Interactions of Saprophytic Yeasts with a nor Mutant of Aspergillus flavus

The nor mutant of Aspergillus flavus has a defective norsolorinic acid reductase, and thus the aflatoxin biosynthetic pathway is blocked, resulting in the accumulation of norsolorinic acid, a bright red-orange pigment. We developed a visual agar plate assay to monitor yeast strains for their ability to inhibit aflatoxin production by visually scoring the accumulation of this pigment of the nor mutant. We identified yeast strains that reduced the red-orange pigment accumulation in the nor mutant. These yeasts also reduced aflatoxin accumulation by a toxigenic strain of A. flavus. These yeasts may be useful for reducing aflatoxin contamination of food commodities.     

黄曲酶尿酸氧化酶突变体的构建、表达、纯化及生物学活性分析

采用融合PCR的方法将黄曲霉尿酸氧化酶(UOX) 基因的307~309 bp的TGC(Cys) 突变为GCC(Ala),将所获得的突变体基因克隆到原核表达质粒pET-42a(+) 后转化大肠杆菌BL21(DE3)。经IPTG诱导,突变体蛋白 (UOX-Ala103) 得到高水平的可溶性表达,目的蛋白占总蛋白含量的45％。疏水柱及阴离子柱纯化后,UOX-Ala103蛋白纯度＞98％。Western blotting分析证实UOX-Ala103能与抗UOX单抗特异结合。与天然型相比较,其体外生物学活性增加约60％,在高尿酸血症小鼠模型体内也有良好的降解尿酸的活性。     

Intracellular hydrolases of Aspergillus parasiticus and Aspergillus flavus

When the distribution profile of hydrolases in mycelial homogenates and culture filtrates of A. parasiticus and A. flavus was examined, six hydrolytic enzymes viz. N-acetyl-beta-glucosaminidase, aryl sulfatase, alkaline proteinase, cathepsin B, cathepsin D and aminopeptidase were detected in homogenate. The culture filtrates were devoid of any activity of these enzymes. The enzyme levels varied with the stage of incubation. The most abundant fungal exopeptidase showing preference for basic amino acid naphthylamides seems to be an aminopeptidase B. Incorporation of CEPA, an ethylene generating compound, stimulated the amino peptidase activity in the mycelium but inhibited the enzyme in vitro. The enzyme was also inhibited by different aflatoxins to varying degree. While aminopeptidase B was located intracellularly, a non-dialysable, heat-stable inhibitor of the enzyme was found to be secreted in the culture filtrate. This peptide inhibitor was however ineffective on the other enzymes.     

Bacterial Antagonists of Aspergillus flavus

In order to search for bacteria capable of reducing the aflatoxin contamination of cottonseed, 892 indigenous bacterial isolates, including 11 that were endophytic to cotton, were screened for their ability to inhibit the growth of Aspergillus flavus on cottonseed in an in vitro bioassay. Only six isolates partially or totally inhibited fungal growth. All antagonistic isolates were recovered from boll, lint or seed surface or from the lint of mature bolls. One was retrieved from mature seeds. None of the endophytic isolates showed activity. In four field trials, the incidence of A. flavus -induced damage to locules inoculated simulteously with A. flavus plus the most A. flavus plus the most effective antagonistic isolate (D1) was reduced by 41-100% relative to locules inoculated with A. flavus alone. The severity of damage to locules inoculated simultaneously with A. flavus and with D1 was reduced by 60-l00% relative to locules inoculated with A. flavus alone. Isolate D1, identified as Pseudomonas cepacia, completely inhibited the growth of A. flavus on synthetic media.     

Color mutants of Aspergillus flavus and Aspergillus parasiticus in a study of preharvest invasion of peanuts.

A comparison of the invasion of flowers, aerial pegs, and kernels by wild-type and mutant strains of Aspergillus flavus or A. parasiticus along with aflatoxin analyses of kernels from different drought treatments have supported the hypothesis that preharvest contamination with aflatoxin originates mainly from the soil. Evidence in support of soil invasion as opposed to aerial invasion was the following. A greater percentage of invasion of kernels rather than flower or aerial pegs by either wild-type A. flavus or mutants. Significant invasion by an A. parasiticus color mutant occurred only in peanuts from soil supplemented with the mutant, whereas adjacent plants in close proximity but in untreated soil were only invaded by wild-type A. flavus or A. parasiticus. Aflatoxin data from drought-stressed, visibly undamaged peanut kernels showed that samples from soil not supplemented with a mutant strain contained a preponderance of aflatoxin B's (from wild-type A. flavus) whereas adjacent samples from mutant-supplemented soil contained a preponderance of B's plus G's (from wild-type and mutant A. parasiticus). Preliminary data from two air samplings showed an absence of propagules of A. flavus or A. parasiticus in air around the experimental facility.     

Production of cyclopiazonic acid by Aspergillus flavus Link.

Production of cyclopiazonic acid by Aspergillus flavus is reported for the first time. A procedure for its production by agitated solid substrate fermentation on red wheat is described along with the isolation procedure and physical and chemical properties of this indole derivative. The compound has been found to exert antibacterial activity.     

Physiology and characterization of a fungal milk-clotting enzyme from Aspergillus flavus.

Aspergillus flavus produced extracellularly an active rennin-like enzyme when grown aerobically in whey media. The enzyme was detected at early stages of growth reaching a maximum after three to four days at 25 degrees. The activity was destroyed by heating to temperatures higher than 50 degrees, whereas the presence of skim milk during heating preserved the enzyme activity, at least, up to 70 degrees. Calcium chloride significantly stimulated the milk-clotting activity up to 1% final concentration. The clotting time was inversely proportional to protein concentration in the range 0.2-0.6 mg/ml and the enzyme exhibited marked stability when stored at 37 degrees at pH 6.     

Aspergillus nomius,a new aflatoxin-producing species related to Aspergillus flavus and Aspergillus tamarii

Aspergillus nomius is described and represents a new aflatoxigenic species phenotypically similar to A. flavus. Strains examined were isolated from insects and agricultural commodities. Separation from A. flavus is based on the presence of indeterminate sclerotia and a lower growth temperature. Comparisons of DNA relatedness show A. nomius to have only relatively recently evolved from A. flavus and A. tamarii.     

Production of cyclopiazonic acid by Aspergillus flavus Link.

Production of cyclopiazonic acid by Aspergillus flavus is reported for the first time. A procedure for its production by agitated solid substrate fermentation on red wheat is described along with the isolation procedure and physical and chemical properties of this indole derivative. The compound has been found to exert antibacterial activity.     

Aspergillus flavus F52菌株鉴定及不同碳源对曲酸产量的影响

【目的】探明豇豆内生真菌F52的分类地位及不同碳源对曲酸产量的影响。【方法】采用形态观察及ITS序列分析对豇豆内生真菌F52进行分类鉴定;通过重结晶获得曲酸高纯度产物,采用核磁共振波谱、高分辨质谱及红外光谱对其进行结构鉴定;采用高效液相色谱检测不同碳源对曲酸产量的影响。【结果】该曲酸产生菌为黄曲霉Aspergillus flavus F52;葡萄糖与蔗糖组成的复合碳源曲酸产量最高,乳糖的存在不利于曲酸的生成;该菌株发酵液中曲酸含量可达24.44 g/L。【结论】Aspergillus flavus F52是一株具有产业化开发价值的曲酸高产菌株。     

Nitrification of Aspartate by Aspergillus flavus

Heterotrophic conversion of l-aspartic acid to nitrification products by Aspergillus flavus was studied in a replacement incubation system. Numerous amino acids supported nitrification; aspartate and glutamate were about equivalent as the best sources of nitrate. Addition of sodium bicarbonate to the incubation system substantially enhanced nitrate formation for all nitrifiable amino acids except aspartic acid, but the basis for the bicarbonate effect is obscure. The yield of nitrate from l-aspartate was not approached by forms of aspartic acid resulting from substitution on the beta carbon, the amino nitrogen, or the gamma carboxyl group or by aspartate presented as the d-configuration. There was no relationship between nitrate formation and the occurrence of such possible intermediates as nitrite, bound hydroxylamine, ammonia, aspergillic acid, and beta-nitropropionic acid. Uniformly labeled (14)C-l-aspartate that was nitrified in replacement incubation led to no accumulation of label in possible nitrification products in the culture filtrate. Label was found in components of the mycelium after acid hydrolysis, with heaviest accumulation in what appeared to be glucosamine and an unidentified compound, possibly acetylglucosamine. Detectable label was redistributed into serine, glycine, and threonine.     

Identification of aflatoxin biosynthesis genes by genetic complementation in an Aspergillus flavus mutant lacking the aflatoxin gene cluster.

Aspergillus flavus mutant strain 649, which has a genomic DNA deletion of at least 120 kb covering the aflatoxin biosynthesis cluster, was transformed with a series of overlapping cosmids that contained DNA harboring the cluster of genes. The mutant phenotype of strain 649 was rescued by transformation with a combination of cosmid clones 5E6, 8B9, and 13B9, indicating that the cluster of genes involved in aflatoxin biosynthesis resides in the 90 kb of A. flavus genomic DNA carried by these clones. Transformants 5E6 and 20B11 and transformants 5E6 and 8B9 accumulated intermediate metabolites of the aflatoxin pathway, which were identified as averufanin and/or averufin, respectively.These data suggest that avf1, which is involved in the conversion of averufin to versiconal hemiacetal acetate, was present in the cosmid 13B9. Deletion analysis of 13B9 located the gene on a 7-kb DNA fragment of the cosmid. Transformants containing cosmid 8B9 converted exogenously supplied O-methylsterigmatocystin to aflatoxin, indicating that the oxidoreductase gene (ord1), which mediates the conversion of O-methylsterigmatocystin to aflatoxin, is carried by this cosmid. The analysis of transformants containing deletions of 8B9 led to the localization of ord1 on a 3.3-kb A. flavus genomic DNA fragment of the cosmid.     

Degradation of aflatoxin by Aspergillus flavus

Aflatoxin degradative activity was demonstrated in 6- to 12-d-old intact mycelium and cell-free extracts of Aspergillus flavus. The addition of cycloheximide, SKF 525-A or metyrapone to cultures of A. flavus prevented subsequent degradation of the aflatoxins, while in cell-free extracts degradation was inhibited by SKF 525-A, metyrapone and cytochrome c but not by KCN. In cell-free extracts, aflatoxin degradation was enhanced by NADPH and NaIO4. The results suggest the involvement of cytochrome P-450 monooxygenases in the aflatoxin degradative activity of A. flavus.     

Aflatoxins in mutants of Aspergillus flavus

Mutants ofAspergillus flavus were recovered following the irradiation of conidia with ultraviolet light. Analysis of the mutants for aflatoxins B₁, B₂, G₁, and G₂ indicated a wide range of variability in aflatoxin levels. None of the isolates produced the G toxins, and four produced little or no aflatoxin B₂. Production of B₁ and B₂ by the mutants ranged from 1.3 µ;g/ml to 967 µg/ml and zero to 30 µg/ml, respectively. The correlation between production of B₁ and B₂ was statistically significant. There was no apparent correlation between nutritional requirement or conidial color and aflatoxin production.