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.     

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.     

Aqueous extracts of Tulbaghia violacea inhibit germination of Aspergillus flavus and Aspergillus parasiticus conidia

Aspergillus flavus and Aspergillus parasiticus are important plant pathogens and causal agents of pre- and postharvest rots of corn, peanuts, and tree nuts. These fungal pathogens cause significant crop losses and produce aflatoxins, which contaminate many food products and contribute to liver cancer worldwide. Aqueous preparations of Tulbaghia violacea (wild garlic) were antifungal and at 10 mg/ml resulted in sustained growth inhibition of greater than 50% for both A. flavus and A. parasiticus. Light microscopy revealed that the plant extract inhibited conidial germination in a dose-dependent manner. When exposed to T. violacea extract concentrations of 10 mg/ml and above, A. parasiticus conidia began germinating earlier and germination was completed before that of A. flavus, indicating that A. parasiticus conidia were more resistant to the antifungal effects of T. violacea than were A. flavus conidia. At a subinhibitory extract dose of 15 mg/ml, hyphae of both fungal species exhibited increased granulation and vesicle formation, possibly due to increased reactivity between hyphal cellular components and T. violacea extract. These hyphal changes were not seen when hyphae were formed in the absence of the extract. Transmission electron microscopy revealed thickening of conidial cell walls in both fungal species when grown in the presence of the plant extract. Cell walls of A. flavus also became considerably thicker than those of A. parasiticus, indicating differential response to the extract. Aqueous preparations of T. violacea can be used as antifungal treatments for the control of A. flavus and A. parasiticus. Because the extract exhibited a more pronounced effect on A. flavus than on A. parasiticus, higher doses may be needed for control of A. parasiticus infections.     

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.     

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.     

Highly sensitive PCR-based detection method specific for Aspergillus flavus in wheat flour

Aspergillus flavus is frequently found in food, producing a wide variety of toxins, aflatoxins being the most relevant in food safety. A specific PCR-based protocol for this species is described which allowed discrimination from other closely related species having different profiles of secondary metabolites from the Aspergillus Section Flavi, particularly A. parasiticus. The specific primers were designed on the multi-copy internal transcribed region of the rDNA unit (ITS1-5.8S-ITS2 rDNA) and were tested in a wide sample of related species and other fungal species commonly found in food. The PCR assay was coupled with a fungal enrichment and a DNA extraction method for wheat flour to enhance the sensitivity of the diagnostic protocol. The results indicated that the critical PCR amplification product was clearly observed for wheat flour contaminated by 10(2) spores after 16 h of incubation.     

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.     

Invertase production of common storage moulds in food and feed grains as a possibility for rapid detection of Aspergillus flavus group and Aspergillus fumigatus

Invertase production of grain storage moulds was studied. Aspergillus spp. and Penicillium spp. were grown in a sucrose based liquid medium, at 37 degrees C. The A. flavus group (A. flavus, A. parasiticus, A. nomius, A. oryzae) and A. fumigatus showed a fast growth and intense invertase activity, while other Aspergillus spp. and Penicillium spp. grew slower and produced less invertase. The pattern of accumulated reducing sugar after 20 and 48 h of incubation was characteristic to the species studied. From inoculation studies the detection limit was calculated as: 1-10 conidia of A. flavus group and A. fumigatus, as compared to 10(3)-10(4) for the other species studied. The method may be recommended as a rapid test for the detection of A. flavus group and A. fumigatus in food and feed grains.     

Studies on Aspergillus section Flavi isolated from maize in northern Italy

In 2003, for the first time in Italy, significant problems arose with colonization and contamination of maize destined for animal feed with Aspergillus section Flavi and aflatoxins (AFs). This resulted in milk and derived products being contaminated with AFM(1) at levels above the legislative limit. There was little knowledge and experience of this problem in Italy. The objectives of this research were thus to study the populations of Aspergillus section Flavi in six northern Italian regions and obtain information on the relative role of the key species, ability to produce sclerotia, production of the main toxic secondary metabolites, aflatoxins and cyclopiazonic acid, and tolerance of key environmental parameters. A total of 70 strains were isolated and they included the toxigenic species A. flavus and A. parasiticus. A. flavus was dominant in the populations studied, representing 93% of the strains. Seventy percent of strains of Aspergillus section Flavi produced AFs, with 50% of strains also producing cyclopiazonic acid. Sixty-two percent of A. flavus strains and 80% of A. parasiticus were able to produce sclerotia at 30 degrees C. Using 5/2 agar, only 1 strain developed S sclerotia and 19 L sclerotia. With regard to ecological studies, growth of Aspergillus section Flavi was optimal at between 25 and 30 degrees C, while AFB(1) production was optimal at 25 degrees C. Regarding water availability (water activity, a(w)), 0.99 a(w) was optimal for both growth and AFs production, while the only aflatoxin produced in the driest condition tested (0.83 a(w)) was AFB(1). This information will be very useful in identifying regions at risk in northern Italy by linking climatic regional information to levels of fungal contamination present and potential for aflatoxin production in maize destined for animal feed. This would be beneficial as part of a prevention strategy for minimising AFs in this product.     

Variability of aflatoxin and cyclopiazonic acid production by Aspergillus section flavi from different substrates in Argentina

Aspergillus section flavi strains isolated from peanuts, wheat and soybean grown in Argentina were screened for aflatoxins (type B and G) and cyclopiazonic acid (CPA) production. Aspergillus flavus was the predominant species in all substrates, although there was almost the same proportion of A. flavus and Aspergillus parasiticus in peanuts. Aspergillus nomius was not found. Incidence of aflatoxigenic A. flavus strains was higher in peanuts (69%) than in wheat (13%) or soybeans (5%) while the ratio of CPA producers A. flavus isolated from all substrates was very high (94% in peanuts, 93% in wheat and 73% in soybeans). Isolates of A. flavus able to produce simultaneously aflatoxins type B and CPA were detected in all substrates, suggesting the possibility of co-occurrence of these toxins. Almost all isolates of A. parasiticus resulted aflatoxins (type B and G) producers but did not produce CPA. Five of sixty-seven strains isolated from peanuts showed an unusual pattern of mycotoxin production (aflatoxins type B and G simultaneously with CPA). These strains also produced numerous small sclerotia like S strains of A. flavus detected in cottonseed in Arizona and in soils of Thailand and West Africa. The atypical strains are not widely distributed in Argentina and were found uniquely in peanuts.     

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.     

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.     

Mold counts and Aspergillus section Flavi populations in rice and its by-products from the Philippines

Mold counts and Aspergillus section Flavi populations in rice and its by-products from the Philippines were examined. The average mold counts of rough rice, brown rice, and locally produced polished rice were 4.1 x 10(3), 1.0 x 10(3), and 1.1 x 10(3) CFU/g, respectively. Average Aspergillus section Flavi counts of the same samples were 3.0 x 10(2), 1.1 x 10(2), and 2.6 x 10(2) CFU/g, respectively. Twenty-seven percent of mold isolates from rough rice, polished rice, and brown rice were section Flavi spp., 31% of which were toxigenic. No section Flavi isolates were obtained from imported rice samples from Thailand and Vietnam. Aspergillus section Flavi was also isolated from rice hull, rice bran, and settled dust from rice milling operations. Toxigenic isolates of both Aspergillus flavus and Aspergillus parasiticus were present in at least one sample of each type of rice and rice by-product except settled dust. Aflatoxins produced in vitro by the isolates ranged from <1 microg/kg to 6,227 microg/kg. A. flavus isolates produced only B aflatoxins, whereas A. parasiticus isolates produced both B and G aflatoxins. Although total mold counts of Philippine rice and its by-products are within tolerable limits, the establishment of maximum limits in counts of potentially aflatoxigenic species in foods and feeds is important because the mere presence of toxin producers is considered a possible risk factor. The results of this research illustrate the need for strict monitoring of rice during both storage and marketing, especially in warm and humid seasons when infestation and consequent production of aflatoxins by Aspergillus section Flavi is expected.     

西藏高原粮油作物曲霉菌污染及菌株产毒力研究

为探明西藏高原粮油作物曲霉菌污染状况及黄曲霉菌产毒能力,连续5年对西藏青稞、小麦、花生3种作物中曲霉菌污染情况进行分析,并对其分离到的黄曲霉菌株开展产毒力研究,结果表明,204份样品中,共分离出15种曲霉菌,曲霉菌污染率呈花生>青稞>小麦。青稞、小麦中曲霉属优势种均为黑曲霉(Aspergillus niger),真菌毒素主要为杂色曲霉毒素和赭曲霉毒素;花生优势种为黄曲霉(A.flavus);仅受黄曲霉毒素污染。来源于不同作物的黄曲霉菌,其产毒类型也有差异,麦类作物产毒菌株以产黄曲霉毒素B₁(AFB₁)、黄曲霉毒素B₂(AFB₂)为主;花生产毒菌株以产AFB₁、AFB₂、AFG₁、AFG₂为主。     

Nannocystis exedens: a potential biocompetitive agent against Aspergillus flavus and Aspergillus parasiticus.

This study examined the potential for controlling toxigenic Aspergillus flavus and Aspergillus parasiticus by biological means using a myxobacterium commonly found in soil. The ability of Nannocystis exedens to antagonize A. flavus ATCC 16875, A. flavus ATCC 26946, and A. parasiticus NRRL 3145 was discovered. Cultures of aflatoxigenic fungi were grown on 0.3% Trypticase peptone yeast extract agar for 14 days at 28 degrees C. When N. exedens was grown in close proximity with an aflatoxigenic mold, zones of inhibition (10 to 20 mm) developed between the bacterium and mold colony. A flattening of the mold colony on the sides nearest N. exedens and general stunting of growth of the mold colony were also observed. When N. exedens was added to the center of the cross-streak of a mold colony, lysis of the colony by the bacterium was observed after 24 h. Microscopic observations revealed that N. exedens grew on spores, germinating spores, hyphae, and sclerotia of the molds. These results indicate that N. exedens may be a potential biocontrol agent against A. flavus and A. parasiticus.     

Fungal population and distribution of aflatoxigenic fungi in commercial almond powder products

The fungal population and distribution of aflatoxin-producing fungi in 30 samples of imported almond powder products purchased from retail markets were examined in this study. Total counts of fungi ranged from under 1.0 x 10 colony-forming units (CFU)/g to 8.5 x 10(3) CFU/g as determined with the dilution plating technique. The predominant fungi in the mould-contaminated almond samples were Aspergillus niger, A. flavus and the related species, Penicillium, Cladosporium and Rhizopus. Aflatoxin-producing ability in the isolates of A. flavus and related fungi were tested by thin layer chromatography using 2% yeast extract and 15% sucrose broth culture. Four different aflatoxigenic fungi were detected in the isolates; aflatoxins B1 and B2 were produced by some strains of A. flavus and A. parvisclerotigenus, and aflatoxins B1, B2, G1, G2 were produced by all tested strains of A. parasiticus and A. nomius. Identification of the strains was based on morphological and metabolic characters.     

Occurrence of Aspergillus spp. and aflatoxin B1 in Malaysian foods used for human consumption

Malaysian population widely consumes the cereal-based foods, oilseeds, nuts, and spices in their daily diet. Mycotoxigenic fungi are well known to invade food products under storage conditions and produce mycotoxins that have threat to human and animal health. Therefore, determining toxigenic fungi and aflatoxin B(1) (AFB1) in foods used for human consumption is of prime importance to develop suitable management strategies and to minimize risk. Ninety-five food products marketed in Penang, Malaysia were randomly collected from different supermarkets and were analyzed for presence of Aspergillus spp. by agar plate assay and AFB1 by enzyme-linked immunosorbent assay (ELISA). A. flavus was the dominant fungi in all foods followed by A. niger. Fifty-five A. flavus strains were tested for their ability to produce aflatoxins on rice grain substrate. Thirty-six (65.4%) strains out of 55 produced AFB1 ranging from 1700 to 4400 μg/kg and 17 strains (31%) produced AFB2 ranging from 620 to 1670 μg/kg. Natural occurrence of AFB1 could be detected in 72.6% food products ranging from 0.54 to 15.33 μg/kg with a mean of 1.95 μg/kg. Maximum AFB1 levels were detected in peanut products ranging from 1.47 to 15.33 μg/kg. AFB1 levels detected in all food products were below the Malaysian permissible limits (<35 μg/kg). Aspergillus spp. and AFB1 was not detected in any cookies tested. Although this survey was not comprehensive, it provides valuable information on aflatoxin levels in foods marketed in Malaysia.     

利用12S rRNA基因的限制性酶切末端片段长度多态性鉴定动物种类

目的：建立一种精确可靠的鉴定常见的1 0 种动物( 猪、狗、牛、山羊、绵羊、马、鸡、鼠、三文鱼和鹿)的方法。方法：利用12S rRNA 基因的限制性酶切末端片段长度多态性(terminal restriction fragment lengthpolymorphism,T-RFLP)鉴别动物种类。将线粒体12S rRNA 基因通过引物的5＇端用FAM 荧光标记,从基因组DNA 中扩增450bp 的目的片段。引物对1(下游引物FAM标记,上游引物不标记)扩增的PCR 产物用限制性内切酶Alu Ⅰ酶切。引物对2(上游引物FAM标记,下游引物不标记)扩增的PCR 产物用限制性内切酶Tru9 Ⅰ酶切。得到的酶切产物分别在遗传分析仪ABI 3100 上进行毛细管电泳,片段大小用Peak Scanner 1.0 软件分析。结果：根据Alu Ⅰ酶切图谱能够区分鸡、马、猪和三文鱼,而鹿和牛、山羊和绵羊、鼠和狗因酶切图谱相同无法分开。根据Tru9 Ⅰ酶切图谱,能够进一步将鹿和牛、山羊和绵羊、鼠和狗分开。同一种动物不同个体的酶切图谱完全相同,结果具有可重复性。没有出现物种内多态的现象。大多数情况下,实际得到的末端片段长度与理论值非常接近,只存在2 ～5bp 的差异。结论：该方法操作简单、结果精确,适用于鉴定动物种类。     

Fungi, aflatoxins, and cyclopiazonic acid associated with peanut retailing in Botswana

Peanuts are important food commodities, but they are susceptible to fungal infestation and mycotoxin contamination. Raw peanuts were purchased from retail outlets in Botswana and examined for fungi and mycotoxin (aflatoxins and cyclopiazonic acid) contamination. Zygomycetes were the most common fungi isolated; they accounted for 41% of all the isolates and were found on 98% of the peanut samples. Among the Zygomycetes, Absidia corymbifera and Rhizopus stolonifer were the most common. Aspergillus spp. accounted for 35% of all the isolates, with Aspergillus niger being the most prevalent (20.4%). Aspergillus flavus/parasiticus were also present and accounted for 8.5% of all the isolates, with A. flavus accounting for the majority of the A. flavus/parasiticus identified. Of the 32 isolates of A. flavus screened for mycotoxin production, 11 did not produce detectable aflatoxins, 8 produced only aflatoxins B1 and B2, and 13 produced all four aflatoxins (B1, B2, G1, and G2) in varying amounts. Only 6 of the A. flavus isolates produced cyclopiazonic acid at concentrations ranging from 1 to 55 microg/kg. The one A. parasiticus isolate screened also produced all the four aflatoxins (1,200 microg/kg) but did not produce cyclopiazonic acid. When the raw peanut samples (n = 120) were analyzed for total aflatoxins, 78% contained aflatoxins at concentrations ranging from 12 to 329 microg/kg. Many of the samples (49%) contained total aflatoxins at concentrations above the 20 microg/kg limit set by the World Health Organization. Only 21% (n = 83) of the samples contained cyclopiazonic acid with concentrations ranging from 1 to 10 microg/kg. The results show that mycotoxins and toxigenic fungi are common contaminants of peanuts sold at retail in Botswana.