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.     

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.     

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.     

二氢杨梅素抑制黄曲霉菌生长的机制

本研究探讨了二氢杨梅素对黄曲霉的抗真菌活性和潜在的抗真菌机制。首先,通过抑菌实验证明,二氢杨梅素对黄曲霉孢子和菌丝的最小抑菌质量浓度（minimum inhibitory concentration,MIC）均为4 mg/mL。通过荧光增白剂（calcofluor white,CW）和碘化丙锭（propidium iodide,PI）染色实验证明,二氢杨梅素处理后黄曲霉细胞壁和细胞膜受损。与对照组相比,1/2 MIC和MIC组黄曲霉细胞内容物释放量（OD260 nm）分别增加了3.14 倍和5.93 倍,细胞外相对电导率和pH值均升高,MIC的二氢杨梅素对黄曲霉的呼吸抑制率达25.82%。这些结果表明,二氢杨梅素通过破坏细胞壁和细胞膜的完整性以及干扰呼吸代谢发挥抗菌活性。此外,二氢杨梅素在MIC时能够完全抑制黄曲霉在花生和玉米籽粒上的萌发,因此,二氢杨梅素可作为一种有效的抗真菌天然化合物应用于粮食及农产品储藏中。     

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.     

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.     

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.     

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.     

In vitro Antifungal Activity of Dehydrozingerone and its Fungitoxic Properties

Abstract: The efficacy of Dehydrozingerone (DZ; dehydroderivative of zingerone) as an antifungal agent and its mode of action against food spoilage fungal pathogens was studied and presented. DZ is a constituent of ginger (Zingiber officinale rhizomes) and structural half analogue of curcumin. Its efficacy against Aspergillus oryzae, Aspergillus flavus, Aspergillus niger, Aspergillus ochraceus, Fusarium oxysporum and Penicillium chrysogenum was evaluated. Effect of DZ on the growth and sporulation of A. ochraceus was also studied. The fungal species were susceptible to DZ and the minimum inhibitory concentration and fungicidal concentration ranged from 755 to 911 μM and 880 to 1041 μM respectively. The mycelial and spore germination was significantly inhibited; reduction in the weight of the cell mass, carbohydrate, protein, DNA and RNA constituents in the cells isolated from cultures of A. ochraceus grown with DZ were observed. Scanning electron microscopy studies revealed morphological observations such as cell lysis, inhibition and morphological alterations in hyphae and sporulation in A. ochraceus on treatment with DZ. Practical application: Current investigations revealed that DZ is a potential antifungal agent and can find application as an additive or adjuvant in food and pharmaceutical industries after appropriate toxicological studies.     

In vitro inhibition of postharvest pathogens of fruit and control of gray mold of strawberry and green mold of citrus by aureobasidin A

Aureobasidin A (AbA), an antifungal cyclic depsipeptide antibiotic produced by Aureobasidium pullulans R106, has previously been shown to be effective against a wide range of fungi and protozoa. Here we report the inhibitory effects of AbA on spore germination, germ tuber elongation and hyphal growth of five pathogenic fungi including Penicillium digitatum, P. italicum, P. expansum, Botrytis cinerea and Monilinia fructicola, which are major pathogens causing postharvest diseases of a variety of fruits. AbA inhibited five pathogenic fungi by reducing conidial germination rates, delaying conidial germination initiation, restricting elongation of germ tuber and mycelium, as well as inducing abnormal alternations of morphology of germ tubes and hyphae of these fungi. The sensitivity of these fungi to AbA was pathogen species-dependent. P. digitatum was the most sensitive and M. fructicola the least. Importantly, AbA at 50 microg/ml was effective in controlling the citrus green mold and in reducing the strawberry gray mold incidence and severity, caused by P. digitatum and B. cinerea, respectively, after artificial inoculation. AbA and/or its analogs, therefore, hold promise as relatively safe and promising fungicide candidates to control postharvest decays of fruits, because AbA targets the inositol phosphorylceramide (IPC) synthase, an enzyme essential for fungi but absent from mammals.     

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.     

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.     

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.     

The effect of beta-sitosterol on spore germination and germ-tube elongation of Aspergillus niger and Botryodiplodia theobromae

The effect of beta-sitosterol on spore germination and prevention of elongation of germ-tube of Aspergillus niger and Botryodiplodia theobromae was studied. The antifungal activity of the compound showed a percentage inhibition, of the fungal spore germination, of about 40% at a concentration of 50 micrograms/ml while inhibition on the elongation of germ-tubes was as high as 65% at the same concentration. The ED50 for inhibition of germ-tube elongation in A. niger was about 31 micrograms/ml. The role of the compound in disease resistance is discussed.     

复配精油对粮油中霉菌防治的增效作用及机理研究

开发新型天然防霉剂控制粮食霉变是保障粮食质量安全的重要途径之一。为研究复配植物精油对粮油储藏过程中常见霉菌赭曲霉(Aspergillus ochrator)、黄曲霉(Aspergillus flavus)和黑曲霉(Aspergillus niger)的防霉效果,挑选活性较强的植物精油进行复配并对联合防霉效果进行评价。通过复配植物精油对霉菌孢子萌发、菌丝干重和细胞完整性的影响,对其防霉机理进行初步探究。结果显示,牛至精油对赭曲霉和黑曲霉的防霉效果最好,抑菌圈直径分别为（27.83±0.58 ）、（15.33±0.29）mm,肉桂醛对黄曲霉的防霉效果最好,抑菌圈直径为（18.50±0.87 ）mm;山苍子精油与牛至精油、肉桂醛与牛至精油复配体积比为2：8时对3种霉菌的防霉效果较优;通过对部分抑菌浓度指数判读,两组植物精油复配对黑曲霉和赭曲霉的防治效果为协同作用,对黄曲霉的防治效果为相加作用;山苍子精油与牛至精油按体积比2：8复配可抑制霉菌孢子萌发和菌丝生长、破坏细胞的完整性、改变孢子和菌丝结构;山苍子精油和牛至精油按体积比2：8复配施用于含黄曲霉的玉米上,可有效降低玉米中黄曲霉毒素B1和赭曲霉毒素的含量。本研究为山苍子精油与牛至精油复配作为防霉剂提供理论支持。     

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.     

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.     

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.