium with 100 % frequention. The largest number of isolates belonged to Penicillium (368 isolates) and Botrytis (334 isolates). For that reason the relative density of both genera were the highest 47 % and 43 %, respectively. The selected isolates - Aspergillus section Nigri, Penicillium crustosum, Penicillium expansum and Penicillium chrysogenum were tested for patulin, citrinin, penitrem A, roquefortin C and ochratoxin A producing ability. A higher percentage of Penicillium crustosum isolates (79 %) were positive for penitrem A and all were positive for roquefortin C. A higher percentage of Penicillium expansum (83 %) were positive for patulin, 72 % for citrinin and they were all positive for roquefortin C. All isolates of Penicillium chrysogenum produced roquefortin C. As would be expected, ochratoxin A was not detected from Aspergillus section Nigri.

Physiological behaviour of gliotoxigenic Aspergillus fumigatus sensu stricto isolated from maize silage under simulated environmental conditions

Environmental conditions play a key role in fungal development. During the silage production process, humidity, oxygen availability and pH vary among lactic-fermentation phases and among different silage sections. The aim of this work was to study the physiological behaviour of gliotoxicogenic Aspergillus fumigatus strains isolated from maize silage under simulated natural physicochemical conditions – different water activities (aW), temperatures (Tº), pH and oxygen pressure – on the growth parameters (growth rate and lag phase) and gliotoxin production. The silage was made with the harvested whole maize plant that was chopped and used for trench-type silo fabrication. Water activity and pH of the silage samples were determined. Total fungal counts were performed on Dichloran Rose Bengal Chloramphenicol agar and Dichloran 18% Glycerol agar. The morphological identification of A. fumigatus was performed with different culture media and at different growth temperature to observe microscopic and macroscopic characteristics. Gliotoxin production by A. fumigatus was determined by HPLC. All strains isolated were morphologically identified as A. fumigatus. Two A. fumigatus strains isolated from the silage samples were selected for the ecophysiological study (A. fumigatus sensu stricto RC031 and RC032). The results of this investigation showed that the fungus grows in the simulated natural physicochemical conditions of corn silage and produces gliotoxin. The study of the physiological behaviour of gliotoxigenic A. fumigatus under simulated environmental conditions allowed its behaviour to be predicted in silage and this will in future enable appropriate control strategies to be developed to prevent the spread of this fungus and toxin production that leads to impairment and reduced quality of silage.

Characterization of Aspergillus section Flavi from pistachio soils in Iran

Abstract During 2012, soil samples from commercial pistachio orchards in three major production regions include Rafsanjan (Kerman Province, center of Iran), Damghan (Semnan Province, north-central Iran) and Feyz-Abad (Khorasan-e Razavi Province, northeastern Iran), were assayed on Dichloran Rose-Bengal Chloramphenicol agar (DRBC) and Aspergillus flavus-parasiticus agar media to quantify populations of Aspergillus species from the section Flavi. The mean propagule density of Aspergillus species from the Flavi section [log10 (CFU/g soil)] was higher in Feyz-Abad (3.06, 2.88–3.24), compared to Damghan (2.55, 2.44–2.65) and Rafsanjan (2.40, 2.26–2.54). A. flavus (69.7, 65.3 and 57.9%), A. parasiticus (19.6, 25.4, and 29.3%), and A. nomius (10.7, 9.3, and 12.8%) were the predominant species in the regions of Rafsanjan, Damghan, and Feyz-Abad, respectively. There were significant differences among sclerotia producing isolates of A. flavus in the sampling regions (p &lt; 0.05). The percentage of sclerotium-producing isolates of A. flavus from Rafsanjan (14.5%) was much lower than Damghan (39.5%) and Feyz-Abad (41.4%). The A. flavus isolates from Damghan, Rafsanjan, and Feyz-Abad were toxigenic at 53.7%, 61.6%, and 60.4%, respectively. In Rafsanjan, aflatoxin B1 (AFB1), and AFB1 + AFB2 (aflatoxin B2) ranged from 274 to 553 ppb (393±17.11) and 394 to 3745 ppb, respectively, while AFB1, and AFB1 + AFB2 ranged from 257 to 392 ppb (285±13.18) and 415 to 1658 ppb, respectively, in Damghan. We found 16 and 20 vegetative compatibility groups (VCGs) for 41 and 37 nit mutant producing isolates of A. flavus from Rafsanjan and Damghan, respectively. From Damghan the VCG diversity for A. flavus isolates was greater (54%) than from Rafsanjan (39%). Because there were a few number of sclerotium-producing isolates of A. flavus, we did not determine the relationships between sclerotium production with VCGs and/or geographical distribution in the three pistachio production regions. This study was the first to determine the strain and VCG diversity of A. flavus soil isolates from Iranian pistachio orchards.

English

Seventy-five species and 3 species varieties belonging to 21 fungal genera were collected from air, soil and leaf surface of broad bean plant on dicholran chloramphenicol malt extract agar (DCMA) and dichloran Rosebengal chloramphenicol agar (DRBC) at 28&deg;C. The results obtained from leaf surface (phyllosphere and phylloplane), soil and atmosphere were basically similar in the two types of media and the most common fungi were: Aspergillus flavus, Aspergillus fumigatus, Aspergillus niger, Cladosporium cladosporioides, Cladosporium sphaerospermum and Drechslera neergaardii. The monthly counts of these fungi on two types of media irregularly fluctuated giving maxima value at various months. A. flavus was the highest fungi that produced both exo- and endo-&szlig;-1,4-glucanases among the 9 tested isolates. Maximum production of two enzymes by A. flavus was 8 and 6 days after incubation at 30&deg;C with culture medium containing glucose and cellulose as carbon sources and sodium nitrate as nitrogen source and initially adjusted to pH 6. Key words: Airborne fungi, soil and leaf surface, broad bean, cellulolytic ability.