Mycoflora and incidence of aflatoxin B1, zearalenone and deoxynivalenol in poultry feeds in Argentina

In Argentina, there is rather little information about the natural occurrence of mycotoxins in feedstuffs. The aim of this work was to determine the fungal flora and natural incidence of aflatoxin B1 (AFB1), zearalenone (ZEA) and deoxynivalenol (DON) in poultry feeds from 5 factories of Río Cuarto, Córdoba. Three hundred samples were taken from May 1995 to May 1996. Fungal counts of poultry feeds ranged 104 to 106 CFU g-1. The lowest counts were obtained on the first months from the sampling (May to September 1995) with mean values significantly different from those found at the last of the sampling (October 1995 to April 1996). The most prevalent species isolated of poultry feed samples belonged to the genera Penicillium that was present in 98% of the samples, Fusarium (87%) and Aspergillus (52%). Fusarium species isolated were: F moniliforme in 73% of the samples, F subglutinans (35%), F graminearum (20%) and within Aspergillus species: A. parasiticus (33%) and A. flavus (8%) were identified. In poultry feeds aflatoxin B1 (AFB1) was the most significant mycotoxin with levels ranging from 17 to 197 ng/g. For deoxynivalenol (DON) the levels ranged from 240 to 410 ng/g. Only three out of 300 samples were contaminated with zearalenone (ZEA) in concentrations of 30, 120 and 280 ng/g. These are preliminary data on this subject in our region. This revised version was published online in June 2006 with corrections to the Cover Date.     

Aspergillus flavus genomics: gateway to human and animal health, food safety, and crop resistance to diseases

Aspergillus flavus is an imperfect filamentous fungus that is an opportunistic pathogen causing invasive and non-invasive aspergillosis in humans, animals, and insects. It also causes allergic reactions in humans. A. flavus infects agricultural crops and stored grains and produces the most toxic and potent carcinogic metabolites such as aflatoxins and other mycotoxins. Breakthroughs in A. flavus genomics may lead to improvement in human health, food safety, and agricultural economy. The availability of A. flavus genomic data marks a new era in research for fungal biology, medical mycology, agricultural ecology, pathogenicity, mycotoxin biosynthesis, and evolution. The availability of whole genome microarrays has equipped scientists with a new powerful tool for studying gene expression under specific conditions. They can be used to identify genes responsible for mycotoxin biosynthesis and for fungal infection in humans, animals and plants. A. flavus genomics is expected to advance the development of therapeutic drugs and to provide information for devising strategies in controlling diseases of humans and other animals. Further, it will provide vital clues for engineering commercial crops resistant to fungal infection by incorporating antifungal genes that may prevent aflatoxin contamination of agricultural harvest.     

Mycotoxins and mycotoxigenic moulds in nuts and sunflower seeds for human consumption

A survey was carried out to obtain data on the occurrence of mycotoxins and the mycotoxin-producing potential of fungi isolated from nuts (almonds, peanuts, hazelnuts, pistachio nuts) and sunflower seeds in Spain. Thin-layer chromatography was used to separate the toxins. Aflatoxins were detected in one sample of almonds (95 ppb aflatoxin B₁ and 15 ppb aflaxtoxin B₂) and in one sample of peanuts at a level below 10 ppb of aflatoxin B1. 100% of samples showed variable incidence of fungal contamination. The predominant fungi present in samples were Penicillium spp, Aspergillus niger, A. flavus, A. glaucus and Rhizopus spp. The results showed that isolates of different species were able to produce aflatoxins B₁, B₂, G₁, and G₂, sterigmatocystin, ochratoxin A, patulin, citrinin, penicillic acid, zearalenone, and griseofulvin.     

Brine Shrimp (Artemia salina L.) Larvae as a Screening System for Fungal Toxins

Concentrations resulting in 50% mortality, determined with brine shrimp (Artemia salina L.) larvae exposed to known mycotoxins for 16 hr, were (mug/ml): aflatoxin G(1), 1.3; diacetoxyscirpenol, 0.47; gliotoxin, 3.5; ochratoxin A, 10.1; and sterigmatocystin, 0.54. 4-Acetamido-4-hydroxy-2-butenoic acid gamma-lactone gave no mortality at 10 mug/ml. Used as a screening system involving discs saturated with solutions of known mycotoxins, the larvae were relatively sensitive to aflatoxin B(1), diacetoxyscirpenol, gliotoxin, kojic acid, ochratoxin A, rubratoxin B, sterigmatocystin, stemphone, and T-2 toxin. Quantities of 0.2 to 2 mug/disc caused detectable mortality. The larvae were only moderately sensitive to citrinin, patulin, penicillic acid, and zearalenone which were detectable at 10 to 20 mug/disc. They were relatively insensitive to griseofulvin, luteoskyrin, oxalic acid, and beta-nitropropionic acid. The disc screening method indicated that 27 out of 70 fungal isolates from foods and feeds grown in liquid or solid media produced chloroform-extractable toxic material. Examination of toxic extracts by thin-layer chromatography for 17 known mycotoxins showed that the toxicity of eight isolates could be attributed to aflatoxin B(1) and B(2), kojic acid, zearalenone, T-2 toxin, or ochratoxin A. Nine out of 32 of these fungal isolates grown in four liquid media yielded toxic culture filtrates from at least one medium. Chemical tests for kojic, oxalic, and beta-nitropropionic acids showed the presence of one or two of these compounds in filtrates of seven of these nine isolates.     

A mitogen-activated protein kinase gene (MGV1) in Fusarium graminearum is required for female fertility,heterokaryon formation,and plant infection

Fusarium graminearum is an important pathogen of small grains and maize in many areas of the world. Infected grains are often contaminated with mycotoxins harmful to humans and animals. During the past decade, F. graminearum has caused several severe epidemics of head scab in wheat and barley. In order to understand molecular mechanisms regulating fungal development and pathogenicity in this pathogen, we isolated and characterized a MAP kinase gene, MGV1, which is highly homologous to the MPS1 gene in Magnaporthe grisea. The MGV1 gene was dispensable for conidiation in F. graminearum but essential for female fertility during sexual reproduction. Vegetative growth of mgv1 deletion mutants was normal in liquid media but reduced on solid media. Mycelia of the mgv1 mutants had weak cell walls and were hypersensitive to cell wall degrading enzymes. Interestingly, the mgv1 mutants were self-incompatible when tested for heterokaryon formation, and their virulence was substantially reduced. The ability of the mutants to accumulate trichothecene mycotoxins on inoculated wheat was also greatly reduced. Our data suggest that MGV1 in F. graminearum is involved in multiple developmental processes related to sexual reproduction, plant infection, and cell wall integrity.     

Influence of multiple metal ions on beta-amyloid aggregation and dissociation on a solid surface

Recently discovered evidences suggest that precipitation of Alzheimer's beta-amyloid (Abeta) peptide and the toxicity in Alzheimer's disease (AD) are caused by abnormal interactions with neocortical metal ions, especially Zn2+, Cu2+, and Fe3+. While many studies had focused on the role of a "single" metal ion and its interaction with Abeta peptides, such studies involving "multiple" metal ions have hardly been explored. Here, to explore the nature of codeposition of different metals, two or more metal ions along with Abeta were incubated over a solid template prepared by immobilizing Abeta42 oligomers. The influence of Zn2+,Cu2+, and Fe3+ on Abeta aggregation was investigated by two approaches: co-incubation and sequential addition. Our results using ex situ AFM, ThT-induced fluorescence, and FTIR spectroscopy indicated that the co-incubation of Cu2+, Zn2+, and Fe3+ significantly altered the morphology of aggregates. A concentration dependence study with mixed metal ions suggested that Zn2+ was required at much lower concentrations than Cu2+ to yield nonfibrillar amorphous Abeta deposits. In addition, sequential addition of Zn2+ or Cu2+ on fibrillar aggregates formed by Fe3+ demonstrated that Zn2+ and Cu2+ could possibly change the conformation of the aggregates induced by Fe3+. Our findings elucidate the coexistence of multiple metal ions through their interactions with Abeta peptides or its aggregates.     

Deoxynivalenol, acetyl deoxynivalenol, and zearalenone formation by Canadian isolates of Fusarium graminearum on solid substrates.

Three isolates of Fusarium graminearum (DAOM 180377, 180378, and 180379) were screened for their ability to produce mycotoxins on the solid substrates corn and rice. They all produced deoxynivalenol and zearalenone on corn. On rice, only DAOM 180378 and 180379 produced significant amounts of these mycotoxins, with levels of deoxynivalenol being much higher than those of zearalenone. The effects of the initial moisture content before autoclaving, incubation temperature, and time were studied with isolate DAOM 180378. At 19.5 degrees C the main product was zearalenone, whereas at 25 degrees C both deoxynivalenol and zearalenone were formed. Higher incubation temperatures (28 degrees C) favored deoxynivalenol formation, the maximum amount being 515 ppm (515 micrograms/g) formed after 24 days at an initial moisture content of 40%. The maximum level of zearalenone produced at the same temperature was 399 ppm, but at an initial moisture content of 35%. Other factors, such as pH, oxygen and carbon dioxide concentrations, and size of the culture flask also appeared to affect the production of mycotoxins.     

Functional analysis of the kinome of the wheat scab fungus Fusarium graminearum

As in other eukaryotes, protein kinases play major regulatory roles in filamentous fungi. Although the genomes of many plant pathogenic fungi have been sequenced, systematic characterization of their kinomes has not been reported. The wheat scab fungus Fusarium graminearum has 116 protein kinases (PK) genes. Although twenty of them appeared to be essential, we generated deletion mutants for the other 96 PK genes, including 12 orthologs of essential genes in yeast. All of the PK mutants were assayed for changes in 17 phenotypes, including growth, conidiation, pathogenesis, stress responses, and sexual reproduction. Overall, deletion of 64 PK genes resulted in at least one of the phenotypes examined, including three mutants blocked in conidiation and five mutants with increased tolerance to hyperosmotic stress. In total, 42 PK mutants were significantly reduced in virulence or non-pathogenic, including mutants deleted of key components of the cAMP signaling and three MAPK pathways. A number of these PK genes, including Fg03146 and Fg04770 that are unique to filamentous fungi, are dispensable for hyphal growth and likely encode novel fungal virulence factors. Ascospores play a critical role in the initiation of wheat scab. Twenty-six PK mutants were blocked in perithecia formation or aborted in ascosporogenesis. Additional 19 mutants were defective in ascospore release or morphology. Interestingly, F. graminearum contains two aurora kinase genes with distinct functions, which has not been reported in fungi. In addition, we used the interlog approach to predict the PK-PK and PK-protein interaction networks of F. graminearum. Several predicted interactions were verified with yeast two-hybrid or co-immunoprecipitation assays. To our knowledge, this is the first functional characterization of the kinome in plant pathogenic fungi. Protein kinase genes important for various aspects of growth, developmental, and infection processes in F. graminearum were identified in this study.     

Mycological survey of Korean cereals and production of mycotoxins by Fusarium isolates

The fungal species isolated from Korean cereals (barley, polished barley, wheat, rye, and malt) were Alternaria spp., Aspergillus spp., Chaetomium spp., Drechslera spp., Epicoccum sp., Fusarium spp., and Penicillium spp., etc. The number of Fusarium strains isolated was 36, and their ability to produce Fusarium mycotoxins on rice was tested. Nivalenol (NIV) was produced by Fusarium graminearum (7 of 9 isolates), Fusarium oxysporum (3 of 10 isolates), and Fusarium spp. (7 of 15 isolates). Of 15 isolates of Fusarium spp., 6 formed deoxynivalenol (DON). Fusarenon-X and 3-acetyl-DON were produced by most NIV- and DON-forming isolates, respectively. Zearalenone was produced by 3 isolates of F. graminearum, 1 isolate of Fusarium equiseti, and 11 isolates of Fusarium spp. T-2 toxin was not produced by any Fusarium isolates. The highest concentrations of mycotoxins produced by Fusarium isolates were 77.4 (NIV), 5.3 (DON), 138.3 (fusarenon-X), 40.6 (3-acetyl-DON), and 23.2 (zearalenone) micrograms/g.     

Mycological survey of Korean cereals and production of mycotoxins by Fusarium isolates.

The fungal species isolated from Korean cereals (barley, polished barley, wheat, rye, and malt) were Alternaria spp., Aspergillus spp., Chaetomium spp., Drechslera spp., Epicoccum sp., Fusarium spp., and Penicillium spp., etc. The number of Fusarium strains isolated was 36, and their ability to produce Fusarium mycotoxins on rice was tested. Nivalenol (NIV) was produced by Fusarium graminearum (7 of 9 isolates), Fusarium oxysporum (3 of 10 isolates), and Fusarium spp. (7 of 15 isolates). Of 15 isolates of Fusarium spp., 6 formed deoxynivalenol (DON). Fusarenon-X and 3-acetyl-DON were produced by most NIV- and DON-forming isolates, respectively. Zearalenone was produced by 3 isolates of F. graminearum, 1 isolate of Fusarium equiseti, and 11 isolates of Fusarium spp. T-2 toxin was not produced by any Fusarium isolates. The highest concentrations of mycotoxins produced by Fusarium isolates were 77.4 (NIV), 5.3 (DON), 138.3 (fusarenon-X), 40.6 (3-acetyl-DON), and 23.2 (zearalenone) micrograms/g.     

金属离子对粪产碱杆菌C16的脱氮和亚硝酸盐积累的影响

【目的】研究不同金属离子对异养氨氧化细菌C16的生长和脱氮性能影响,探讨适于C16生长和脱氮的金属离子及其浓度。【方法】实验选用Mg2+、Mn2+、Fe2+、Cu2+、Zn2+5种金属离子,对C16的生长﹑脱氮性能﹑亚硝酸盐氮积累以及相关酶活性进行研究。【结果】Mg2+明显促进C16的生长和NH4+-N氧化速率;较高浓度Mn2+使得C16无法生长;原培养基中缺少Fe2+会抑制C16的生长和NH4+-N氧化速率;在原培养基中加入0.1 mmol/L的Cu2+对C16的生长和脱氮具有一定的促进作用,Cu2+使得培养基中基本无NO2--N和NH2OH的积累;不同浓度的Zn2+对C16的生长和氨氮去除有抑制作用。酶活实验结果显示,0.1 mmol/L Mg2+促进了羟胺氧化还原酶(HAO)的活性;0.1 mmol/L Cu2+促进了硝酸盐还原酶(Nar)和亚硝酸盐还原酶(Nir)的活性。【结论】Mg2+是C16生长和脱氮过程中的一种重要金属离子;加入Cu2+可避免过量亚硝酸盐积累。     

The mitochondrial glycine cleavage system: differential inhibition by divalent cations of glycine synthesis and glycine decarboxylation in the glycine-CO2 exchange

The exchange of glycine carboxyl carbon with CO2 catalyzed by the combination of chicken liver glycine decarboxylase (P-protein) and aminomethyl carrier protein (H-protein) was markedly inhibited by various divalent cations, although extents of inhibition by individual metal ions varied considerably. Cu2+ and Zn2+, at 100 microM, inhibited the reaction almost completely, and the inhibitions by Co2+ and Ni2+ were also significant, while Mg2+ and Mn2+ did not appreciably affect the reaction. The inhibition by Zn2+ was competitive with both bicarbonate and H-protein and non-competitive with glycine. Of the two reactions involved in the glycine-CO2 exchange, decarboxylation of glycine yielding the H-protein-bound aminomethyl moiety was not significantly affected by 100 microM Zn2+ or Cu2+, but carboxylation of the H-protein-bound aminomethyl moiety to form glycine was strongly inhibited by either Zn2+ or Cu2+. Various degrees of inhibition of the glycine-CO2 exchange by other divalent metal ions could also be accounted for by the inhibition of the carboxylation step of the exchange reaction. The primary site of the action of divalent metal ions is likely to be not P-protein but H-protein, and the binding of metal ions with the H-protein-bound intermediate of glycine decarboxylation was assumed to account for the observed marked inhibition.     

Novel inhibition of some pathogenic fungal and bacterial species by new synthetic phytochemical coumarin derivatives

novel antifungal and antibacterial activities of new synthesized phytochemical coumarin compounds [H2L1, HL2 and H2L3] and their copper (II) complexes [L1Cu], [L2Cu(OAc)] and [(L3)Cu2(H2O)4(OAc)2] were evaluated against nine pathogenic fungal species (Alternaria alternata, Aspergillus flavus, Botrytis cinerea, Cladosporium herbarum, Fusarium moniliforme, Helminthosporium tetramera, Penicillium expansum, Rhizopus stolonifer andVerticillium albo-atrum) and eight pathogenic bacterial species, from which four Gram-positive bacteria (Bacillus subtilis, Micrococcus luteus, Staphylococcus citrus andStreptococcus pneumoniae) and four Gram-negative bacteria (Enterobacter aerogenes, Escherichia coli, Pseudomonas aeruginosa andSalmonella typhi). The phytochemical copper (II) complex [L2Cu(OAc)] was the most effective derivative, where it reaches to 90 and 100% inhibition in the most sensitive pathogens (B. subtilis and A. flavus), respectively accompanied with a significant reduction in pectinolytic and cellulytic enzyme activities in all tested pathogenic species. Addition of [L2Cu(OAc)] complex leading to leakage of sugars and electrolytes from the most sensitive microbial cells accompanied with collapsed hyphae ofA. Flavus and membrane blobbing ofB. Subtilis. The production of mycotoxins decreased with the extension exposure to [L2Cu(OAc)] complex reaching to a minimum values for the mycelium originating from the inoculum exposed to the minimum inhibitory concentration (2%). Both aflatoxin (AFB1) and citrinin were the most sensitive toxins.     

Substrate-induced lipase gene expression and aflatoxin production in Aspergillus parasiticus and Aspergillus flavus

AIMS: To establish a relationship between lipase gene expression and aflatoxin production by cloning the lipA gene and studying its expression pattern in several aflatoxigenic and nontoxigenic isolates of Aspergillus flavus and A. parasiticus. METHODS AND RESULTS: We have cloned a gene, lipA, that encodes a lipase involved in the breakdown of lipids from aflatoxin-producing A. flavus, A. parasiticus and two nonaflatoxigenic A. flavus isolates, wool-1 and wool-2. The lipA gene was transcribed under diverse media conditions, however, no mature mRNA was detected unless the growth medium was supplemented with 0.5% soya bean or peanut oil or the fungus was grown in lipid-rich medium such as coconut medium. The expression of the lipase gene (mature mRNA) under substrate-induced conditions correlated well with aflatoxin production in aflatoxigenic species A. flavus (SRRC 1007) and A. parasiticus (SRRC 143). CONCLUSIONS: Substrate-induced lipase gene expression might be indirectly related to aflatoxin formation by providing the basic building block 'acetate' for aflatoxin synthesis. No direct relationship between lipid metabolism and aflatoxin production can be ascertained, however, lipase gene expression correlates well with aflatoxin formation. SIGNIFICANCE AND IMPACT OF THE STUDY: Lipid substrate induces and promotes aflatoxin formation. It gives insight into genetic and biochemical aspects of aflatoxin formation.     

qPCR quantification of Sphaerodes mycoparasitica biotrophic mycoparasite interaction with Fusarium graminearum: in vitro and in planta assays

Sphaerodes mycoparasitica, a biotrophic mycoparasite of Fusarium species, improved wheat seed germination and seedling growth in vitro compared to Trichoderma harzianum, a necrotrophic mycoparasite. However, under phytotron conditions, both S. mycoparasitica and T. harzianum had positive impact on wheat seedlings growth in the presence of F. graminearum. Once exposed to the mycoparasites, the DNA quantity of F. graminearum in wheat root decreased. Observed shifts in DNA quantity using qPCR, a set of newly designed Sphaerodes-specific SmyITS primers, as well as Trichoderma-TGP4 and Fusarium-Fg16 N primers, demonstrated the mycoparasite's biocontrol effectiveness in planta. In the presence of F. graminearum, the concentration of S. mycoparasitica DNA remained stable in the root, whereas the amount of T. harzianum DNA decreased. The toxicity assays indicated that S. mycoparasitica's mycelia withstand higher concentrations of deoxynivalenol, 3-acetyldeoxynivalenol, and zearalenone mycotoxins than T. harzianum mycelia. This study compares the ability of two fungi to improve the wheat growth, decrease the root colonization of Fusarium, and withstand mycotoxins.