Mycoflora and ochratoxin A producing strains of Aspergillus in Algerian wheat

Wheat is a basic staple food for very large segments of the population of Algeria. The aim of this study is to analyse ochratoxin A (OTA)-producing mould and OTA-contaminated wheat. To evaluate the mycoflora and the potential for OTA production by Aspergillus strains, a total of 85 samples of wheat destined for human consumption were collected from two regions in Algeria (Tizi Ouzou and Setif) during the following phases: preharvest, storage in silos, and after processing. The mean value counts of fungi ranged from 275 to 1277 CFU g(-1). The dominant genus was Aspergillus, predominantly A. flavus, A. niger and A. versicolor. The other isolated species were A. ochraceus, A. alliaceus, A. carbonarius, A. terreus, A. fumigatus, A. candidus and Aspergillus spp. The occurrence and the levels of the genus Penicillium, Fusarium, Alternaria and Mucor were substantially lower than those of Aspergillus. The storage in silos shows high levels of Aspergillus (66 to 84%), especially A. flavus, but A. niger and other fungi were isolated at relatively low percentages. Equal distribution of the fungal contamination into the bran, flour and semolina fractions was observed from Flour Mill and Semolina Mill. The genus Aspergillus remained present at high levels at several phases of the production process. In addition, the ability to produce OTA by 135 isolates belonging to eleven species of Aspergillus and 23 isolates of Penicillium spp. was analyzed using fluorescent detection-based HPLC. Thus, it was found that 51 isolates (32.3%) were ochratoxigenic. All isolated strains of A. ochraceus (12) and A. alliaceus (6) produced OTA at concentrations ranging from 0.23 to 11.50 microg g(-1). Most of the A. carbonarius strains (80%) were OTA producers (0.01 to 9.35 microg g(-1)), whereas A. terreus (50%), A. niger (28%), A. fumigatus (40%), A. versicolor (18%) and Penicillium spp. (21.7%) were low level producers (0.01 to 0.07 microg g(-1)). The concentration of OTA was determined in 30 samples of wheat. OTA was detected in 12 (40%) of the samples at levels ranging from 0.21 to 41.55 microg kg(-1).     

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.     

Characterization of filamentous fungi isolated from Moroccan olive and olive cake: toxinogenic potential of Aspergillus strains

During the 2003 and 2004 olive oil production campaigns in Morocco, 136 samples from spoiled olive and olive cake were analyzed and 285 strains were isolated in pure culture. Strains included 167 mesophilic strains belonging to ten genera: Penicillium, Aspergillus, Geotrichum, Mucor, Rhizopus, Trichoderma, Alternaria, Acremonium, Humicola, Ulocladium as well as 118 thermophilic strains isolated in 2003 and 2004, mainly belonging to six species: Aspergillus fumigatus, Paecilomyces variotii, Mucor pusillus, Thermomyces lanuginosus, Humicola grisea, and Thermoascus aurantiacus. Penicillium and Aspergillus, respectively, 32.3 and 26.9% of total isolates represented the majority of mesophilic fungi isolated. When considering total strains (including thermotolerant strains) Aspergillus were the predominant strains isolated; follow-up studies on mycotoxins therefore focused primarily on aflatoxins (AFs) and ochratoxin A (OTA) from the latter strains. All isolated Aspergillus flavus strains (9) and Aspergillus niger strains (36) were studied in order to evaluate their capacity to produce AFs and OTA, respectively, when grown on starch-based culture media. Seven of the nine tested A. flavus strains isolated from olive and olive cake produced AF B1 at concentrations between 48 and 95 microg/kg of dry rice weight. As for the A. niger strains, 27 of the 36 strains produced OTA.     

Effect of water activity and temperature on growth of three Penicillium species and Aspergillus flavus on a sponge cake analogue.

This study compared the effect of temperature and water activity and their interactions on the rate of mycelial growth of Penicillium aurantiogriseum, P. chrysogenum, P. corylophilum and Aspergillus flavus on a sponge cake analogue. As expected, growth rates showed dependence on a(w), and temperature. However, no significant differences were observed in the growth rates of different isolates. The minimum a(w) values for growth of the Penicillium spp. was 0.85-0.90. A. flavus was able to grow at 0.90 a(w) when the temperature was above 15 degrees C. This study has shown that fungal growth by these species on a sponge cake analogue, with a composition similar to usual bakery products, is prevented if the a(w) is kept at < 0.85.     

Aspergillus Flavus, Aspergillus Niger, and Penicillium Corylophilum Spoilage Prevention of Bakery Products by Means of Weak-Acid Preservatives

ABSTRACT: Apart from Eurotium species, Aspergillus and Penicillium isolates are common contaminants of bakery products. This paper deals with the use of weak-acid preservatives (potassium sorbate, calcium propionate, and sodium benzoate) to prevent spoilage by Aspergillus niger, A. flavus , and Penicillium corylophilum in analogs of a bakery product. A hurdle technology approach has been considered in which factors other than preservatives are pH and water activity. Potassium sorbate has been found to be the most effective in preventing fungal spoilage of this kind of products at the maximum concentration tested (0.3%). Suboptimal doses (0.03%) of all preservatives tested led to an enhancement of growth of Aspergillus and Penicillium isolates. The characteristics of the products involved must be carefully considered before making the decision of adding weak-acid preservatives; moreover, they must be added at the right concentrations. Furthermore, more research is needed on the use of alternative natural preservatives, such as essential oils.     

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.     

Odor volatiles associated with microflora in damp ventilated and non-ventilated bin-stored bulk wheat

Western hard red spring wheat, stored at 20 and 25% moisture contents for 10 months during 1985-86, was monitored for biotic and abiotic variables in 10 unheated bins in Winnipeg, Manitoba. The major odor volatiles identified were 3-methyl-1-butanol, 3-octanone and 1-octen-3-ol. The production of these volatiles was associated and correlated with microfloral infection. Ventilation, used for cooling and drying of grain, disrupted microfloral growth patterns and production of volatiles. The highest levels of 3-methyl-1-butanol occurred in 25% moisture content wheat infected with bacteria, Penicillium spp. and Fusarium spp. In non-ventilated (control) bins with 20% moisture content wheat, 3-methyl-1-butanol was correlated with infection by members of the Aspergillus glaucus group and bacteria. In control bins, 1-octen-3-ol production was correlated with infection of wheat of both moisture contents by Penicillium spp. The fungal species, isolated from damp bin-stored wheat and tested for production of odor volatiles on wheat substrate, included Alternaria alternata (Fr.) Keissler, Aspergillus repens (Corda) Saccardo, A. flavus Link ex Fries, A. versicolor (Vuill.) Tiraboschi, Penicillium chrysogenum Thom, P. cyclopium Westling, Fusarium moniliforme Sheldon, F. semitectum (Cooke) Sacc. In the laboratory, fungus-inoculated wheat produced 3-methyl-1-butanol; 3-octanone and 1-octen-3-ol were also produced, but less frequently. Two unidentified bacterial species isolated from damp wheat and inoculated on agar produced 3-methyl-1-butanol.     

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.     

Survey of the mycobiota of Spanish malting barley and evaluation of the mycotoxin producing potential of species of Alternaria, Aspergillus and Fusarium

The present work deals with the toxigenic mycobiota occurring in Spanish malting barley and the capability for producing mycotoxins by several important toxigenic fungi. One hundred and eighty seven samples of malting barley were gathered from Spanish breweries before processing. One hundred and fifty kernels per sample were surface-sanitized with a 2% sodium hypochlorite solution and incubated on three culture media. The most abundant fungi were species of Alternaria, Aspergillus, Penicillium and Fusarium, which were present in 93%, 82.3%, 57.8% and 27.8% of the samples, respectively. To evaluate their mycotoxin producing potential a number of isolates belonging to each genus, except Penicillium, were randomly selected and incubated on culture media known to be appropriate for production of mycotoxins. Alternariol and alternariol monomethyl ether were produced by 26.7% of Alternaria spp. isolates (all belonged to Alternaria alternata). All tested isolates of F. verticillioides produced fumonisin B(1) (FB(1)) and 61.3% of them produced fumonisin B(2) (FB(2)), whereas FB(1) was synthesized by 83.3% and FB(2) by 77.8% of F. proliferatum isolates. Twenty percent of the isolates of the Aspergillus flavus/A. parasiticus group had the capability to produce aflatoxin B(1) and aflatoxin B(2). Thirty out of 34 isolates of F. graminearum produced deoxynivalenol and zearalenone whereas the other 4 isolates produced nivalenol. Ochratoxin A was detected in 75% and 15% of isolates of Aspergillus section Nigri and A. ochraceus, respectively. This is the first survey carried out in Spain on the toxigenic mycobiota contaminating malting barley in breweries and the mycotoxin producing capacity of several species. The information obtained is useful for assessing the risk of mycotoxins in beer.     

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.     

Mycological condition of maize products.

Maize and maize-related products were investigated in a collaborative study for viable moulds and antigenic extracellular polysaccharides (EPS) produced by Aspergillus and Penicillium species. In addition, the samples were tested for the presence of aflatoxin B1. All maize products, with the exception of the heat processed products, contained viable moulds on an average of (log10 values) 3.3 +/- 0.7 colony-forming units per gram. In most samples a mixed mould flora was present. Species of the genus Fusarium were dominant, followed by Aspergillus, Eurotium and Penicillium. The mould colony count correlated positively with the presence of antigenic extracellular polysaccharides produced by species of Aspergillus and Penicillium. Gamma irradiation did not affect the detection of antigenic extracellular polysaccharides. Aflatoxin B1 was detected in two out of 35 samples; these contained 0.6 and 0.8 microgram/kg. From one of these aflatoxin B1-containing samples, Aspergillus flavus was isolated.     

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。     

Occurrence of pathogenic fungal species in Tunisian vineyards

Mycotoxins are secondary metabolites produced by filamentous fungi detected in food, such are grapes. OTA was evaluated in ten handle musts from different Tunisian vineyard. This mycotoxin was found at levels 1.1 mug/L to 4.3 mug/L. A survey was conducted to assess the contamination of the Tunisian vineyard with pathogenic fungal species, in particular those responsible of the OTA production. The results were evaluated for the first time in parcels cultivated in the North, in the Centre and in the South of the country. Italia Muscate and Superior Seedless varieties were concerned at three developmental stages of the berry, setting, veraison and maturity. Carigon variety was used as positive control for musts contaminating by OTA. The main fungal species isolated were Aspergillus spp. (33.32%), Botrytis cinerea (23.32%), Alternaria spp. (12.80%), Cladosporium spp. (10.59%) and Penicillium spp. (8.3%). The isolates of the Aspergillus genus were identified as Aspergillus niger aggregate (77%), Aspergillus carbonarius (15%) and Aspergillus flavus (8%). Their presence was characterized by a significant decrease in the Centre during the veraison and a slight increase in the North and the South during the maturity stage. Furthermore, when comparing Superior Seedless and Italia Muscate cultivated in the same area, the aspergilli were particularly less abundant at the setting stage in the case of Superior Seedless. There is no correlation between the OTA amount in musts and the contamination by Aspergillus species in different vineyards and for grape varieties studied.     

Microbial quality and presence of moulds in Kuflu cheese

The chemical and microbial qualities, including fungal flora, of 30 samples of Kuflu cheese randomly purchased from different markets in Turkey were investigated. The gross composition of the cheese samples ranged between 37.65-53.65% moisture, 6.21-40.09% fat-in-dry matter, 4.70-10.07% salt-in-moisture and 26.18-44.85% protein. The mean pH value of the cheeses was 6.29+/-0.28 and pH values ranged from 5.52 to 7.22. Variations between the samples in terms of their gross composition suggested a lack of quality standards in cheesemilk, cheesemaking procedure and ripening conditions. The levels of main microbial groups including total mesophilic and coliform bacteria, yeasts and moulds and the presence of some potentially pathogenic microorganisms (E. coli, Salmonella spp. and Staphylococcus aureus) were determined. The high numbers of all microbial groups and presence of potentially pathogenic organisms in the cheese samples suggested that the production and maturation of Kuflu cheese should be improved by better hygiene. Moulds at the cheese surface were isolated and identified. A total of 24 different mould species were detected and the genus most frequently isolated was Penicillium spp. which represented 70.25% of total isolates. Penicillium commune, P. roqueforti and P. verrucosum were the most abundant species in the cheeses sampled. The other dominant fungal groups were Geotrichum candidum, Penicillium expansum and P. chrysogenum. Other genera isolated from the cheese were Acremonium, Alternaria, Aspergillus, Cladosporium, Geotrichum, Mucor, Rhizopus and Trichoderma. The potentially toxigenic species, including some Penicillum spp. and Aspergillus flavus, were also detected.     

Current importance of ochratoxin A-producing Aspergillus spp

Ochratoxin A (OA) is receiving attention worldwide because of the hazard it poses to human and animal health. OA contamination of commodities, such as cereals or pork and poultry meat, is well recognized. Nevertheless, there is an increasing number of articles reporting OA contamination in other food commodities, such as coffee, beer, wine, grape juice, and milk, in the last few years. This continuous and increasing exposure to OA that humans experience is reflected in the high incidence of OA in both human blood and milk in several countries. OA was believed to be produced only by Aspergillus ochraceus and closely related species of section Circumdati and by Penicillium verrucosum; however, in the genus Aspergillus, the production of OA has been recently reported by species outside the section Circumdati. Thus, it has been clearly established as a metabolite of different species of the section Nigri, such as Aspergillus niger and Aspergillus carbonarius. OA production ability by Aspergillus spp. is more widespread than previously thought; therefore, there is the possibility that unexpected species can be new sources of this mycotoxin in their natural substrates.     

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.     

EFFECT OF GAMMA IRRADIATION ON AFLATOXIN B1 PRODUCTION BY ASPERGILLUS FLAVUS IN GROUND BEEF STORED AT 5C

The effect of γ-irradiation for controlling the production of aflatoxin B₁ by Aspergillus flavus in ground beef stored at 5C for 2 weeks was investigated. Aspergillus, Penicillium, Cladosporium, Mucor, Scopulariopsis, Candida and Rhodotorula were the most common fungal genera contaminating ground beef. A. flavus and A. niger were the most common Aspergillus spp. Aspergillus flavus isolates were able to produce aflatoxin B₁ in ground beef. Only 3 (20%) samples of ground beef were contaminated with aflatoxin B₁ (25–45 μg/Kg). Gamma irradiation dose levels resulted in an immediate reduction in the total numbers of A. flavus. No growth or aflatoxin B₁ production occurred at 1.50 kGy during storage.     

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。     

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.     

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.