Flavus Cultures Research Articles

Determination of fungicidal effect of disinfectant ‘SanDezVet’ on sanitary significant test cultures of mold micromycetes of the genus Aspergillus Mich.

Studying the properties of new disinfectants is a crucial aspect of disinfectology research. During the investigation of the fungicidal properties of the ‘SanDezVet’ disinfectant, it was discovered that the recommended concentrations of 0.1% and 0.5%, at a temperature of 20.0 ± 0.5 °C and an exposure time of 60, 120, and 180 minutes, do not inhibit the growth of Aspergillus fumigatus, Aspergillus flavus, and Aspergillus niger test cultures. This conclusion was reached as continuous growth of micromycetes was observed in all drug dilutions. The disinfectant ‘SanDezVet’ at a concentration of 3.0% displayed fungistatic properties by significantly delaying the growth of experimental test cultures. A 5.0% solution of the disinfectant resulted in a complete delay in growth of museum strains of Aspergillus fumigatus, Aspergillus flavus, and Aspergillus niger. This indicates fungicidal properties when compared to the positive control. Based on the obtained results, it was observed that the optimal exposure times for disinfection measures with ‘SanDezVet’ are 60 and 120 minutes. ‘SanDezVet’ can be effectively used for fungal infections at different veterinary facilities

High voltage atmospheric cold plasma treatment inactivates Aspergillus flavus spores and deoxynivalenol toxin

Fungal contamination is a concern for the food industry. Fungal spores resist food sterilization treatments and produce mycotoxins that are toxic for animals and humans. Technologies that deactivate spores and toxins without impacting food quality are desirable. This study demonstrates the efficiency of a high voltage atmospheric cold plasma (HVACP) technology using air to generate reactive oxygen (ROS) and nitrogen (RNS) species for the degradation of Aspergillus flavus cultures and the deoxynivalenol (DON) mycotoxin. Optical emission and absorption spectroscopy demonstrate ionization of hydroxyl groups, atomic oxygen and nitrogen, and confirm production of ROS and RNS, e.g. O3, NO2, NO3, N2O4, and N2O5. Fungal cultures show a depletion in pigmentation and an ~50% spore inactivation after 1-min treatments. Treated spores show surface ablation and membrane degradation by scanning electron microscopy. Twenty-minute direct HVACP treatments of 100 μg of DON in one mL aqueous suspensions resulted in a greater than 99% reduction in DON structure and rescued over 80% of Caco-2 cell viability; however, the same treatment on 100 μg of powdered DON toxin only showed a 33% reduction in DON and only rescued 15% of cell viability. In summary, HVACP air treatment can inactivate both fungal spores and toxins in minutes.

Biofilm Mode of Cultivation Leads to an Improvement of the Entomotoxic Patterns of Two Aspergillus Species.

Two fungi, i.e., Aspergillus flavus Link and Aspergillus oryzae (Ahlb.) E. Cohn, were cultivated according to two methodologies, namely submerged and biofilm cultures with the primary aim to use their secondary metabolites the supernatant CL50, and CL90 varied between 1.3% (v/v) to 12.7% (v/v) for incubation times from 24 to 72 h. While the A. flavus supernatant entomotoxicity was higher than this of A. oryzae, the biofilm culture application increased the efficiency of the former. Proteomic analysis of the supernatants revealed discrepancies among the two species and modes of cultivation. Furthermore, the secondary metabolite profiles of both Aspergillus cultures were verified. Aspergillic acid, beta-cyclopiazonic acid, cyclopiazonic acid, ferrineospergillin, flavacol, and spermadin A were most predominant. Generally, these secondary metabolites were present in higher concentrations in the supernatants of A. flavus and biofilm cultures. These molecular identifications correlated positively with entomotoxic activity. Noteworthy, the absence of carcinogenic aflatoxins was remarkable, and it will allow further valorization to produce A. flavus to develop potential biopesticides.

Staphylococcus saprophyticus L-38 produces volatile 3,3-dimethyl-1,2-epoxybutane with strong inhibitory activity againstAspergillus flavus germination and aflatoxin production

Controlling proliferation and aflatoxin production byAspergillus flavus is a pressing challenge for global food safety and security. Marine bacteriumStaphylococcus saprophyticus strain L-38 showed excellent antifungal activity towardA. flavus in vitro andin vivo. In sealed, non-contact confrontation assays, L-38 completely inhibited conidial germination and mycelial growth ofA. flavus through the production of volatile organic compounds (VOCs). Gas chromatography-mass spectrometry identified 3,3-dimethyl-1,2-epoxybutane (3-DE) as the most abundant VOC (32.61% of total peak area, 78% matching). Exposure ofA. flavus cultures to synthetic 3-DE similarly demonstrated strong inhibition of growth. Moreover, culture of L-38 in a sealed chamber with maize or peanuts artificially inoculated withA. flavus, at high water activity, resulted in significant inhibition ofA. flavus germination and aflatoxin biosynthesis. Scanning electron microscopy of these samples revealed severe damage to conidial cells and hyphae compared to samples not exposed to L-38. L-38 also showed broad and effective antifungal activity toward eight other phytopathogenic fungi includingAspergillus niger, Fusarium verticillioides, Fusarium graminearum, Sclerotinia sclerotiorum, Rhizoctonia solani, Alternaria alternata, Monilinia fructicola, andBotrytis cinerea. This work introducesS. saprophyticus L-38 as a potential biocontrol agent and demonstrates the efficacy of the volatile 3-DE in the control ofA. flavus and other destructive plant pathogens for post-harvest food safety.

Aspergillus flavus Secondary Metabolites: More than Just Aflatoxins.

Aspergillus flavus is best known for producing the family of potent carcinogenic secondary metabolites known as aflatoxins. However, this opportunistic plant and animal pathogen also produces numerous other secondary metabolites, many of which have also been shown to be toxic. While about forty of these secondary metabolites have been identified from A. flavus cultures, analysis of the genome has predicted the existence of at least 56 secondary metabolite gene clusters. Many of these gene clusters are not expressed during growth of the fungus on standard laboratory media. This presents researchers with a major challenge of devising novel strategies to manipulate the fungus and its genome so as to activate secondary metabolite gene expression and allow identification of associated cluster metabolites. In this review, we discuss the genetic, biochemical and bioinformatic methods that are being used to identify previously uncharacterized secondary metabolite gene clusters and their associated metabolites. It is important to identify as many of these compounds as possible to determine their bioactivity with respect to fungal development, survival, virulence and especially with respect to any potential synergistic toxic effects with aflatoxin.

Unravelling the Diversity of the Cyclopiazonic Acid Family of Mycotoxins in Aspergillus flavus by UHPLC Triple-TOF HRMS.

Cyclopiazonic acid (α-cyclopiazonic acid, α-CPA) is an indole-hydrindane-tetramic acid neurotoxin produced by various fungal species, including the notorious food and feed contaminant Aspergillus flavus. Despite its discovery in A. flavus cultures approximately 40 years ago, its contribution to the A. flavus mycotoxin burden is consistently minimized by our focus on the more potent carcinogenic aflatoxins also produced by this fungus. Here, we report the screening and identification of several CPA-type alkaloids not previously found in A. flavus cultures. Our identifications of these CPA-type alkaloids are based on a dereplication strategy involving accurate mass high resolution mass spectrometry data and a careful study of the α-CPA fragmentation pattern. In total, 22 CPA-type alkaloids were identified in extracts from the A. flavus strains examined. Of these metabolites, 13 have been previously reported in other fungi, though this is the first report of their existence in A. flavus. Two of our metabolite discoveries, 11,12-dehydro α-CPA and 3-hydroxy-2-oxo CPA, have never been reported for any organism. The conspicuous presence of CPA and its numerous derivatives in A. flavus cultures raises concerns about the long-term and cumulative toxicological effects of these fungal secondary metabolites and their contributions to the entire A. flavus mycotoxin problem.

Fungal Aflatoxins Reduce Respiratory Mucosal Ciliary Function.

Aflatoxins are mycotoxins secreted by Aspergillus flavus, which can colonize the respiratory tract and cause fungal rhinosinusitis or bronchopulmonary aspergillosis. A. flavus is the second leading cause of invasive aspergillosis worldwide. Because many respiratory pathogens secrete toxins to impair mucociliary immunity, we examined the effects of acute exposure to aflatoxins on airway cell physiology. Using air-liquid interface cultures of primary human sinonasal and bronchial cells, we imaged ciliary beat frequency (CBF), intracellular calcium, and nitric oxide (NO). Exposure to aflatoxins (0.1 to 10 μM; 5 to 10 minutes) reduced baseline (~6–12%) and agonist-stimulated CBF. Conditioned media (CM) from A. fumigatus, A. niger, and A. flavus cultures also reduced CBF by ~10% after 60 min exposure, but effects were blocked by an anti-aflatoxin antibody only with A. flavus CM. CBF reduction required protein kinase C but was not associated with changes in calcium or NO. However, AFB2 reduced NO production by ~50% during stimulation of the ciliary-localized T2R38 receptor. Using a fluorescent reporter construct expressed in A549 cells, we directly observed activation of PKC activity by AFB2. Aflatoxins secreted by respiratory A. flavus may impair motile and chemosensory functions of airway cilia, contributing to pathogenesis of fungal airway diseases.

Detection of seedborne Aspergillus flavus from rice cultivars using molecular markers

Aflatoxins are carcinogenic metabolites produced by Aspergillus flavus. Rice seeds may be contaminated by it at the time of harvesting or during storage. Detection of aflatoxigenic strains by TLC and analysis of genetic variability among 50 A. flavus cultures from seeds of various rice genotypes by PCR using 18 RAPD primers is reported here. About 58% isolates were aflatoxigenic whereas 42% were non-toxic. There were 246 bands and 48 haplotypes. Approximately 99% bands were polymorphic, and thus high degree of genetic variability was observed. All the primers were informative with PIC values of range 0.75–0.95. Seventeen RAPD markers were not found to be useful for the detection of aflatoxigenic A. flavus. However, one RAPD primer 3B could differentiate aflatoxigenic and non-toxigenic isolates to some extent. One allele of Primer 3B at approximately 480 bp was present in 85% aflatoxin non-producers and in 6% aflatoxigenic isolates. This information may be utilised for designing markers for differentiating toxic/non-toxic isolates of A. flavus.

English

The objective of the present work was to evaluate the capacity of two isolates ofAspergillus flavus from sugar cane (SC), SC-3 and SC-5 to produce aflatoxin under different culture conditions. Aflatoxin B1 (AFB1) production was determined in four different media and culture conditions (temperature and pH) were varied to achieve maximal toxin production. AFB1 was extracted directly from the cultures with chloroform. Thin layer chromatography (TLC) and high performance liquid chromatography (HPLC) were used to identify AFB1. It was observed that A. flavus cultures SC-3 and SC-5 produced highest AFB1 when grown in potato dextrose medium (PD) and maximum mycelium production in sabauroud dextrose medium (SD). Supplementation of potato dextrose medium with peptone (PD+P) and sodium nitrate (PD+SN), AFB1 production increased by 27 and 20% respectively when compared to the control. The effect of pH and temperature on aflatoxin production by A. flavus was examined in peptone containing PD medium. The results concluded that AFB1 production and mycelial growth was maximum at pH 5.6 when incubated at 27°C in potato dextrose medium supplemented with peptone. Key words: Aflatoxin B1, Aspergillus flavus, mycotoxins

Validation and application of HPLC–ESI-MS/MS method for the quantification of RBBR decolorization, a model for highly toxic molecules, using several fungi strains

A novel analytical method using HPLC–MS/MS operating in selected reaction monitoring (SRM) for evaluation of fungi efficacy to decolorize Remazol Brilliant Blue R (RBBR) dye solution was developed, validated and applied. The method shows high sensibility allowing the detection of 4.6pM of RBBR. Four fungal strains were tested in liquid medium, three strains of Aspergillus (Aspergillus aculeatus, Aspergillus flavus and Aspergillus fumigatus) and Phanerochaete chrysosporium. All fungi were able to degrade the dye, with efficiencies ranging from 40% for P. chrysosporium up to 99% for A. flavus during a 30-day incubation period. During the experiment, increased accumulation of degradation products was observed in A. flavus cultures containing RBBR. Through the use of full scan HPLC–MS technique it was possible to propose the biogenesis of the microbial metabolic degradation pathway. Screening using microorganisms and RBBR may be hereafter used to investigate microbial biodegradation of high toxicity molecules such as dioxins.

Present and future directions of translational research on aflatoxin and hepatocellular carcinoma. A review

The aflatoxins were discovered in toxic peanut meal causing “turkey X” disease, which killed large numbers of turkey poults, ducklings and chicks in the UK in the early 1960s. Extracts of toxic feed induced the symptoms in experimental animals, and purified metabolites with properties identical to aflatoxins B1 and G1 (AFB1 and AFG1) were isolated from Aspergillus flavus cultures. Structure elucidation of aflatoxin B1 was accomplished and confirmed by total synthesis in 1963. AFB1 is a potent liver carcinogen in rodents, non-human primates, fish and birds, operating through a genotoxic mechanism involving metabolic activation to an epoxide, formation of DNA adducts and, in humans, modification of the p53 gene. Aflatoxins are unique among environmental carcinogens, in that elucidation of their mechanisms of action combined with molecular epidemiology provides a foundation for quantitative risk assessment; extensive evidence confirms that contamination of the food supply by AFB1 puts an exposed population at increased risk of developing hepatocellular carcinoma (HCC). Molecular biomarkers to quantify aflatoxin exposure in individuals were essential to link aflatoxin exposure with liver cancer risk. Biomarkers were validated in populations with high HCC incidence in China and The Gambia, West Africa; urinary AFB1–N 7-Guanine excretion was linearly related to aflatoxin intake, and levels of aflatoxin–serum albumin adducts also reflected aflatoxin intake. Two major cohort studies employing aflatoxin biomarkers identified their causative role in HCC etiology. Results of a study in Shanghai men strongly support a causal relationship between HCC risk and the presence of biomarkers for aflatoxin and HBV infection, and also show that the two risk factors act synergistically. Subsequent cohort studies in Taiwan confirm these results. IARC classified aflatoxin as a Group 1 human carcinogen in 1993, based on sufficient evidence in humans and experimental animals indicating the carcinogenicity of naturally occurring mixtures of aflatoxins, aflatoxin B1, G1 and M1. Aflatoxin biomarkers have also been used to show that primary prevention to reduce aflatoxin exposure can be achieved by low-technology approaches at the subsistence farm level in sub-Saharan Africa. Also, in residents of Qidong, China, oral dosing with chlorophyllin, a chlorophyll derivative, prior to each meal led to significant reduction in aflatoxin–DNA biomarker excretion, supporting the feasibility of preventive measures to reduce HCC risk in populations experiencing unavoidable aflatoxin exposure. The systematic, comprehensive approach used to create the total aflatoxin database justifies optimism for potential success of preventive interventions to ameliorate cancer risk attributable to aflatoxin exposure. This strategy could serve as a template for the development, validation and application of molecular and biochemical markers for other carcinogens and cancers as well as other chronic diseases resulting from environmental exposures.

Absence of the aflatoxin biosynthesis gene, norA, allows accumulation of deoxyaflatoxin B1 in Aspergillus flavus cultures

Biosynthesis of the highly toxic and carcinogenic aflatoxins in select Aspergillus species from the common intermediate O-methylsterigmatocystin has been postulated to require only the cytochrome P450 monooxygenase, OrdA (AflQ). We now provide evidence that the aryl alcohol dehydrogenase NorA (AflE) encoded by the aflatoxin biosynthetic gene cluster in Aspergillus flavus affects the accumulation of aflatoxins in the final steps of aflatoxin biosynthesis. Mutants with inactive norA produced reduced quantities of aflatoxin B(1) (AFB(1)), but elevated quantities of a new metabolite, deoxyAFB(1). To explain this result, we suggest that, in the absence of NorA, the AFB(1) reduction product, aflatoxicol, is produced and is readily dehydrated to deoxyAFB(1) in the acidic medium, enabling us to observe this otherwise minor toxin produced in wild-type A. flavus.

Serial passage of the opportunistic pathogen Aspergillus flavus through an insect host yields decreased saprobic capacity

To study the early stages of the effect of host restriction on pathogen evolution, we subjected the opportunistic fungus Aspergillus flavus to a serial propagation scheme, whereby insect-virulent conidia were selected for repeated passage through an insect host (Galleria mellonella larvae) for 5 generations. Of the 35 lineages promulgated through this scheme, there were no consistent changes in virulence, which was measured by percent mortality of the larvae. There were, however, increases in the number of conidia on the insect cadavers (9 of 35 lineages) and decreases in the number of days between death and the appearance of fungal growth on the cadavers (4 of 35 lineages). Notably, most of the lineages (28 of 35 lineages) demonstrated a statistically significant decrease in the diameter of the colonies subcultured onto artificial media, indicating a decreased ability to grow saprobically. Conversely, most of the A. flavus cultures successively grown on agar media (9 of 10 lineages) exhibited no change in colony diameter after 15 rounds of subculturing. Propagation of the opportunist A. flavus through the insect host G. mellonella resulted in a diminished capacity to grow on an alternate substrate, while maintaining or increasing its ability to use the host as a nutrient supply.

Biflavonoids inhibit the production of aflatoxin by Aspergillus flavus.

The biflavonoids 6,6"-bigenkwanin, amenthoflavone, 7,7"-dimethoxyagastisflavone and tetradimethoxybigenkwanin isolated from Ouratea species were tested for inhibitory activity on Aspergillus flavus cultures. Suspensions of Aspergillus flavus spores were inoculated into 50 ml of YES medium at different biflavonoid concentrations: 5 and 10 microg/ml for 6,6"-bigenkwanin, amenthoflavone and 7,7"-dimethoxyagastisflavone, and 5, 10, 15 and 20 microg/ml for tetradimethoxybigenkwanin. The four biflavonoids showed inhibitory activity on aflatoxin B1 and B2 production (P<0.001), but did not inhibit fungal growth at the concentration tested (P>0.05). These results show that biflavonoids can be used for the development of agents to control aflatoxin production.

Raffinose content may influence cottonseed susceptibility to aflatoxin contamination

AbstractThe effect of the cotton storage trisaccharide raffinose and cottonseed storage protein (CSP) in combination on aflatoxin production by Aspergillus flavus was investigated. The ability of ground whole cottonseed and water‐extracted cottonseed meal to support fungal biosynthesis of aflatoxin also was assessed in vitro. Dose response data showed that utilization of raffinose as a sole carbon source supported growth and aflatoxin production by A. flavus. When raffinose was a carbon source and CSP was the sole nitrogen source, aflatoxin levels were stimulated up to fourfold above those in raffinose reference media. Results with ground whole cottonseed as a sole carbon/nitrogen source demonstrated the capacity for aflatoxin production in A. flavus cultures. Lipid extraction of ground whole seed severely reduced aflatoxigenesis potential; however, lipid extraction followed by water extraction of ground whole seed restored much of the aflatoxin biosynthetic potential, suggesting the presence of a water soluble inhibitory factor. Accessible carbon appeared to be the limiting resource in water‐extracted meal, as a raffinose supplement stimulated aflatoxin production. Either cottonseed storage lipid or raffinose was capable of providing accessible carbon to A. flavus. Raffinose and CSP in developing and mature cottonseed may predispose seed to potentially high levels of aflatoxin contamination by A. flavus upon seed infection.

Selective expression of a major allergen and cytotoxin, Asp f I, in Aspergillus fumigatus. Implications for the immunopathogenesis of Aspergillus-related diseases.

Asp f I is a major 18-kDa Aspergillus fumigatus allergen and a member of the mitogillin family of cytotoxins. The nucleotide sequence of the Asp f I gene was determined by sequencing polymerase chain reaction products amplified from A. fumigatus spore DNA. The entire 678-bp DNA includes an 81-bp leader sequence, preceding the N-terminal alanine codon, a 52-bp intron, and a 444-bp open reading frame, encoding a 149-amino acid protein (M(r) 16,899), which is 99% homologous to mitogillin from Aspergillus restrictus. A mAb-based ELISA was used to compare Asp f I levels in spores, mycelia, and culture filtrate, and to determine the kinetics of allergen production. Disrupted hyphae or spore extracts had a 1000-fold lower level of Asp f I than culture filtrate, suggesting that germination of spores and growth of the fungus are essential for allergen production. Asp f I levels in A. fumigatus and A. restrictus peaked at day 3 (0.87 to 12.1 micrograms/ml), however, the allergen was not detected in Aspergillus flavus, Aspergillus niger, Aspergillus terreus, and Aspergillus nidulans cultures (< 1.5 ng/ml) on either days 3 or 8. Northern analysis confirmed that Asp f I mRNA was detected only in A. fumigatus and A. restrictus, but not in the other four Aspergillus spp. Asp f I-specific DNA was generated after polymerase chain reaction amplification of genomic mycelial DNA obtained from A. fumigatus and A. restrictus, but not from the other Aspergillus spp. The results show that Asp f I is selectively expressed in A. fumigatus, and suggest that this cytotoxin could be a specific virulence factor for A. fumigatus.

Changes in cotton leaf chemistry induced by volatile elicitors

Cotton ( Gossypium hirsutum) leaves were exposed for 7 days to volatile chemicals originating from Aspergillus flavus-infected cotton leaves, A. flavus cultures or mechanically damaged cotton leaves. Volatiles from A. flavus-infected leaves triggered significant increases of 52 and 34% in phloroglucinol-reactive compounds in wounded or undamaged cotton leaves, respectively. Increased production of heliocides (C 25 terpenoid aldehydes) were found in the volatile recepient wounded or undamaged cotton leaves. The heliocides are natural insecticides presumed localized in the subepidermal pigment glands in leaves. Myrcene, a volatile precursor of heliocide H 2, also caused significant increases in heliocide production when leaves were exposed to the volatilized chemical.

Mineralisation dans le sol de materiaux microbiens marques au carbone 14 et a l'azote 15: Quantification de l'azote de la biomasse microbienne

The mineralization of microbial material of different C-to-N ratios (5.2, 7.9, 10.2, 12.7) was followed in fumigated soil. The microbial materials used were from Aspergillus flavus cultures, grown in liquid media and labelled with [ 14C]glucose and ( 15NN4)3804. Three contrasting soils were used and the microbial materials incubated with the fumigated soils for 28 days at 28°C. The evolution of the added organic microbial C was fast: 80% of the [ 14C]CO 2 produced during the whole 28 days incubation was evolved in the first week. Microbial C mineralization was mainly related to soil type; the C-to-N ratio had small effect on the ratio (mineralized microbial carbon-to-added microbial carbon). Calculation of the K c - coefficient (the fraction of the added microbial C mineralized in 7 days) shows that K c values lie between 0.38 and 0.43 in the 3 soils. Organic N in the added microbial material also breaks down quickly: between 60 and 100% of the organic nitrogen mineralized was evolved during the first week of incubation. Mineralization kinetics are related to soil type and to the C-to-N ratio of the microbial material. The proportion of N mineralized in 7 days was lower in an acid soil than in near neutral soils and lower with high C-to-N ratio material than with low C-to-N ratio material. The ratio (mineralized microbial N-to-added microbial N) depends on soil type and is negatively correlated with the C-to-N ratio of the microbial material. The K N value (the fraction of the added microbial N mineralized in 7 days) lies between 0.22 and 0.47 for the three soils and four materials investigated. The added microbial material induced a priming effect on soil native N: materials with C-to-N ratios of 10.2 and 12.7 produced negative priming effects whereas materials with C-to-N ratios of 5.2 and 7.9 sometimes produced a positive priming action. From the relationship between the C-to-N ratio of the added material and the (mineralized microbial C-to-mineralized microbial N) ratio, the soil native microbial biomass was estimated using the fiush-C-to-flush-N ratio. Biomass nitrogen was then calculated from the formula biomass-N = biomassC/(biomass C-to-N ratio). Calculated in this way, 2–4% of the total nitrogen in the three soils was in microbial biomass.

Polychlorinated biphenyls as unjustified or gratuitous inducers in Aspergillus flavus cultures.

From the toxicological premises discussed the main aspect appears to be the contrasting signs of metabolic activation (high RNA levels, increased oxygen consumption) that the microorganism develops in the presence of PCBs, and the fact that such signs are not accompanied by a higher rate of biomass synthesis but quite the opposite.

Differential Production of Aflatoxin on Natural and Heat-Treated Cocoa Beans

Costa Rican-type cocoa beans were tested as a substrate for two known aflatoxigenic fungal species, Aspergillus flavus NRRL 3251 and Aspergillus parasiticus NRRL 2999. Natural floral growth in both cooked (autoclaved) and raw cultures failed to show aspergilli and aflatoxin as analyzed by thin layer chromatography. Raw, ground cocoa bean medium inoculated with both species of aspergilli had mycelial growth, sporulation, but no detectable levels of aflatoxin. Similarly treated but cooked (autoclaved) cocoa medium also provided for mycelial growth, and sporulation, but total aflatoxin levels were 388.1 μg/g of substrate for the A. parasiticus cultures and 2.2 μg/g of substrate for the A. flavus cultures. The general results appear to support the low level of a limited number of reports of toxin contamination in natural cocoa. The simplified extraction methods used herein provided results similar to other methods.