Interconversion of aflatoxin B1 and aflatoxicol by several fungi

Four fungal strains, namely, Aspergillus niger, Eurotium herbariorum, a Rhizopus sp., and non-aflatoxin (AF)-producing Aspergillus flavus, which could convert AF-B1 to aflatoxicol (AFL), could also reconvert AFL to AF-B1. The interconversion of AF-B1 to AFL and of AFL to AF-B1 was ascertained to occur during proliferation of the fungi. These reactions were distinctly observed in cell-free systems obtained from disrupted mycelia of A. flavus and the Rhizopus sp., but they were not observed in culture filtrates from intact (nondisrupted) mycelia of the same strains. The interconversion activities of AF-B1 and AFL were not observed when the cell-free systems were preheated at 100 degrees C. These findings strongly suggest that the interconversion of AF-B1 and AFL is mediated by intracellular enzymes of A. flavus and the Rhizopus sp. In addition, the isomerization of AFL-A to AFL-B observed in culture medium was also found to occur by the lowering of the culture pH.     

Fungal degradation of aflatoxin B1

A number of fungal cultures were screened to select an organism suitable to be used in the detoxification of aflatoxin B1. They were co-cultured in Czapek-Dox-Casamino acid medium with aflatoxin B1 producing Aspergillus flavus. Several fungal cultures were found to prevent synthesis of aflatoxin B1 in liquid culture medium. Among these Phoma sp., Mucor sp., Trichoderma harzianum, Trichoderma sp. 639, Rhizopus sp. 663, Rhizopus sp. 710, Rhizopus sp. 668, Alternaria sp. and some strains belonging to the Sporotrichum group (ADA IV B14(a), ADA SF VI BF (9), strain 720) could inhibit aflatoxin synthesis by > or =90%. A few fungi, namely ADA IV B1, ADA F1, ADA F8, also belonging to the Sporotrichum group, were less efficient than the Phoma sp. The Cladosporium sp. and A. terreus sp. were by far the least efficient, registering <10% inhibition. The cultures which prevent aflatoxin biosynthesis are also capable of degrading the preformed toxin. Among these, Phoma sp. was the most efficient destroying about 99% of aflatoxin B1. The cell free extract of Phoma sp. destroyed nearly 50 microg aflatoxin B1 100 ml(-1) culture medium (90% of the added toxin), and this was more effective than its own culture filtrate over 5 days incubation at 28+/-2 degrees C. The degradation was gradual: 35% at 24 h, 58% at 48 h, 65% at 72 h, 85% at 96 h and 90% at 120 h. The possibility of a heat stable enzymatic activity in the cell free extract of Phoma is proposed.     

Bioconversion of poultry wastes. I--Factors influencing the assay and productivity of crude uricase by three uricolytic filamentous fungi

The optimum temperature for biomass yield and uricase production by uricolytic fungi, Aspergillus terreus, A. flavus and Trichoderma sp. was at 30 degrees C. The time required for maximum production of uricase and biomass yield was 4 days for two Aspergillus species and 6 days for Trichoderma sp. The optimum pH was at 6.4 for A. terreus and pH 6.6 for A. flavus and Trichoderma sp. The maximum fungal biomass yield was achieved in medium supplemented with 4% poultry waste. The best carbon sources for the production of uricase and mycelia yield were glycerol, sucrose and maltose by A. terreus, A. flavus and Trichoderma sp., respectively. Uric acid was found to be the best nitrogen source for production and activity of uricase by the three tested fungi. The addition of some vitamins to the culture media increased the maximum biomass yield of all the isolates, although no significantly increased uricase production was found.     

Bioconversion of poultry wastes I-factors influencing the assay and productivity of crude uricase by three uricolytic filamentous fungi

The optimum temperature for biomass yield and uricase production by uricolytic fungi, Aspergillus terreus. A. flavus and Trichoderma sp. was at 30 degrees C. The time required for maximum production of uricase and biomass yield was 4 days for two Aspergillus species and 6 days for Trichoderma sp. The optimum pH was at 6.4 for A. terreus and pH 6.6 for both A. flavus and Trichoderma sp. The maximum fungal biomass yield was achieved in medium supplemented with 4% poultry waste. The best carbon sources for the production of uricase and mycelia yield were glycerol, sucrose and maltose by A. terreus, A. flavus and Trichoderma sp., respectively. Uric acid was found to be the best nitrogen source for production and activity of uricase by the three tested fungi. The addition of some vitamins to the culture media increased the maximum biomass yield of all the isolates, but did not significantly increase uricase production.     

T-2 toxin degradation by micromycetes

The biodegradation of T-2 toxin was studied by strains of micromycetes which were isolated from the environment. The 26 tested strains were divided into three groups. Group contains strains which degraded T-2 toxin very fast. This toxin could not be chromatographically determined in the medium even after 48 hours of incubation and the antifungal activity of residua against Kluyveromyces fragilis CCY-51-1-2 was low or zero. There were strains of Alternaria sp., Ulocladium sp., Aspergillus candidus, Cladosporium cladosporioides, Rhodotorula sp., Aspergillus flavus and Cladosporium macrocarpum. Group II contains with a low activity and in group III the results were variable and non stable.     

Isolation, identification and antifungal susceptibility of lemon pathogenic and non pathogenic fungi

Numerous species of filamentous fungi were isolated from lemon on different plantations in the province of Tucuman, Argentina. The techniques suggested by the Subcommittee of Antifungal Susceptibility of the National Committee for Clinical Laboratory Standards, (USA) were adapted. The effect of three different concentrations of the fungicides imazalil, guazatine, SOPP and thiabendazole on the fungi Fusarium oxysporum, Fusarium moniliforme, Aspergillus niger, Aspergillus flavus, Aspergillus clavatus, Geotrichum candidum, Rhizopus sp, Penicillium sp, Penicillium digitatum and Mucor sp were studied. All the tested strains were resistant to thiabendazole. We assayed a mixture of SOPP (5%), guazatine (350 ppm) and imazalil (100 ppm), which showed a synergic effect on Rhizopus sp. Mucor sp was the only fungus resistant to the four fungicides tested as well as to the above mentioned mixture.     

Detection of fungal infectous agent of wheat grains in store-pits of Markazi province, Iran

Wheat is an economic and important crop that provides approximately 20% of food calorie in the world. It is first crop in Iran and cultivated in the most areas of this country. Store-pit fungi make undesirable changes in quality and appearance of wheat grains. Even, some fungi produce different mycotoxins which are toxic to human and livestock's that use wheat grains as source of food. In this study, several samples were randomly collected from each of five store-pits located in different areas of Markazi Province including: Arak, Mahallat, Khomein, Saveh and Sarband. Grains were treated on PDA, and blotter, agar and washing test also used for isolating and detection of fungi. At least 100 grains per each sample were randomly used for each test and treatment. The fungi that determined in this study were Cochliobolus australiensis, Cladosporium herbarum, Epicoccum sp., Tilletia leavis, Aspergillus flavus, A. niger, A. fumigatus, Alternaria alternata, Alternaria sp., Penicillium italicum, P. digitatum, Fusarium sp., Rhizopus sp., Ustilago tritici, Scytalidium sp. Among these fungi the most isolates were belonged to Cladosporium, Alternaria, Rhizopus and Fusarium. Cladosporium herbarum was the most common in different sampling areas. Tilletia laevis and Ustilago tritici were just recovered in washing test. This study revealed that different fungi are associated with wheat grains in store-pits in Markazi Province. Some of them like Aspergillus flavus normally produce aflatoxin, a very toxic and carcinogenic mycotoxin that is harmful for human.     

应用反向线点杂交技术鉴定临床常见曲霉属和毛霉目真菌

目的应用反向线点杂交技术(reverse line blot hybridization,RLB)快速鉴定临床常见的曲霉属和毛霉目真菌。方法收集我院真菌和真菌病研究中心保存的5种曲霉菌(烟曲霉、黄曲霉、黑曲霉、土曲霉、构巢曲霉)和7种毛霉目真菌(冻土毛霉菌、总状毛霉菌、卷枝毛霉菌、少根根霉、小孢根霉、微小根毛霉、伞状犁头霉),共计98株菌株。利用真菌通用引物ITS1和ITS4对菌株进行PCR扩增,用12个真菌种特异性探针与扩增后产物进行反向线点杂交。将RLB结果与真菌传统形态学鉴定结果、ITS区DNA测序结果进行比较。结果 RLB可以正确鉴定98株实验菌株,与形态学方法和ITS区测序方法鉴定结果100%一致,种特异性探针之间未见交叉杂交,显示出该方法的高度敏感性和特异性。8株阴性对照菌株(白念珠菌、茄病镰刀菌、尖端赛多孢、马尔尼菲青霉、疣状瓶霉、棒曲霉、日本曲霉以及雅致小克银汉霉),使用RLB方法无法鉴定。通过烟曲霉基因组DNA浓度10倍倍比稀释法验证RLB的敏感性为1.8×10-3 ng/μL。结论 RLB技术为实验室早期快速诊断、鉴定临床常见的曲霉属和毛霉目真菌提供参考。     

Factors effecting chitinase activity of <Emphasis Type="Italic">Rhizobium</Emphasis> sp. from <Emphasis Type="Italic">Sesbania sesban</Emphasis>

Twenty six Rhizobium strains isolated from root nodules of Sesbania sesban were studied for chitinase activity on chitin agar plates. Among them, only 12 strains showed chitinase activity. The strain showing the highest chitinase activity was selected based on maximum clear zone/colony size ratio on chitin agar plates and chitinase activity in culture filtrate. The strain was identified as Rhizobium sp. which showed a high degree of similarity with Rhizobium radiobacter (= Agrobacterium radiobacter). The cultural and nutritional conditions were optimized for maximum chitinase activity. The Rhizobium sp. exhibited maximum chitinase activity after 36 h of incubation, at neutral pH. Among the different nutritional sources, arabinose and yeast extract were found to be good inducers for chitinase activity. Rhizobium sp. could degrade and utilize dead mycelia of Aspergillus flavus, Aspergillus niger, Curvularia lunata, Fusarium oxysporum and Fusarium udum.     

Anaerobic growth ability and alcohol fermentation activity of microscopic fungi

The method proposed in this study was used to isolate fungi grown under anaerobic conditions and to reveal distinctions in their abundance and species composition in different habitats. The ability of micromycetes of different taxa to grow under anaerobic conditions and ensure alcohol fermentation was determined for a representative sample (344 strains belonging to more than 60 species). The group of fungi growing under anaerobic conditions included species with high, moderate, and low fermentation activity. The ability for anaerobic growth and fermentation depended on the taxonomic affiliation of fungi. In some cases, the expression of these characteristics depended on the habitat from which the strain was isolated. The maximum level of ethanol accumulation in culture liquid (1.2–4.7%) was detected for Absidia spinosa, Aspergillus sp. of group flavus, Aspergillus terreus, Acremonium sp., Mucor circinelloides, Mucor sp., Fusarium oxysporum, F. solani, F. sambucinum, Rhizopus arrhizus var. arrhizus, Trichoderma atroviride, and Trichoderma sp.     

Synthesis, antimicrobial evaluation and QSAR studies of novel piperidin-4-yl-5-spiro-thiadiazoline derivatives

In an attempt to find a new class of antimicrobial agents, a series of new 1,3,4-thiadiazolines were synthesized from 2,6-diarylpiperidin-4-ones, via the corresponding 4'-phenylthiosemicarbazones. All the synthesized compounds (23-39) were virtually screened against bacterial (Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and Salmonella typhi) and fungal strains (Candida albicans, Rhizopus sp, Aspergillus niger and Aspergillus flavus) by serial dilution method. QSAR study indicated that the increase in weakly polar component of solvent accessible surface area will favour antibacterial activity while increase in polarizability and decrease in ionisation potential and hydrogen bond donor will favour antifungal activity.     

Isolation and identification of marine chitinolytic bacteria and their potential in antifungal biocontrol

Chitinolytic marine bacterial strains (30) were isolated from the sea dumps at Bhavnagar, India. They were screened as chitinase producers on the basis of zone of clearance on chitin agar plates incorporated with calcofluor white M2R for the better resolution. Out of these, three strains namely, Pseudomonas sp., Pantoea dispersa and Enterobacter amnigenus showed high chitinase production. They were also found to produce proteases and therefore have a good potential for use as antifungal biocontrol agents for the control of fungal plant pathogens. These strains could degrade and utilize the mycelia of Macrophomina phaseoliena (Tassi) Goidanich and Fusarium sp. In vitro, these strains could inhibit the growth of Fusarium sp. and M. phaseolina. The culture filtrate inhibiting hyphal elongation was observed microscopically.     

Intrafungal distribution of aflatoxins among conidia and sclerotia of Aspergillus flavus and Aspergillus parasiticus

This research examines the distribution of aflatoxins among conidia and sclerotia of toxigenic strains of Aspergillus flavus Link and Aspergillus parasiticus Speare cultured on Czapek agar (21 days, 28 degrees C). Total aflatoxin levels in conidia and sclerotia varied considerably both within (intrafungal) and among strains. Aspergillus flavus NRRL 6554 accumulated the highest levels of aflatoxin (conidia: B1, 84000 ppb; G1, 566000 ppb; sclerotia: B1, 135000 ppb; G1, 968000 ppb). Substantial aflatoxin levels in conidia could place at risk those agricultural workers exposed to dust containing large numbers of A. flavus conidia. Cellular ratios of aflatoxin B1 to aflatoxin G1 were nearly identical in conidia and sclerotia even though levels of total aflatoxins in these propagule types may have differed greatly. Aflatoxin G1 was detected in sclerotia of all A. flavus strains but in the conidia of only one strain. Each of the A. parasiticus strains examined accumulated aflatoxin G1 in both sclerotia and conidia. These results are examined in the context of current evolutionary theory predicting an increase in the chemical defense systems of fungal sclerotia, propagules critical to the survival of these organisms.     

Stimulation of aflatoxin biosynthesis by lipophilic epoxides

Epoxy fatty acids added to the culture media either with the inoculum or at the end of exponential growth phase stimulated aflatoxin production by toxigenic strains of Aspergillus flavus and Aspergillus parasiticus. This effect did not appear when the unsaturated fatty acids used for the synthesis of the epoxides and the polyhydroxyacids (which can be considered to be derived from the opening of the oxirane ring) replaced the epoxides in the culture media. No significant differences were detected in the lipid fractions (diglycerides, sterols, triglycerides, free fatty acids, sterol esters) extracted from the mycelia grown in the presence of any of the fatty acid derivates.     

Influence of microbial co-inhabitants on aflatoxin synthesis of Aspergillus flavus on maize kernels

The co-inhabiting mycoflora with Aspergillus flavus observed on individual maize kernels was evaluated for its influence on aflatoxin synthesis. All 13 types of associations of different fungal species inhibited aflatoxin B₁ and G₁ production at different levels (34·3–100%). Inhibition of radial growth of A. flavus by Fusarium moniliforme (59·8%), Trichoderma viride (72·5%) and Rhizopus nigricans (42%) could be directly correlated to the per cent inhibition of aflatoxin production. High levels of inhibition of aflatoxin elaboration were noted in competition of A. flavus with other toxigenic moulds.     

Isolation and characterization of acid- and Al-tolerant microorganisms

Acid- and aluminum (Al)-tolerant microorganisms were isolated from tea fields, from which six strains were selected and identified as Cryptococcus humicola, Rhodotorula glutinis, Aspergillus flavus Link, Penicillium sp., Penicillium janthinellum Biourge and Trichoderma asperellum. They were tolerant to Al up to 100-200 mM and could grow at low pH, 2.5-2.2. In a glucose medium (pH 3.5) the pH of the spent medium decreased to below 3.0. The toxic inorganic monomeric Al in the spent medium decreased with three strains (A. flavus F-6b, Penicillium sp. F-8b and P. janthinellum F-13), but the total Al remained constant for all strains. In a soil extract medium (pH 3.5), the pH of the spent medium of all strains increased to around 6.0-7. 2 and total Al in the spent medium was removed by precipitation due to pH increase. Thus, different tolerance mechanisms were suggested in glucose and soil extract media.     

Biological detoxification of coffee husk by filamentous fungi using a solid state fermentation system

Studies were carried out on detoxification of coffee husk in solid state fermentation using three different strains of Rhizopus, Phanerochaete, and Aspergillus sp. Fungal strains were selected by their ability to grow on a coffee husk extract-agar medium. Using R. arrizus LPB-79, the best results on the degradation of caffeine (87%) and tannins (65%) were obtained with pH 6.0 and moisture 60% in 6 days. When P. chrysosporium BK was used, maximum degradation of caffeine and tannins were 70.8 and 45%, respectively, with coffee husk having 65% moisture and pH 5.5 in 14 days. The Aspergillus strain, isolated from the coffee husk, showed best biomass formation on coffee husk extract-agar medium. Optimization assays were conducted using factorial design, and surface response experiments with Aspergillus sp. The best detoxification rates achieved were 92% for caffeine and 65% for tannins. The results showed good prospects of using these fungal strains, in particular Aspergillus sp., for the detoxification of coffee husk.     

Alteration of cell wall composition leads to amphotericin B resistance in Aspergillus flavus

An amphotericin B (AmB)-resistant mutant was isolated from a wild-type AmB-susceptible strain of Aspergillus flavus by serial transfer of conidia on agar plates containing stepwise increased concentrations of AmB up to 100 microg ml-1. The acquired resistance of mycelia was specific for polyene-antibiotics AmB, nystatin and trichomycin. Spheroplasts derived from the resistant mycelia were as susceptible to AmB as the wild-type. Chemical analysis of the cell wall revealed that levels of alkali-soluble and -insoluble glucans were significantly higher in the resistant mycelia as compared to those in the wild-type. When resistant mycelia were treated with SDS, they adsorbed as much AmB as wild-type mycelia. These results suggest that alterations in the cell wall components of mycelia, especially 1,3-alpha-glucan and protein complex in the outermost wall layer, lead to AmB resistance in A. flavus.     

Biotransformation of benzaldehyde into (R)-phenylacetylcarbinol by filamentous fungi or their extracts

Extracts of 14 filamentous fungi were examined regarding their potential for production of (R)-phenylacetylcarbinol [(R)-PAC], which is the chiral precursor in the manufacture of the pharmaceuticals ephedrine and pseudoephedrine. Benzaldehyde and pyruvate were transformed at a scale of 1.2 ml into PAC by cell-free extracts of all selected strains, covering the broad taxonomic spectrum of Ascomycota, Zygomycota and Basidiomycota. Highest final PAC concentrations were obtained with the extracts of Rhizopus javanicus and Fusarium sp. [78-84 mM (11.7-12.6 g/l) PAC within 20 h from initial substrate concentrations of 100 mM benzaldehyde and 150 mM pyruvate]. (R)-PAC was in about 90-93% enantiomeric excess. Rhizopus javanicus had the advantage of faster growth than Fusarium sp. Rhizopus javanicus mycelia were used as an example in a biotransformation process based on whole cells and benzaldehyde and glucose as substrates. The substrate pyruvate was generated through the fungal fermentation of glucose. Only 19 mM PAC (2.9 g/l) were produced within 8 h from 80 mM benzaldehyde. with evidence of significant benzyl alcohol production.     

Design, synthesis, characterization and in vitro antimicrobial evaluation of 4,6-diaryl-4,5-dihydro-2-phenyl-2H-indazol-3-ols

New 4,6-diaryl-4,5-dihydro-2-phenyl-2H-indazol-3-ols 25-32 were designed, synthesized and in vitro microbially evaluated using clinically isolated bacterial strains viz Staphylococcus aureus, beta-Heamolytic streptococcus, Vibreo cholerae, Salmonella typhii, Shigella felxneri and fungal strains viz Aspergillus flavus, Mucor, Rhizopus and Microsporum gypsuem. Results of this study showed that the nature of the substituents on the phenyl rings viz., methyl, methoxy, chloro, nitro as well as the bromo functions at the meta and para positions of the aryl moieties determined the nature and extent of the activity of the fused indazolonol compounds 25-32.