Aflatoxin reduction in corn through field application of competitive fungi.

Soil in corn plots was inoculated with nonaflatoxigenic strains of Aspergillus flavus and A. parasiticus during crop years 1994 to 1997 to determine the effect of application of the nontoxigenic strains on preharvest aflatoxin contamination of corn. Corn plots in a separate part of the field were not inoculated and served as controls. Inoculation resulted in significant increases in the total A. flavus/parasiticus soil population in treated plots, and that population was dominated by the applied strain of A. parasiticus (NRRL 21369). In the years when weather conditions favored aflatoxin contamination (1996 and 1997), corn was predominately colonized by A. flavus as opposed to A. parasiticus. In 1996, colonization by wild-type A. flavus was significantly reduced in treated plots compared with control plots, but total A. flavus/parasiticus colonization was not different between the two groups. A change to a more aggressive strain of A. flavus (NRRL 21882) as part of the biocontrol inoculum in 1997 resulted in a significantly (P < 0.001) higher colonization of corn by the applied strain. Weather conditions did not favor aflatoxin contamination in 1994 and 1995. In 1996, the aflatoxin concentration in corn from treated plots averaged 24.0 ppb, a reduction of 87% compared with the aflatoxin in control plots that averaged 188.4 ppb. In 1997, aflatoxin was reduced by 66% in treated corn (29.8 ppb) compared with control corn (87.5 ppb). Together, the data indicated that although the applied strain of A. parasiticus dominated in the soil, the nonaflatoxigenic strains of A. flavus were more responsible for the observed reductions in aflatoxin contamination. Inclusion of a nonaflatoxigenic strain of A. parasiticus in a biological control formulation for aflatoxin contamination may not be as important for airborne crops, such as corn, as for soilborne crops, such as peanuts.     

Ethylene inhibits aflatoxin biosynthesis in Aspergillus parasiticus grown on peanuts

The filamentous fungi Aspergillus parasiticus and Aspergillus flavus synthesize aflatoxins when they grow on a variety of susceptible food and feed crops. These mycotoxins are among the most carcinogenic naturally occurring compounds known and they pose significant health risks to humans and animals. We previously demonstrated that ethylene and CO2 act alone and together to reduce aflatoxin synthesis by A. parasiticus grown on laboratory media. To demonstrate the potential efficacy of treatment of stored seeds and grains with these gases, we tested ethylene and CO2 for ability to inhibit aflatoxin accumulation on Georgia Green peanuts stored for up to 5 days. We demonstrated an inverse relationship between A. parasiticus spore inoculum size and the level of toxin accumulation. We showed that ethylene inhibits aflatoxin synthesis in a dose-dependent manner on peanuts; CO2 also inhibits aflatoxin synthesis over a narrow dose range. Treatments had no discernable effect on mold growth. These observations support further exploration of this technology to reduce aflatoxin contamination of susceptible crops in the field and during storage.     

Aflatoxin-producing Aspergillus species from Thailand

Aflatoxin-producing Aspergillus species were isolated from soil samples from ten different regions within Thailand. Aspergillus flavus was present in all of the soil samples. Unlike previous studies, we found no A. parasiticus or A. flavus capable of both B- and G-type aflatoxin production in any of the samples. A. pseudotamarii, which had not been previously reported from Thailand, was found in four soil samples. In two of the samples A. nomius was determined to be the most abundant aflatoxin-producing species. Based on sequence alignments for three DNA regions (Taka-amylase A (taa), the rRNA internal transcribed spacer (ITS), and the intergenic region for the aflatoxin biosynthesis genes aflJ and aflR) the A. nomius isolates separated into three well-supported clades. Isolates from one of the A. nomius clades had morphological properties similar to those found for S-type isolates capable of B and G aflatoxin production and could easily be mistaken for these isolates. Our results suggest that such unusual A. nomius isolates could be a previously unrecognized agent for aflatoxin contamination in Thailand.     

PCR-restriction fragment length analysis of aflR gene for differentiation and detection of Aspergillus flavus and Aspergillus parasiticus in maize

Contamination of food and feedstuffs by Aspergillus species and their toxic metabolites is a serious problem as they have adverse effects on human and animal health. Hence, food contamination monitoring is an important activity, which gives information on the level and type of contamination. A PCR-based method of detection of Aspergillus species was developed in spiked samples of sterile maize flour. Gene-specific primers were designed to target aflR gene, and restriction fragment length polymorphism (RFLP) of the PCR product was done to differentiate Aspergillus flavus and Aspergillus parasiticus. Sterile maize flour was inoculated separately with A. flavus and A. parasiticus, each at several spore concentrations. Positive results were obtained only after 12-h incubation in enriched media, with extracts of maize inoculated with A. flavus (101 spores/g) and A. parasiticus (104 spores/g). PCR products were subjected to restriction endonuclease (HincII and PvuII) analysis to look for RFLPs. PCR-RFLP patterns obtained with these two enzymes showed enough differences to distinguish A. flavus and A. parasiticus. This approach of differentiating these two species would be simpler, less costly and quicker than conventional sequencing of PCR products.     

黄曲霉群体感应研究进展

曲霉属真菌(Aspergillus)如黄曲霉、寄生曲霉侵染玉米、花生等富含油脂的作物种子后产生的黄曲霉毒素(aflatoxin)具有强致癌作用,严重威胁食品安全和人类健康。群体感应(quorum sensing,QS)曾经认为只存在于细菌中,但是在真菌中也存在QS系统,菌体的形态建成和次级代谢产物的产生都与细胞的群体密度有关。黄曲霉拥有类似群体感应的机制,菌核到分生孢子的转换受细胞密度和脂肪氧合酶调控。氧脂素作为信号分子通过密度依赖机制可抑制或促进黄曲霉的生长及黄曲霉毒素的生物合成,本文综述了黄曲霉群体感应及信号通路的研究进展,旨在从群体感应的角度抑制黄曲霉毒素的产生,为微生物与食品安全的研究提供指导。     

Detection of aflatoxin-producing molds in Korean fermented foods and grains by multiplex PCR

An assay based on multiplex PCR was applied for the detection of potential aflatoxin-producing molds in Korean fermented foods and grains. Three genes, avfA, omtA, and ver-1, coding for key enzymes in aflatoxin biosynthesis, were used as aflatoxin-detecting target genes in multiplex PCR. DNA extracted from Aspergillus flavus, Aspergillus parasiticus, Aspergillus oryzae, Aspergillus niger, Aspergillus terreus, Penicillium expansum, and Fusarium verticillioides was used as PCR template to test specificity of the multiplex PCR assay. Positive results were achieved only with DNA that was extracted from the aflatoxigenic molds A. flavus and A. parasiticus in all three primer pairs. This result was supported by aflatoxin detection with direct competitive enzyme-linked immunosorbent assay (DC-ELISA). The PCR assay required just a few hours, enabling rapid and simultaneous detection of many samples at a low cost. A total of 22 Meju samples, 24 Doenjang samples, and 10 barley samples commercially obtained in Korea were analyzed. The DC-ELISA assay for aflatoxin detection gave negative results for all samples, whereas the PCR-based method gave positive results for 1 of 22 Meju samples and 2 of 10 barley samples. After incubation of the positive samples with malt extract agar, DC-ELISA also gave positive results for aflatoxin detection. All Doenjang samples were negative by multiplex PCR and DC-ELISA assay, suggesting that aflatoxin contamination and the presence of aflatoxin-producing molds in Doenjang are probably low.     

Nonaflatoxigenic Aspergillus flavus TX9-8 competitively prevents aflatoxin accumulation by A. flavus isolates of large and small sclerotial morphotypes

Aflatoxins are a family of highly toxic and carcinogenic toxins produced by several Aspergillus species. Aflatoxin contamination of agricultural commodities both pre- and postharvest is a serious food safety issue and a significant economic concern. Using nonaflatoxigenic A. flavus isolates to competitively exclude toxigenic A. flavus isolates in agricultural fields has become an adopted approach to reduce aflatoxin contamination. From screening subgroups of nonaflatoxigenic A. flavus, we identified an A. flavus isolate, TX9-8, which competed well with three A. flavus isolates producing low, intermediate, and high levels of aflatoxins, respectively. TX9-8 has a defective polyketide synthase gene (pksA), which is necessary for aflatoxin biosynthesis. Co-inoculating TX9-8 at the same time with large sclerotial (L strain) A. flavus isolates at a ratio of 1:1 or 1:10 (TX9-8:toxigenic) prevented aflatoxin accumulation. The intervention of TX9-8 on small sclerotial (S strain) A. flavus isolates varied and depended on isolate and ratio of co-inoculation. At a ratio of 1:1 TX9-8 prevented aflatoxin accumulation by A. flavus CA28 and reduced aflatoxin accumulation 10-fold by A. flavus CA43. No decrease in aflatoxin accumulation was apparent when TX9-8 was inoculated 24 h after toxigenic L- or S strain A. flavus isolates started growing. The competitive effect likely is due to TX9-8 outgrowing toxigenic A. flavus isolates.     

Polymerase chain reaction-mediated characterization of molds belonging to the Aspergillus flavus group and detection of Aspergillus parasiticus in peanut kernels by a multiplex polymerase chain reaction

The Aspergillus flavus group covers species of A. flavus and Aspergillus parasiticus as aflatoxin producers and Aspergillus oryzae and Aspergillus sojae as koji molds. Genetic similarity among these species is high, and aflatoxin production of a culture may be affected by cultivation conditions and substrate composition. Therefore, a polymerase chain reaction (PCR)-mediated method of detecting the aflatoxin-synthesizing genes to indicate the degree of risk a genotype has of being a phenotypic producer was demonstrated. In this study, 19 strains of the A. flavus group, including A. flavus, A. parasiticus, A. oryzae, A. sojae, and one Aspergillus niger, were subjected to PCR testing in an attempt to detect four genes, encoding for norsolorinic acid reductase (nor-1), versicolorin A dehydrogenase (ver-1), sterigmatocystin O-methyltransferase (omt-1), and a regulatory protein (apa-2), involved in aflatoxin biosynthesis. Concurrently, the strains were cultivated in yeast-malt (YM) broth for aflatoxin detection. Fifteen strains were shown to possess the four target DNA fragments. With regard to aflatoxigenicity, all seven aflatoxigenic strains possessed the four DNA fragments, and five strains bearing less than the four DNA fragments did not produce aflatoxin. When peanut kernels were artificially contaminated with A. parasiticus and A. niger for 7 days, the contaminant DNA was extractable from a piece of cotyledon (ca. 100 mg), and when subjected to multiplex PCR testing using the four pairs of primers coding for the above genes, they were successfully detected. The target DNA fragments were detected in the kernels infected with A. parasiticus, and none was detected in the sound (uninoculated) kernels or in the kernels infected with A. niger.     

Interaction of water activity and temperature on aflatoxin production by Aspergillus flavus and A. parasiticus in irradiated maize seeds.

The effects of aw (0.90, 0.95, 0.98) and temperature (25 degrees C, 30 degrees C, 35 degrees C) on aflatoxin production by Aspergillus flavus and Aspergillus parasiticus growing on irradiated maize seeds, were examined. Highest levels of aflatoxin were produced by A. parasitious at 25 degrees C and 0.98 aw and by A. flavus at 30 degrees C at 0.95 and 0.98 aw. At 0.90 aw toxin production was consistently low for both species at all temperatures. Temperature cycling of A. flavus between 25 degrees C and 35 degrees C each for 12 h resulted in higher aflatoxin synthesis than when incubated either at 25 degrees C or 35 degrees C.     

Microbiological and aflatoxin evaluation of Brazil nut pods and the effects of unit processing operations

Harvesting of Brazil nuts not only helps to preserve the Amazon rainforest but also provides income to individuals who would otherwise have little means of making a livelihood. Recently, the European Community has tightened the quality requirements for Brazil nuts, particularly with regard to aflatoxin levels and microbiological contamination. The objectives of this research were to gain a better understanding of the origin of aflatoxins on Brazil nuts and to microbiologically evaluate some of the operations involved in processing. In this regard, five Brazil nut pods were aseptically picked from trees located in each of three concessions of the Peruvian Amazon rainforest (Madre de Dios province). The exteriors of the pods and the nuts were examined for yeast and molds, including Aspergillus flavus and Aspergillus parasiticus, and for bacteria, including Salmonella and Escherichia coli. Brazil nuts obtained from various commercial process operations located in Peru were similarly evaluated. Exteriors of all Brazil nut pods did not contain A. parasiticus, and only pods from one concession yielded A. flavus isolates. All isolates tested were aflatoxigenic (630 to 915 ppb total aflatoxin). Coliforms, E. coli, and salmonellae were not recovered from any of the pods. Whole, in-shell nuts obtained after opening the pods yielded no A. flavus or A. parasiticus. Aflatoxins were not detected (detection limit 1.75 ppb) in any of the nuts. Whole, in-shell and shelled nuts from various process operations were all positive for A. flavus but negative for E. coli and salmonellae. Soaking of whole, in-shell nuts before cracking or shelling increased coliform numbers, whereas levels of A. flavus decreased. In order to gain a better understanding of the sanitary performance of the unit process operations, additional evaluations should be conducted on product lots processed on different days. Also, the microbiology of product processed from common lots should be followed through the various unit operations and compared.     

脂氧合酶与作物黄曲霉毒素污染抗性关系研究进展

曲霉属真菌（Aspergillus）侵染玉米、花生等富含油脂的作物种子后产生的黄曲霉毒素（aflatoxin）具有强致癌作用,严重威胁食品安全和人类健康。脂氧合酶（LOX）及其代谢衍生物在曲霉菌一种子互作中具有重要调节作用。LOX活性的增加可提高植物对细菌、真菌和病毒等病原物的抵抗力,同时由于其催化不饱和脂肪酸代谢生成的脂氧合物如茉莉酸甲酯以及挥发性醛类等物质,可影响黄曲霉菌的生长及黄曲霉毒素的生物合成,因而LOX在作物黄曲霉毒素污染抗性遗传改良中具有潜在的利用价值。本文评述了脂氧合酶及其代谢产物与黄曲霉毒素污染抗性关系的研究进展,旨在从植物一病原菌互作的角度揭示作物黄曲霉毒素污染抗性机制,为下一步的研究提供指导。     

Distribution of aflatoxin-producing Aspergillus flavus and Aspergillus parasiticus in sugarcane fields in the southernmost islands of Japan

The distribution of Aspergillus flavus and Aspergillus parasiticus in sugarcane field soils and on harvested sugarcane stems was studied on seven islands of Okinawa and Kagoshima Prefectures, the southernmost prefectures in Japan. With the use of a combination of dilution plate and plant debris plate techniques, the fungi were detected on all seven islands studied and in 74% of 53 soil samples. The fungi were also found on the cut surfaces of sugarcane stems from one of the islands. A. parasiticus was the predominant fungus, although many atypical A. parasiticus isolates that produced metulated conidial heads were also obtained. The proportions of isolates testing positive for aflatoxin production were ca. 89% (146 of 164) of all isolates and ca. 69% of A. flavus isolates. More than 40% of A. flavus isolates also produced G aflatoxins. Scanning electron microscopic observation of conidial wall texture was useful in distinguishing A. parasiticus from A. flavus. Cyclopiazonic acid, an indole mycotoxin, was never synthesized by any of the A. parasiticus or G aflatoxin-producing A. flavus isolates tested.     

Identification and toxigenic potential of the industrially important fungi, Aspergillus oryzae and Aspergillus sojae

Mold strains belonging to the species Aspergillus oryzae and Aspergillus sojae are highly valued as koji molds in the traditional preparation of fermented foods, such as miso, sake, and shoyu, and as protein production hosts in modern industrial processes. A. oryzae and A. sojae are relatives of the wild molds Aspergillus flavus and Aspergillus parasiticus. All four species are classified to the A. flavus group. Strains of the A. flavus group are characterized by a high degree of morphological similarity. Koji mold species are generally perceived of as being nontoxigenic, whereas wild molds are associated with the carcinogenic aflatoxins. Thus, reliable identification of individual strains is very important for application purposes. This review considers the pheno- and genotypic markers used in the classification of A. flavus group strains and specifically in the identification of A. oryzae and A. sojae strains. Separation of A. oryzae and A. sojae from A. flavus and A. parasiticus, respectively, is inconsistent, and both morphologic and molecular evidence support conspecificity. The high degree of identity is reflected by the divergent identification of reference cultures maintained in culture collections. As close relatives of aflatoxin-producing wild molds, koji molds possess an aflatoxin gene homolog cluster. Some strains identified as A. oryzae and A. sojae have been implicated in aflatoxin production. Identification of a strain as A. oryzae or A. sojae is no guarantee of its inability to produce aflatoxins or other toxic metabolites. Toxigenic potential must be determined specifically for individual strains. The species taxa, A. oryzae and A. sojae, are currently conserved by societal issues.     

Reduction of aflatoxin B1 in stored peanuts (Arachis hypogaea L.) using Saccharomyces cerevisiae

Aflatoxin B(1) is a toxigenic and carcinogenic compound produced by Aspergillus flavus and Aspergillus parasiticus. To inhibit aflatoxin contamination of peanuts, seeds of two peanut breeds, IAC Caiapó and IAC Runner 886, were inoculated with A. parasiticus (1.0 × 10(6) spores per ml) and the yeast Saccharomyces cerevisiae (3.2 × 10(7) cells per ml) and incubated at 25°C for 7 and 15 days. Two experiments were conducted for each incubation period separately. The treatments were completely randomized, with three replications per treatment. Treatments included the two cultivars and three types of inoculation (pathogen alone, yeast and pathogen, and yeast 3 h before pathogen). Aflatoxin B(1) was quantified with a densitometer at 366 nm after thin layer chromatography. Aflatoxin B(1) contamination in peanuts was reduced after the addition of S. cerevisiae. The concentration of aflatoxin B(1) decreased by 74.4 and 55.9% after 7 and 15 days, respectively. The greatest aflatoxin reduction was observed when S. cerevisiae was inoculated 3 h before the pathogen in IAC Caiapó seeds and incubated for 7 days at 25°C. The use of S. cerevisiae is a promising strategy for biological control of aflatoxin contamination in peanuts.     

Quality control of Aspergillus flavus and A. parasiticus agar and comparison with dichloran 18% glycerol agar: a collaborative study

AFPA culture medium, which is used for recognition of Aspergillus flavus and A. parasiticus, has been validated in a collaborative study including nine laboratories located in Australia, Brazil, Denmark, The Netherlands, Sweden and United Kingdom. Three freeze-dried fungal mixtures, containing A. flavus/A. parasiticus and background fungi, were produced and checked for homogeneity. The coefficients of variance were low, ranging from 0.81% to 1.09% for total fungal counts and between 2.50% and 2.72% for counts of A. flavus/A. parasiticus. The laboratories analysed the contents of two vials of each mixture on commercial A. flavus and A. parasiticus agar (AFPA), in-house-made AFPA, and on a standard media, dichloran 18% glycerol agar (DG18). Reproducibility values for counts of A. flavus/A. parasiticus indicated no differences between the commercial AFPA and the in-house-made AFPA. Variation between laboratories was low, indicating that the medium was effective in use. Reproducibility values for DG18 were higher. There were no differences in counts of A. flavus/A. parasiticus on AFPA and DG18. However, DG18 gave slightly higher total fungal counts compared to AFPA.     

INCIDENCE OF AFLATOXIGENIC ISOLATES OF ASPERGILLUS FLAVUS/PARASITICUS OBTAINED FROM GEORGIA CORN PROCESSING PLANTS

Two corn processing facilities within Georgia were evaluated in order to determine the incidence of Aspergillus flavus or A. parasiticus within the plant and in corn harvested and processed in 1984 and 1985. Conidia of A. flavus/parasiticus were found in all corn samples evaluated as well as in settled dust samples taken within these processing facilities. Isolates were obtained by using the differential/selective medium Aspergillus flavus/parasiticus agar. Upon subsequent culture only 55% of the selected isolates were confirmed as belonging to A. flavus/parasiticus group. Some of these isolates were randomly chosen and their ability to produce aflatoxins B₁, B₂, G₁, or G₂ evaluated. Thirty-two percent of the A. flavus/parasiticus isolates cultured for aflatoxin production were found to be aflatoxigenic.     

Inhibition of aflatoxin-producing fungi by Welsh onion extracts.

Welsh onion ethanol extracts were tested for their inhibitory activity against the growth and aflatoxin production of Aspergillus flavus and A. parasiticus. The survival of spores of A. flavus and A. parasiticus depended on both the extract concentration and the exposure time of the spores to the Welsh onion extracts. The mycelial growth of two tested fungi cultured on yeast extract-sucrose broth was completely inhibited in the presence of the Welsh onion ethanol extract at a concentration of 10 mg/ml during 30 days of incubation at 25 degrees C. The extracts added to the cultures also inhibited aflatoxin production at a concentration of 10 mg/ml or permitted only a small amount of aflatoxin production with extract concentration of 5 mg/ml after 2 weeks of incubation. Welsh onion ethanol extracts showed more pronounced inhibitory effects against the two tested aflatoxin-producing fungi than did the same added levels of the preservatives sorbate and propionate at pH values near 6.5.     

Detection of moulds producing aflatoxins in maize and peanuts by an immunoassay

An enzyme-linked immunosorbent assay (ELISA) was developed to detect moulds producing aflatoxins in maize and peanuts by an antibody produced to extracellular antigen from Aspergillus parasiticus. This antibody recognized species with phenotypic similarities to A. parasiticus, A. flavus and the domesticated species A. sojae and A. oryzae. For maize samples that were naturally contaminated with aflatoxins, low and high levels of aflatoxin corresponded with low and high ELISA readings for mould antigens, respectively. Maize and peanuts inoculated with 10(2) spores ml(-1) of A. parasiticus and incubated at 15 degrees C for 18 days or 21 degrees C for 7 days were analyzed for mould antigens and aflatoxin levels. At 15 degrees C, mould antigens were detected by day 4 in maize when 0.16 ng g(-1) of aflatoxin was detected by ELISA but not by thin layer chromatography (TLC). Antigens were detected in peanuts by day 4 before aflatoxin was found. Likewise, at 21 degrees C, antigens were detected by day 4 in maize when less than 1 ng g(-1) of aflatoxin was detected by ELISA but not by TLC, but by day 2 in peanuts when no aflatoxin was detected. A. parasiticus could be detected before it could produce aflatoxins. Therefore, this ELISA shows potential as an early detection method for moulds that produce aflatoxins.