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.     

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.     

Identification of species in Aspergillus section Flavi based on sequencing of the mitochondrial cytochrome b gene.

The partial sequences of the mitochondrial (mt) cytochrome b gene (402 bp) were determined for species of Aspergillus section Flavi. On the basis of identities of DNA sequences, 77 strains were divided into seven DNA types, from D-1 to D-7. The type strains of A. sojae, A. parasiticus, A. flavus and A. oryzae together, A. tamarii, and A. nomius were placed in DNA types D-1. D-2, D-4, D-5 and D-7, respectively. These species could be differentiated from each other. Furthermore, two other DNA types, D-3 and D-6 were found. DNA type D-3 was closely related to A. parasiticus (D-2) and included one strain that deposited as A. flatus var. flavus and produced aflatoxins B and G. DNA types D-6 included one strain named A. flavus and closely related to A. tamarii. The observations of conidial wall texture by SEM (Scanning Electron Microscopy) supported the relationships derived from the cytochrome b gene. The production of aflatoxins was also examined. Using the DNA sequence of cytochrome b gene, several strains were reidentified. The derived amino acids sequences were all the same in the studied strains. The mt cytochrome b gene is useful and reliable in distinguishing and identifying the species in Aspergillus section Flavi.     

黑曲霉抗产毒黄曲霉作用的初步研究

采用90 × 2.6 × 1013N+/cm2 注入黑曲霉筛选能抗黄曲霉(Aflavus)生长的突变菌株,以利发酵中控制被黄曲霉污染的原材料的再污染。进行产毒黄曲霉与被离子注入的黑曲霉混合对峙、原黑曲霉菌株与黄曲霉单独培养生长及混合对峙培养实验。结果显示经离子注入的菌株及未注入菌株均对黄曲霉产生抑制作用,但后者仅有微弱抑制,前者不仅表现出几乎不能使黄曲霉生长,且已长出的黄曲霉菌丝体较瘦小,并呈灰白色。从培养基中提取物检验结果显示,黄曲霉组表现出有较明显的荧光反应,而黑曲霉菌株对峙培养物提取物中有微弱的荧光反应,其黑曲霉突变株对峙培养物未见荧光反应检出。这表明黑曲霉原菌株虽然能对黄曲霉只有微弱抑制,但表现出黄曲霉产毒和合成色素能力下降。与对照组相比,突变株有较强抑制黄曲霉生长能力。     

INFLUENCE OF OTHER FUNGI ON AFLATOXIN PRODUCTION BY ASPERGILLUS FLAVUS IN MAIZE KERNELS

Experiments were designed to determine whether certain nontoxigenic fungi commonly isolated from maize kernels can affect aflatoxin B₁ development when inoculated with A. flavus onto individual unsterilized, and autoclaved maize kernels . Trichoderma viride and Aspergillus niger were found to be strongly antagonist inhibiting the growth of A. flavus by 87 and 66% respectively, whereas Aspergillus versicolor, Fusarium moniliforme, Paecilomyces variotii and Emericella quadrillineata inhibited the growth of A. flavus by less than 51%. Less aflatoxin B₁ was detected when A. flavus was paired with A. niger or T. viride than with the other test fungi. When A. niger or T. viride was introduced onto the kernels 72 h before inoculation with A. flavus, no aflatoxin B₁ was detected in unsterilized kernels and the levels of aflatoxin B₁ were greatly reduced from 700 ppb to 160 and 140 ppb in autoclaved kernels, respectively. When inoculation of A. flavus followed 72 h of incubation of either A. niger and T. viride, no aflatoxin B₁ was detected. However, when both A. niger and T. viride were introduced 72 h after inoculation with A. flavus, the levels of aflatoxin B₁ were reduced to 385 and 560 ppb, respectively in unsterilized and autoclaved maize kernels . Trichoderma viride and Aspergillus niger may be useful in biological control of aflatoxin contamination of maize kernels .; Accepted for Publication June 11, 1997     

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.     

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.     

High genetic variability in non-aflatoxigenic A. flavus strains by using Quadruplex PCR-based assay

Aflatoxigenic Aspergillus flavus isolates always show, by using a multiplex PCR-system, four DNA fragments specific for aflR, nor-1, ver-1, and omt-A genes. Non-aflatoxigenic A. flavus strains give variable DNA banding pattern lacking one, two, three or four of these genes. Recently, it has been found and reported that some aflatoxin non-producing A. flavus strains show a complete set of genes. Because less is known about the incidence of structural genes aflR, nor-1, ver-1 and omt-A in aflatoxin non-producing strains of A. flavus, we decided to study the frequencies of the aflatoxin structural genes in non-aflatoxigenic A. flavus strains isolated from food and feed commodities. The results can be summarized as following: 36.5% of the examined non-aflatoxigenic A. flavus strains showed DNA fragments that correspond to the complete set of genes (quadruplet pattern) as found in aflatoxigenic A. flavus. Forty three strains (32%) showed three DNA banding patterns grouped in four profiles where nor-1, ver-1 and omt-A was the most frequent profile. Twenty five (18.7%) of non-aflatoxigenic A. flavus strains yielded two DNA banding pattern whereas sixteen (12%) of the strains showed one DNA banding pattern. In one strain, isolated from poultry feed, no DNA bands were found. The nor-1 gene was the most representative between the four aflatoxin structural assayed genes. Lower incidence was found for aflR gene. Our data show a high level of genetic variability among non-aflatoxigenic A. flavus isolates that require greater attention in order to design molecular experiment to distinguish true aflatoxigenic from non-aflatoxigenic A. flavus strains.     

高大平房仓稻谷粮层霉菌量与优势霉菌的差异性研究

采用传统培养法对湖南与湖北两省粮库中的稻谷进行研究,对高大平房仓粮仓上中下三层的稻谷霉菌量及优势霉菌进行研究。研究结果表明:湖南省储藏一年稻谷与新入库稻谷中层霉菌数分别为6.4×10~3 CFU/g、1.3×10~3 CFU/g,相比上、下层,中层最多;湖北省储藏一年稻谷下层霉菌数为1.4×10~4 CFU/g,相比上、中层,下层最多,湖北省新入库稻谷上层霉菌数为1.4×10~4 CFU/g,相比中、下层,上层最多。通过传统的菌落培养及菌丝、孢子观察,初步判断上中下三层的优势霉菌,并结合分子生物学的方法对其ITS序列进行分析,通过PCR扩增,将扩增出来的基因序列,在GenBank进行BLAST,最终鉴定优势菌株为黄曲霉、白曲霉、聚多曲霉、内生真菌、黑曲霉。     

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.     

Differentiation between Aspergillus flavus and Aspergillus parasiticus from pure culture and aflatoxin-contaminated grapes using PCR-RFLP analysis of aflR-aflJ intergenic spacer

Aflatoxins (AFs) represent the most important single mycotoxin-related food safety problem in developed and developing countries as they have adverse effects on human and animal health. They are produced mainly by Aspergillus flavus and A. parasiticus. Both species have different aflatoxinogenic profile. In order to distinguish between A. flavus and A. parasiticus, gene-specific primers were designed to target the intergenic spacer (IGS) for the AF biosynthesis genes, aflJ and aflR. Polymerase chain reaction (PCR) products were subjected to restriction endonuclease analysis using BglII to look for restriction fragment length polymorphisms (RFLPs). Our result showed that both species displayed different PCR-based RFLP (PCR-RFLP) profile. PCR products from A. flavus cleaved into 3 fragments of 362, 210, and 102 bp. However, there is only one restriction site for this enzyme in the sequence of A. parasiticus that produced only 2 fragments of 363 and 311 bp. The method was successfully applied to contaminated grapes samples. This approach of differentiating these 2 species would be simpler, less costly, and quicker than conventional sequencing of PCR products and/or morphological identification.     

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.     

Mycoflora and toxigenic Aspergillus flavus in Spanish dry-cured ham

Sixty-five dry-salted hams were analysed. Aspergillus and Penicillium were the dominant genera. In general, the mould flora was dominated by Aspergillus spp. and primarily A. glaucus, A. fumigatus, A. niger and A. flavus. A flavus was found in 16 hams and 9 out of 16 strains examined produced aflatoxins 'in vitro'. Surface samples of dry-salted hams showed growth of inoculated A. parasiticus NRRL-2999 strains and production of aflatoxins in low levels at 25 and 30 degrees C. It is concluded that the presence of toxigenic strains in Spanish dry-salted ham does not constitute a health risk.     

黄曲霉菌株的分离、鉴定及产毒能力分析

对几株从发霉粮食中分离出的黄曲霉菌菌株进行形态学和分子生物学鉴定,并进行发酵培养和产毒能力的HPLC测定。结果表明:试验分离菌株均为黄曲霉菌株且含有黄曲霉毒素产生的关键基因aflR;黄曲霉菌株之间产毒能力差异巨大:黄曲霉菌株3.4408产毒量最高,黄曲霉菌株HDWS产毒量最低,黄曲霉菌株3.2572甚至不产生黄曲霉毒素;产生黄曲霉毒素菌株中部分黄曲霉菌株产生4种黄曲霉毒素AFB1、AFB2、AFG1、AFG2,黄曲霉菌株HDWH只产生黄曲霉毒素AFB1、AFB2。     

Mycoflora of hazelnut (Corylus avellana L.) and aflatoxin content in hazelnut kernels artificially infected with Aspergillus parasiticus

In this research, 30 hazelnut samples were used to determine their internal mould populations and their aflatoxin contents. The results showed that Aspergillus was the most common genus in the experimental samples (96.6% of all samples). Thirty-one% of isolates were classified as Aspergillus flavus and none of the samples contained aflatoxins. In the second part of the experiment, the kernels were inoculated with conidia of Aspergillus parasiticus and incubated at three different humidified conditions and two different temperatures for 45 days. Aflatoxin contents of the samples kept in 98% relative humidity and at 28 degrees C were higher (904.6 micrograms/kg) than that of the other samples. On the other hand, no aflatoxin was detected in the control samples that were not inoculated but kept at the same conditions.     

PCR detection of aflatoxin producing fungi and its limitations

Unlike bacterial toxins that are primarily peptides and are therefore encoded by a single gene, fungal toxins such as the aflatoxins are multi-ring structures and therefore require a sequence of structural genes for their biological synthesis. There is therefore no specific PCR for any one of the four biologically produced aflatoxins. Unfortunately, the structural genes presently in use for PCR detection of aflatoxin producing fungi are also involved in the synthesis of other fungal toxins such as sterigmatocystin by Aspergillus versicolor and Aspergillus nidulans and therefore lack absolute specificity for aflatoxin producing fungi (Table?1). In addition, the genomic presence of several structural genes involved in aflatoxin biosynthesis does not guarantee the production of aflatoxin by all isolates of Aspergillus flavus and Aspergillus parasiticus. The most widely used DNA target regions for discriminating Aspergillus species are those of the rDNA complex, mainly the internal transcribed spacer regions 1 and 2 (ITS1 and ITS2) and the variable regions in the 5'-end of the 28S rRNA gene. Since these sequence regions are unrelated to the structural genes involved in aflatoxin biosynthesis there successful amplification can be used for species identification but do not confirm aflatoxin production. This review therefore presents the various approaches and limitations in the use of the PCR in attempting to detect aflatoxin producing fungi.