A note on the screening of dried shrimps, shrimp paste and raw groundnut kernels for aflatoxin-producing Aspergillus flavus

All of 33 samples of dried shrimps, shrimp paste, peanut butter and raw groundnut kernels were contaminated with fungi. Aspergillus and Penicillium spp. were the predominant types in dried shrimps and raw groundnut kernels but no Aspergillus spp. were present in peanut butter or shrimp paste samples. Among 81 Aspergillus isolates obtained from dried shrimps and raw groundnut kernels, 10 were A. flavus/A. parasiticus, of which five were potential aflatoxin-producing A. flavus strains. No aflatoxins were detected in the food samples although some were visibly mouldy and some had high mould counts. The occurrence of aflatoxin-producing strains of A. flavus in dried shrimps and raw groundnut kernels warrants further investigation of these foods and their products as potentially significant sources of aflatoxins.     

Genetic transformation system for the aflatoxin-producing fungus Aspergillus flavus

A heterologous transformation system was developed for Aspergillus flavus with efficiencies greater than 20 stable transformants per micrograms of DNA. Protoplasts of uracil-requiring strains of the fungus were transformed with plasmid and cosmid vectors containing the pyr-4 gene of Neurospora crassa. Transformants were selected for their ability to grow and sporulate on medium lacking uracil. Vector DNA appeared to integrate randomly into the genome of A. flavus with a tendency for multiple, tandem insertion. Transformants with single or multiple insertions were stable after five consecutive transfers on medium containing uracil. Uracil-requiring recipient strains were obtained either by UV-irradiating conidia and selecting colonies resistant to 5-fluoroorotic acid or by transferring the mutated pyr locus to strains by parasexual recombination. This is the first report of a transformation system for an aflatoxin-producing fungus. The transformation system and the availability of aflatoxin-negative mutants provide a new approach to studying the biosynthesis and regulation of aflatoxin.     

Twenty-four microsatellite markers for the aflatoxin-producing fungus Aspergillus flavus

Aspergillus flavus infects both plants and humans and contaminates diverse agricultural crops with aflatoxins, highly carcinogenic fungal metabolites. We describe 24 microsatellite markers developed to assess genetic diversity and recombination within and between three vegetative compatibility groups (VCGs) of Aspergillus flavus. These loci are polymorphic within at least one VCG or between VCGs. For loci polymorphic across all three VCGs, the number of alleles ranged from two to 19. These markers will be useful for genetic studies of this economically important pathogen.     

Aspergillus flavus and other mycoflora of groundnut kernels in Israel and the absence of aflatoxin

More than 300 groundnut (peanut) samples collected from different regions of Israel were examined by ELISA for aflatoxin contamination. Samples were designated for export, local consumption or for sowing. None of the samples were contaminated with the toxin. However, when kernels were kept at high humidity (RH?99%), aflatoxin could be frequently detected seven days after incubation and the toxin was not uniformly distributed among kernels.Aspergillus niger, A flavus, Penicillium citrinum andP pinophilum were the dominant fungi and no differences were observed among cultivars. Almost half of the commercial samples examined were devoid ofA flavus. Other fungi identified wereA tamaril, A amstelodami, P rubrum, Rhizoctonia solani, Macrophomina phaseolina, Rhizopus spp., Sclerotium rolfsll, Fusarium andAlternaria spp; the two last ones comprising a group of low incidence. Although groundnut samples that containA flavus—infected kernels are moderately common, the local climate and agrotechniques In use in Israel are not conducive to aflatoxin accumulation. Nevertheless infected kernels may become a threat to health if stored under inadequate conditions.     

Genetic transformation system for the aflatoxin-producing fungus Aspergillus flavus.

A heterologous transformation system was developed for Aspergillus flavus with efficiencies greater than 20 stable transformants per micrograms of DNA. Protoplasts of uracil-requiring strains of the fungus were transformed with plasmid and cosmid vectors containing the pyr-4 gene of Neurospora crassa. Transformants were selected for their ability to grow and sporulate on medium lacking uracil. Vector DNA appeared to integrate randomly into the genome of A. flavus with a tendency for multiple, tandem insertion. Transformants with single or multiple insertions were stable after five consecutive transfers on medium containing uracil. Uracil-requiring recipient strains were obtained either by UV-irradiating conidia and selecting colonies resistant to 5-fluoroorotic acid or by transferring the mutated pyr locus to strains by parasexual recombination. This is the first report of a transformation system for an aflatoxin-producing fungus. The transformation system and the availability of aflatoxin-negative mutants provide a new approach to studying the biosynthesis and regulation of aflatoxin.     

Aspergillus nomius,a new aflatoxin-producing species related to Aspergillus flavus and Aspergillus tamarii

Aspergillus nomius is described and represents a new aflatoxigenic species phenotypically similar to A. flavus. Strains examined were isolated from insects and agricultural commodities. Separation from A. flavus is based on the presence of indeterminate sclerotia and a lower growth temperature. Comparisons of DNA relatedness show A. nomius to have only relatively recently evolved from A. flavus and A. tamarii.     

Differentiation of aflatoxin-producing and non-producing strains of Aspergillus flavus group

AIMS: Three conventional methods and a multiplex PCR procedure with a set of four primers (Quadruplex-PCR) were used to differentiate between aflatoxin-producing and non-producing strains of the Aspergillus flavus group. METHODS AND RESULTS: By combining sets of primers for aflR, nor-1, ver-1 and omt-A genes of the aflatoxin biosynthetic pathway, Quadruplex-PCR showed that aflatoxinogenic strains gave a quadruplet pattern, indicating the presence of all the genes involved in the aflatoxin biosynthetic pathway which encode for functional products. Non-aflatoxinogenic strains gave varying results with one, two, three or four banding patterns. A banding pattern in three non-aflatoxinogenic strains resulted in non-differentiation between these and aflatoxinogenic strains. CONCLUSION AND SIGNIFICANCE AND IMPACT OF THE STUDY: Because conventional methods are time-consuming, further studies are needed to develop a rapid and objective technique that permits complete differentiation between aflatoxin-producing and non-producing strains of the A. flavus group.     

Relationships betweenAspergillus flavus,A. niger and some other fungi in the mycoflora of groundnut kernels

Summary The relationships betweenAspergillus flavus, A. niger, Penicillium funiculosum, P. rubrum, andFusarium solani have been studied in 234 samples and 5850 plates from fresh and stored kernels derived from 2 years' groundnut crops in Israel.Numerical relations between these fungi, as obtained by totalling the number of colonies developing in all the 25 platings made for each sample, did not always give reliable indications of potential antagonism between the species. But consideration of the number of colonies developing in individual plates showed pronounced antagonism betweenA. flavus andA. niger, and slightly less but still marked antagonism between each of these species andP. funiculosum, P. rubrum andF. solani.This research is supported by Grant Number FG-Is-161 of the United States Department of Agriculture to whom the author is indebted.     

Screening for resistance to Aspergillus flavus and aflatoxin production in groundnut

All the varieties, advanced breeding lines, germplasm lines, and wild species used in the experiments differed significantly for their ability to allow invasion and aflatoxin production by an aflatoxigenicAspergillus flavus strain. Infection and colonisation were strongly correlated (r = 0.82), while there was no relation between infection and aflatoxin content or colonisation and aflatoxin content (r = 0.15). The varieties ICGS11 and S 206 supported less infection and colonisation (range 35 to 40%). Lowest aflatoxin content was recorded in Chitra (3,200 ppb), while it was highest in Kaushal (38,250 ppb). A cross derivative of GAUG1 × NC Ac 17133 R F showed lowest infection and colonisation (86,3 and 25,28%, respectively), and also supported moderate aflatoxin production (4,000 ppb). Among germplasm lines spancross supported lowest aflatoxin production (2,026 ppb) while both the wild species vz. ICG 8127 and ICG 8128 were highly susceptible to infection, colonisation, and aflatoxin production.     

The presence of aflatoxin in kernels from five years groundnut crops and ofAspergillus flavus isolates from kernels and soils

Summary In T.L.C. tests for 605 samples of groundnut kernels from 5 years' yield, the percentage of fresh kernels in which aflatoxin was present was very low (up to 6.4%), while that of stored kernels ranged from 0 to 32.0%. But the intensity of toxicity was invariably very low (up to 125 ppb). Of 1626Aspergillus flavus isolates from groundnut kernels rhizosphere and geocarposphere, and from soil in which groundnuts grew, about 90% were found capable of forming aflatoxin. In quantitative tests with 750 isolates 60% of the isolates produce aflatoxin in excess of 25,000 ppb. This research is supported by Grant Number FG-Is-161 of the United States Department of Agriculture to whom the author is indebted.     

Influence of microbial co-inhabitants on aflatoxin synthesis of Aspergillus flavus on maize kernels

The co-inhabiting mycoflora with Aspergillus flavus observed on individual maize kernels was evaluated for its influence on aflatoxin synthesis. All 13 types of associations of different fungal species inhibited aflatoxin B₁ and G₁ production at different levels (34·3–100%). Inhibition of radial growth of A. flavus by Fusarium moniliforme (59·8%), Trichoderma viride (72·5%) and Rhizopus nigricans (42%) could be directly correlated to the per cent inhibition of aflatoxin production. High levels of inhibition of aflatoxin elaboration were noted in competition of A. flavus with other toxigenic moulds.     

Evaluation of bacteria and Trichoderma for biocontrol of pre-harvest seed infection by Aspergillus flavus in groundnut

Pre- and post harvest aflatoxin contamination of groundnut caused by Aspergillus flavus is a major problem in the semi-arid tropics. Fluorescent Pseudomonas, Bacillus and Trichoderma spp. potentially antagonistic to A. flavus were isolated from the geocarposphere (pod-zone) of groundnut and used successfully for the control of pre-harvest groundnut seed infection by A. flavus. In greenhouse and field experiments, inoculation of selected antagonistic strains on groundnut resulted in significant reduction of seed infection by A. flavus, and it also reduced >50% of the A. flavus populations (as cfu) in the geocarposphere of groundnut.     

抑黄曲霉乳酸菌的筛选及菌种鉴定

目的筛选具有抑制黄曲霉(Aspergillus flavus)生长的乳酸菌。方法以各地泡菜、实验室自制泡菜、豆浆渣以及新鲜猪肠、鸡肠道内容物为材料,采用牛津杯法筛选所需菌株。对筛选出的菌株进行生理生化及16S rRNA基因序列同源性分析。结果分离得到756株乳酸菌,其中有6株菌株对黄曲霉的生长有明显的抑制作用。结论实验获得的6株产酸菌,3株为植物乳杆菌(Lactobacillus plantarum),2株为消化乳杆菌(L.alimentari-us),1株为亚利桑那乳杆菌(L.arizonenensis)。     

A simple test tube screening method for identifying aflatoxin-producing strains of Aspergillus sp. using coconut milk agar medium

Most agricultural commodities are susceptible to Aspergillus sp. infestation and aflatoxin elaboration. A simple test-tube screening procedure using fresh coconut milk agar medium (CMAM), for identifying toxigenic strains of Aspergillus sp., based on u.v. fluorescence (365 nm) and visual detection has been proposed and evaluated.     

Restriction enzyme analysis of mitochondrial DNA of the Aspergillus flavus group: A. flavus, A. parasiticus, and A. nomius

Mitochondrial DNA restriction fragment length polymorphisms were identified that clearly distinguish Aspergillus flavus, A. parasiticus, and A. nomius. Mitochondrial DNAs of A. flavus and A. parasiticus were found to be circular, and their size was estimated size to be 32 kilobases. A restriction map was constructed for the mitochondrial genome of an A. parasiticus isolate by using four restriction endonucleases. Four genes tested were found to have the same order as in the mitochondrial genome of A. nidulans. The mitochondrial genome of A. nomius was estimated to be 33 kilobases.     

An improved medium for the detection of Aspergillus flavus and A. parasiticus

An effective selective medium for the enumeration of Aspergillus flavus and Aspergillus parasiticus has been developed by modification of Bothast and Fennell's Aspergillus Differential Medium. Results can be obtained with the new medium, Aspergillus flavus and parasiticus Agar (AFPA), after 42 h incubation at 30 degrees C. The medium is thus suitable for use in quality control as a guide to the presence of A. flavus and, potentially, of aflatoxins. AFPA has been extensively tested on peanuts and soils. Results were reproducible and comparable with those on standard fungal enumeration media incubated for much longer periods. A very low percentage of false positive or negatives was found.     

An improved medium for the detection of Aspergillus flavus and A. parasiticus

An effective selective medium for the enumeration of Aspergillus flavus and Aspergillus parasiticus has been developed by modification of Bothast and Fennell's Aspergillus Differential Medium. Results can be obtained with the new medium, Aspergillus flavus and parasiticus Agar (AFPA), after 42 h incubation at 30°C. The medium is thus suitable for use in quality control as a guide to the presence of A. flavus and, potentially, of aflatoxins. AFPA has been extensively tested on peanuts and soils. Results were reproducible and comparable with those on standard fungal enumeration media incubated for much longer periods. A very low percentage of false positives or negatives was found.     

黄曲霉和米曲霉的多相鉴定方法

【背景】黄曲霉(Aspergillus flavus)和米曲霉(Aspergillus oryzae)形态特征相近,基因组高度相似,较难区分。【目的】旨在总结一套准确鉴别二者的分类方法。【方法】利用22株标准菌株对传统形态学、产毒培养基、酶联免疫毒素检测、系统发育分析、产毒基因检测等5种鉴别方法分别进行验证。【结果】各鉴定方法的结果存在异同,单一的鉴定方法容易出现假阴性或假阳性结果。【结论】利用单一方法区分黄曲霉和米曲霉具有潜在风险,多相鉴定方法可以准确鉴别二者。