Mycotoxins producing fungi and mycoflora of air-dust from Taif,Saudi Arabia

Using the dilution plate method, 70 species and 31 genera were collected from 20 dust samples on glucose (28 genera and 64 species) and cellulose Czapek's agar (22 genera and 46 species) at 28° C. The most common fungi were Aspergillus niger, A. flavus, A. flavus var. columnaris, Phoma glomerata, Fusarium oxysporum, Penicillium chrysogenum and Mucor racemosus; and A. nidulans, Phoma humicola, Drechslera spicifera and Stachybotrys chartarum on the two media, respectively.Toxicity test showed that about 85% of the isolates tested were toxic to brine shrimp (Artemia salina). Thin layer chromatographic analysis revealed that 13 out of 23 toxic isolates produced known mycotoxins. Toxins identified were: aflatoxins B₁ and B₂, Kojic acid and trichodermine.     

Microcalorimetric study of aerobic growth of Cellulomonas sp. 21399 on various carbohydrates

Summary Microcalorimetry was used to study the energetic aerobic growth of Cellulomonas sp. 21399 on glucose, cellobiose and amorphous and crystalline cellulose. The thermochemical aspect of growth on glucose was established with regard to the anabolic contribution. The results obtained allowed the use of glucose as a reference substrate for cellulose degradation. The experimental enthalpy change and the maximum catabolic activity, calculated from the maximum power evolved by the culture, were, respectively,-1079 kJ/mol and 0.85 mmol glucose per hour per dry weight of cells. The growth response on amorphous cellulose was equivalent to that demonstrated on glucose. However, on crystalline cellulose media, Cellulomonas sp. 21399 exhibited eight times less power and the quantity of heat evolved during growth showed that 50% of the cellulose was degraded. Quantitative results and the shape of power-time curves achieved indicate that the structural features of cellulose strongly influence its microbial degradability.     

Soil and entomopathogenic fungi with potential for biodegradation of insecticides: degradation of flubendiamide in vivo by fungi and in vitro by laccase

Purpose

Flubendiamide is a highly toxic and persistent insecticide that causes loss of insect muscle functions leading to paralysis and death. The objective was to screen for filamentous fungi in soils where insecticides had been applied, to isolate entomopathogenic fungi from insect larva (Anticarsia gemmatalis) that infest soybean crops, and to use these in biodegradation of insecticides.

Method

Filamentous fungi were isolated from soils, and growth inhibition was evaluated on solid medium containing commercial insecticides, Belt® (flubendiamide) and Actara® (thiamethoxam). A total of 133 fungi were isolated from soil and 80 entomopathogenic fungi from insect larva. Based on growth inhibition tests, ten soil fungi, 2 entomopathogenic fungi, and Botryosphaeria rhodina MAMB-05 (reference standard) were selected for growth on commercial insecticides in solid media. Fungi were grown in submerged fermentation on media containing commercial insecticides and assayed for laccase activity.

Result

Isolates JUSOLCL039 (soil), JUANT070 (insect), and MAMB-05 performed best, and were respectively inhibited by 48.41%, 75.97%, and 79.23% when cultivated on 35 g/L Actara®, and 0.0, 5.42%, and 43.39% on 39.04 g/L Belt®. JUSOLCL039 and JUANT070 were molecularly identified as Trichoderma koningiopsis and Neurospora sp., respectively. The three fungal isolates produced laccase constitutively, albeit at low activities. Fungal growth on pure flubendiamide and thiamethoxam resulted in only thiamethoxam inducing high laccase titers (10.16 U/mL) by JUANT070. Neurospora sp. and B. rhodina degraded flubendiamide by 27.4% and 9.5% in vivo, while a crude laccase from B. rhodina degraded flubendiamide by 20.2% in vitro.

Conclusion

This is the first report of fungi capable of degrading flubendiamide, which have applications in bioremediation.

     

Fungal Decomposition of Oat Straw during Liquid and Solid-State Fermentation

White rot fungi (Coriolus hirsutus, Coriolus zonatus, and Cerrena maxima from the collection of the Komarov Botanical Institute of the Russian Academy of Sciences) and filamentous fungi (Mycelia sterilia INBI 2-26 and Trichoderma reesei6/16) were grown on oat straw–based liquid and solid media, as well as in a bench-scale reactor, either individually or as cocultures. All fungi grew well on solid agar medium supplemented with powdered oat straw as the sole carbon source. Under these conditions, the mold Trichoderma reesei fully suppressed the growth of all basidiomycetes studied; conversely,Mycelia sterilia neither affected the development of any of the cultures, nor did it show any substantial susceptibility to suppression by their presence. Pure solid cultures of basidiomycetes, as well as the coculture of Coriolus hirsutus andCerrena maxima,caused a notable bleaching of the oat straw during its consumption. When grown on the surface of oat straw–based liquid medium, the basidiomycetes consumed up to 40% of the polysaccharides without measurable lignin degradation (a concomitant process). Under these conditions, Mycelia sterilia decomposed no more than 25% of the lignin in 60 days, but this was observed only after polysaccharide exhaustion and biomass accumulation. In contrast, during solid-state straw fermentation, white rot fungi consumed up to 75% of cellulose and 55% of lignin in 83 days (C. zonarus), whereas the corresponding consumption levels for cocultures ofMycelia sterilia and Trichoderma reesei equaled 70 and 45%, respectively (total loss of dry weight ranged from 55 to 60%). Carbon dioxide–monitored solid-state fermentation of oat straw by the coculture of filamentous fungi was successfully performed in an aerated bench-scale reactor.     

Mycoflora and trichothecene toxins of paddy grains from Egypt

120 species and 38 genera were collected from 64 samples of paddy grains on glucose- and cellulose-Czapek's agar at 28 °C. The total count of glycophilic and cellulose-decomposing fungi fluctuated between 216–29760; and 124–11320 colonies/g paddy grains on the two media, respectively.On glucose agar, the most common species were Aspergillus niger, A. flavus, A. sydowi, A. terreus, A. fumigatus, A. ochraceus, Penicillium chrysogenum, P. corylophilum, Fusarium oxysporum, Alternaria alternata, Cladosporium cladosporioides, Trichoderma viride and Mucor racemosus.On cellulose agar with pH 5.5 & 8.0, the most prevalent fungi were Stachybotrys chartarum, S. bisybi, Aspergillus niger, A. flavus, Fusarium oxysporum, Alternaria alternata, Drechslera sativus and Acremonium strictum.Extracts from 64 paddy samples were tested against brine shrimp larvae (Artemis salina). Of these 9 displayed varying degrees of toxicity. Trichothecene-toxins were detected in the extracts of three paddy samples only. Diacetoxyscirpenol and T-2 toxin were detected in two samples and only T-2 toxin in the other.     

Cellulase production on glucose-based media by the UV-irradiated mutants of Trichoderma reesei

From 22,791 mutants of a cellulase hyper-producing strain of Trichoderma reesei (Hypocrea jecorina), ATCC66589, as the parent, we selected two mutants, M2-1 and M3-1, that produce cellulases in media containing both cellulose and glucose. The mutation enabled the mutants to produce cellulases, which were measured as p-nitrophenyl β-d-lactopyranoside-hydrolyzing activities, in media with glucose as a sole carbon source, although M2-1 exhibited different sensitivities to glucose from M3-1. When the mutants were grown for 8 days on a medium with cellulose as a sole carbon source, the filter-paper-degrading activities (FPAs) per gram of cellulose were 257 and 281 U for M2-1 and M3-1, respectively, values that were 1.1–1.2 times higher than that of the parental strain. Cellulase production by M2-1 and M3-1 on a medium with a continuously fed mixture of glucose and cellobiose resulted in 214 and 210 U of FPA/gram carbon sources, respectively, whereas less efficient production (140 U of FPA/gram carbon source) was achieved by the parental strain. The improved cellulase productivity of the mutants allows us to use glucose as a carbon source for efficient on-site production of cellulases with quality/quantity-controlled feeding of soluble carbon sources and inducers.     

Changes in organic matter composition of forest soil treated with a large amount of urea to promote ammonia fungi and the abilities of these fungi to decompose organic matter

Organic matter composition (lignin, holocellulose, 50% (v/v) methanol extract, water-soluble carbohydrate (WSC) and phenolics (WSP), petroleum ether extract, and ash) of A₀ layer soil treated with 700 g/m² of urea to promote ammonia fungi was investigated in a Japanese red pine (Pinus densiflora) forest. Nine species of fungi were found during the study period of 18 months after the treatment. Of these, seven species belong to the ammonia fungi. WSC content of the treated soil was lower than that of the control. Methanol extract content increased initially after the treatment, then decreased to below the control level. There were no consistent differences in other components between the treated plot and the control. The abilities to decompose cellulose, lignin, chitin, protein and lipid in 18 strains in 10 species of the ammonia fungi were also screened. Cellulose was not lysed byPseudombrophila deerata, Hebeloma spp. andLaccaria bicolor. Strong lignolytic activity was shown byLyophyllum tylicolor, Coprinus echinosporus andP. deerata. Chitin was decomposed byAmblyosporium botrytis, L. tylicolor, C. echinosporus andHebeloma vinosophyllum. All strains possessed proteolytic and lipolytic activities. Supply of glucose to the culture media resulted in weaker enzyme activities except for lignolytic ability.     

Use of carbohydrate-supplemented distillery spent wash as a medium for ethanol production by a thermotolerant strain of yeast at 45°C

Summary Spent wash from the Old Bushmill's Distillery Co. Ltd. was supplemented with either glucose (10% [w/v]) or cellulose (5% [w/v]) and used as a medium for the thermotolerant yeast strain Kluyveromyces marxianus IMB3. There was no significant difference in ethanol production during growth on these media at 45° C, compared with that produced during growth on conventional, pre-defined laboratory media. On glucose supplemented spent wash ethanol yields were in the region of 45 g/L, representing 87% of the maximum theoretical yield. Analysis of spent media from the glucose-containing fermentations demonstrated that the total organic carbon (TOC) content was reduced by 36%. The results suggest a novel means of utilizing whiskey distiller spent wash.     

Hydrothermal processing and enzymatic hydrolysis of sorghum bagasse for fermentable carbohydrates production

Untreated and hydrothermally treated sorghum bagasse (SB) was hydrolyzed to simple sugars by the synergistic action of cellulases and hemicellulases produced by the fungi Fusarium oxysporum and Neurospora crassa. Synergism between the two lignocellulolytic systems was maximized with the application of higher fraction of N. crassa enzymes.Hydrothermolysis of SB was studied at a wide range of treatment times and temperatures. At intense pretreatment conditions (210 °C for 20 min; logR₀ = 4.54), the residual hemicellulose percentage was 17.45%, while formation of inhibitory products, 5-hydromethyl-furfural (HMF), furfural, acetic and formic acid, (0.21, 0.51, 3.36 and 1.80 g/l, respectively) remained in acceptable levels.Maximum conversion of cellulose and total polysaccharides of the untreated SB were 23.18% and 18.79%, respectively. Combining hydrothermal treatment and enzymatic hydrolysis of released oligosaccharides and insoluble solids resulted in improvement of cellulose (approximately 15% increase) and total polysaccharides (two fold) hydrolysis compared to that of untreated SB.     

Fermentation of cellulose and production of cellulolytic and xylanolytic enzymes by anaerobic fungi from ruminant and non-ruminant herbivores

Four anaerobic fungi were grown on filter paper cellulose and monitored over a 7–8 days period for substrate utilisation, fermentation products, and secretion of cellulolytic and xylanolytic enzymes. Two of the fungi (N1 and N2) were Neocallimastix species isolated from a ruminant (sheep) and the other two fungi were Piromyces species (E2 and R1) isolated from an Indian Elephant and an Indian Rhinoceros, respectively. The tested anaerobic fungi degraded the filter paper cellulose almost completely and estimated cellulose digestion rates were 0.25, 0.13, 0.21 and 0.18 g · 1^-1 · h^-1 for strains E2, N1, N2, R1, respectively. All strains secreted cellulolytic and xylanolytic enzymes, including endoglucanase, exoglucanase, -glucosidase and xylanase. Strain E2 secreted the highest levels of enzymes in a relatively short time. The product formation on avicel by enzymes secreted by the four fungi was studied. Both in the presence and absence of glucurono-1,5--lactone, a specific inhibitor of -glucosidase, mainly glucose was formed but no cellobiose. Therefore the exoglucanase secreted by the four fungi is probably a glucohydrolase.     

三种白腐菌生物学特性与木质纤维素酶基因遗传多样性

以采自东北林业大学帽儿山实验林场的3种白腐真菌——木蹄层孔菌(Fomes fomentarius)、鲍姆鲍姆木层孔菌(Phellinus baumiibaumii)和火木层孔菌(Phellinus igniarius)为材料,用菌落直径测量法比较3种白腐菌在马铃署葡萄糖固体培养基上的生长速度,采用菌丝体干重法比较其在马铃署葡萄糖液体培养基中的生物量变化。结果显示:马铃薯葡萄糖固体培养基上3种白腐菌均为快速生长类型,其生长速度木蹄层孔菌火木层孔菌鲍姆鲍姆木层孔菌;马铃署葡萄糖液体培养基中生物量增长速度木蹄层孔菌鲍姆鲍姆木层孔菌火木层孔菌。用比色法测量其木质纤维素酶活性,结果显示:木蹄层孔菌产锰过氧化物酶和漆酶量较高,鲍姆鲍姆木层孔菌和火木层孔菌产木质素过氧化物酶量较高;木蹄层孔菌、鲍姆鲍姆木层孔菌和火木层孔菌3种白腐菌的3种主要木质素酶(锰过氧化物酶、漆酶和木质素过氧化物酶)的表达量,种间差异显著(F=3.75*、5.20**、3.01*),白桦木屑诱导处理与对照间差异显著(F=3.84*、4.19*、5.28*);两种主要纤维素酶(葡聚糖内切酶、葡聚糖外切酶)的表达量,种间差异不显著,受碳源影响作用显著(F=3.99*、4.04*)。筛选29对引物组合,对3种白腐菌几种主要木质纤维素酶基因进行TRAP-PCR分子标记检测,比较3菌种间遗传差异,扩增总条带数为357条,多态性条带数为255条,多态性条带的比例为71.43%,其中木质素降解酶基因总多态位点比率为73.77%,纤维素降解酶基因总多态位点比率为68.97%。3种白腐菌的木质纤维素降解酶基因在种间均存在较高的遗传差异。因此,特定基因的TRAP分子标记可以用于木腐菌的遗传变异分析。     

Different carbonic supplements induced changes of microflora in two types of compost teas and biocontrol efficiency against Pythium aphanidermatum

ABSTRACT

Considering the different carbonic source utilisation abilities of microbial species, we used six carbonic supplements to acclimate the microorganisms in two types of compost teas (named CT1 and CT2). The effects of the different carbonic supplements on the physicochemical and biological parameters of the acclimated microbial broth were investigated. The influence of these supplements on the biocontrol effect of the broth against cucumber damping off caused by Pythium aphanidermatum was also assessed. Results showed that treatment with glucose, sucrose, or starch reduced the pH and weakened the biocontrol effect of the acclimated microbial broth. The glucose treatment increased the population size of fungi and yeast. The chitin, cellulose, and wheat straw treatments significantly elevated the pH and electrical conductivity (P?<?0.05), increased the bacterial population size, and positively affected the biocontrol effect of the acclimated microbial broth. The glucose treatment had higher (75.3% and 79.4% for the CT1 and CT2, respectively) whereas the wheat straw treatment had lower (34.6% and 25.6% for the CT1 and CT2, respectively) disease incidence relative to the blank control (85.6%), bacterial population size (r?=?0.64 and 0.69) and number of colony types (r?=?0.82 and 0.76) highly correlated with the biocontrol effect of CT1 and CT2 while the reducing sugar content negatively correlated with the biocontrol effect (r?=??0.57 and ?0.86).     

Survey on the mycoflora of Egyptian wheat grains and their lemmae and paleae

The plating technique has been used to study the fungus floras of covered and uncovered wheat grains and their lemmae and paleae on glucose-cellulose and 40% sucrose-Czapek's agar at 28° C. Seventy-two species and 28 genera were collected from the three microhabitats on the three types of media. On glucose-Czapek's agar the most frequent species were Aspergillus niger, A. flavus, A. terreus, A. nidulans, Alternaria alternata, Cladosporium herbarum and Fusarium Oxysporum. On cellulose and 40% sucrose-Czapek's agar, the composition of fungal floras of the three substrates and the frequency of prevalence of the individual fungi were basically similar to those obtained on glucose agar, but the frequency of some species was promoted or decreased.     

A study of fungal spore poupulations in the atmosphere of Kuwait

The fungal air-spora of Kuwait was investigated using the Petri-dish trapping technique. During the period from 1st April, 1974 to 30th June, 1975, a total of 3685 colonies were recorded from 2 % malt agar plates. Fifty-five genera and 116 species were identified. Alternaria occupied the first place in the order of percentage incidence, being represented by 18.3% of the entire catch, followed by Aspergillus (17.1%), Penicillium (14%), Cladosporium (13.6%), Drechslera (13.3%), and Ulocladium (7.1%). The widest spectrum of species was displayed by Aspergillus (possessed 19 species) followed by Penicillium (17 species), Alternaria (6 species), Drechslera (5 species), and Ulocladium (4 species). The monthly total number of fungi showed marked seasonal periodicity with the greatest number of colonies occurring in March–April and November. The lowest counts were recorded in mid-summer (July) and mid-winter (January). Only 220 colonies and 17 species of thermophilous fungi were reported from plates incubated at 45 °C. The most common species were Aspergillus fumigatus and A. terreus.     

Comparison of four media for the isolation ofAspergillus flavus group fungi

Four agar media used to isolate aflatoxin producing fungi were compared for utility in isolating fungi in theAspergillus flavus group from agricultural soils collected in 15 fields and four states in the southern United States. The four media wereAspergillus flavus andparasiticus Agar (AFPA, 14), the rose bengal agar described by Bell and Crawford (BCRB; 3), a modified rose bengal agar (M-RB), and Czapek's-Dox Agar supplemented with the antibiotics in BC-RB (CZ-RB). M-RB was the most useful for studying the population biology of this group because it permitted both identification of the greatest number ofA. flavus group strains and growth of the fewest competing fungi. M-RB supported an average of 12% moreA. flavus group colonies than the original rose bengal medium while reducing the number of mucorales colonies and the number of total fungi by 99% and 70%, respectively. M-RB was successfully employed to isolate all three aflatoxin producing species,A. flavus, A. parasiticus andA. nomius, and both the S and L strains ofA. flavus. M-RB is a defined medium without complex nitrogen and carbon sources (e.g. peptone and yeast extract) present in BC-RB. M-RB should be useful for studies on the population biology of theA. flavus group.Abbreviations M-RB Modified Rose Bengal Agar - CZ-RB Czapeks Rose Bengal Agar - BC-RB Bell and Crawford's Rose Bengal Agar - AFPA Aspergillus flavus andparasiticus agar     

Gas Chromatographic Determination of Optical Purity of a Chiral Derivatization Reagent,N-Acyl-l-prolyl Chloride

Two endo-type cellulases, tentatively called carboxymethyl cellulases (CMCases) I and II, were purified by gel filtration, ion-exchange chromatography, affinity chromatography, and chromato-focusing from a culture supernatant of Penicillium purpurogenum. Their homogeneity was verified by analytical polyacrylamide gel electrophoresis. The molecular weights of CMCases I and II, estimated by gel filtration, were 72,000 and 50,000, respectively. CMCases I and II contained about 12% and 8% carbohydrate, and had isoelectric points of 4.3 and 3.9, respectively. CMCase I produced cellobiose, glucose, and a trace amount of cellotriose from H₃PO₄-swollen cellulose and Avicel (microcrystalline cellulose), while CMCase II produced cellobiose and cellotriose with a small amount of glucose and cellotetraose. The products from reduced cellopentaose by both enzymes were released predominantly in the β-configuration. CMCase II appeared to act in more random fashion than I against carboxymethyl cellulose. These results suggest that both enzymes attack insoluble cellulose randomly, although there are some differences in the mode of hydrolytic action.     

Degradation of nitrocellulose by fungi

Three lignocellulolytic fungi, Trametes versicolor, Pleurotus ostreatus, and Coprinus cinereus, and two cellulolytic fungi Trichoderma reesei andChaetomium elatum were tested for their ability to degrade nitrocellulose. They were provided with different carbon and nitrogen sources in liquid cultures. Nitrocellulose (N content above 12%) was added as nitrogen source (in solution in acetone) alongside amino acids or as sole N source. Either starch or carboxy-methyl cellulose were provided as carbon sources. After 28 days of growth the highest decrease of nitrocellulose was observed with Chaetomium elatum when up to 43% was degraded in a medium containing nitrocellulose as the only nitrogen source. Coprinus cinereus caused a 37% decrease of nitrocellulose when provided with amino acids and starch as co-substrate. In cultures of Trametes versicolor, Pleurotus ostreatus andTrichoderma reesei, only 10%–22% decrease of nitrocellulose was measured in all media. In the presence of nitrocellulose with N content below 12% supplied as 3 mm pellets as the only carbon source, or with nitrocellulose with carboxy-methyl cellulose, the release of nitrite and nitrate from liquid cultures of Chaetomium elatum was measured. Between 6 and 9 days of growth in these media, an increase in both nitrite and nitrate was observed with a loss in weight of nitrocellulose up to 6% achieved after 34 days. The physical nature of the NC pellets may have reduced the rate of degradation in comparison with supplying NC in solution in the cultures.     

Production of extracellular keratinases by keratinophilic fungal species inhabiting feathers of living poultry birds (Gallus domesticus): A comparison

Fourteen species of keratinophilic fungi belonging to ten genera (Chrysoporium, Malbranchea, Chaetomium,Sepedonium, Microascus, Scopulariopsis, Curvularia, Fusarium, Aspergillus, Penicillium) were isolated from feathers of about one hundred living poultry birds. The isolated fungi were compared for their keratinase activity after growing them on two different media: (1) basal salts solution containing natural keratin (human hair) as the only source of carbon and nitrogen; (2) the medium was supplemented with a minor amount of readily assimilable source of carbon along with natural keratin. All the test fungi could grow on keratinous material, degrading it and releasing sulphydryl containing compounds detected as cysteine, total proteins and extracellular keratinase. Maximum enzyme release by these fungi occurred in the broth supplemented with glucose and vitamins, thereby indicating a correlation between the mycelial biomass and production of proteolytic keratinases. This revised version was published online in June 2006 with corrections to the Cover Date.     

Effect of trehalose on the spawn storage in some edible mushroom fungi (2): Effect on preservation in the freezer

 We describe the effects of trehalose on spawn storage in a home freezer (average temperature, −16°C) where edible fungi usually do not survive. When the mycelia of Lentinula edodes were stored in a freezer for 3 days, the survival rate of mycelia cultivated on 2% glucose medium was 30%, whereas those on media containing 2% and 5% trehalose were 50% and 60%, respectively. Addition of trehalose to the culture was more effective in Pleurotus ostreatus. These results suggest that trehalose played the role of a stress protectant against freezing, because the mycelia cultured on a trehalose medium grew more rapidly and produced more fruiting bodies compared to those cultured on glucose. Received: February 6, 2002 / Accepted: October 1, 2002 Acknowledgments This work was partially supported by a Grant in Aid for Scientific Research (c) (2) No. 12660156 from the Japan Society for the Promotion of Science. We also gratefully acknowledge a grant from Hokuto Foundation for the Promotion of Biological Science. Correspondence to:T. Terashita     

桃儿七茎叶组织内生真菌多样性

通过用两种传统培养基(PDA、SDA)分离方法对5个自然野生分布的桃儿七群落(分布于青海省、甘肃省和四川省)茎叶组织内的内生真菌多样性进行研究,并用形态学与分子生物学的方法鉴定菌株。实验结果显示,720个茎叶组织块中共分离到141株内生真菌。依据真菌在培养基上的形态初步划分为52个分类单元,经鉴定归属于19属,其中茎组织中16属叶组织中6属,桃儿七茎叶组织中的真菌优势属为拟青霉属,相对分离频率为26.34%。5个采样点间内生真菌的香浓维纳多样性指数为0.71—1.41,Sorenson相似性系数为0.13—0.50,定殖率为14.58%—28.47%。通过对PDA、SDA两种培养基以及茎、叶组织的定殖率进行统计,结果显示:PDA(17.50%)SDA(21.67%),茎(29.72%)叶(9.44%)。研究结果表明,桃儿七茎叶组织中内生真菌的多样性较低,不同采样点间宿主植物内内生真菌群落的相似性较低,真菌群落结构存在差异。研究为进一步扩大从桃儿七中筛选产鬼臼毒素等活性物质内生真菌提供了一种新的思路。