Biological Control Of Aflatoxin Contamination Research Articles

Application of Non-Aflatoxigenic Aspergillus flavus for the Biological Control of Aflatoxin Contamination in China.

Biological control through the application of competitive non-aflatoxigenic Aspergillus flavus (A. flavus) to the soil during peanut growth is a practical method for controlling aflatoxin contamination. However, appropriate materials need to be found to reduce the cost of biocontrol products. In this study, a two-year experiment was conducted under field conditions in China, using a native non-aflatoxigenic strain to explore its effect. After three months of storage under high humidity, aflatoxin levels remained low in peanuts from fields treated with the biocontrol agent. Three types of substrates were tested with the biocontrol agent: rice grains, peanut meal (peanut meal fertilizer) and peanut coating. Compared to untreated fields, these formulations resulted in reductions of 78.23%, 67.54% and 38.48%, respectively. Furthermore, the ratios of non-aflatoxigenic A. flavus recovered in the soils at harvest in the treated fields were between 41.11% and 96.67% higher than that in untreated fields (25.00%), indicating that the rice inoculum was the most effective, followed by the peanut meal fertilizer and peanut coating. In 2019, the mean aflatoxin content of freshly harvested peanuts in untreated fields was 19.35 µg/kg higher than that in the fields treated with 7.5 kg/ha rice inoculum, which was 1.37 µg/kg. Moreover, no aflatoxin was detected in the two other plots treated with 10 and 15 kg/ha rice inoculum. This study showed that the native Chinese non-aflatoxigenic strain of A. flavus (18PAsp-zy1) had the potential to reduce aflatoxin contamination in peanuts. In addition, peanut meal can be used as an alternative substrate to replace traditional grains, reducing the cost of biocontrol products.

Rapid isolation of non-aflatoxigenicAspergillus flavus strains

In the present study, a method for screening non-aflatoxigenicAspergillus flavus in soil samples collected from major peanut-growing regions of China was developed. The single colonies were picked and cultured onAspergillus flavus andparasiticus agar (AFPA). If the reverse side of the colony on AFPA was orange-coloured, it was consideredA. flavus orAspergillus parasiticus. After the genomic DNA of each strain was extracted, 28S rRNA and calmodulin were amplified and sequenced to determine the species. The key gene,aflR, was amplified and digested via polymerase chain reaction-restriction fragment length polymorphism. The aflatoxigenicA. flavus and the non-aflatoxigenicA. flavus andA. parasiticus were distinguished by enzyme digestion ofaflR. 156 strains ofA. flavus were screened, which consisted of 135 aflatoxigenic and 21 non-aflatoxigenic strains. The aflatoxin producing ability of each strain was confirmed using solid-state fermentation experiments. Using the method developed in the present study, we confirmed that the non-aflatoxigenicA. flavus strains isolated lost their capacity to produce aflatoxins. Considering there could be some alterations in other functional genes, some non-aflatoxigenic strains could be identified inaccurately as aflatoxigenic strains, although that did not occur in the present study. The growth of non-aflatoxigenicA. flavus was observed, and the most rapidly growing non-aflatoxigenic strain was selected for plate confrontation assays and toxic mixed culture experiments. The inhibition rate of non-aflatoxigenicA. flavus against aflatoxigenicA. flavus was 55.4 and 72.6% in potato dextrose agar (PDA) plate and natural soybean medium, respectively. The screened non-aflatoxigenicA. flavus strains provide a microbial resource for biological control of aflatoxin contamination.

Effects of postharvest handling practices on quality of groundnuts and aflatoxin contamination

The increased cases of aflatoxin contamination are exacerbated by poor post-harvest management practices, coupled with adverse climatic conditions at harvest and post-harvest stages. This study therefore was carried out to improve safety and quality of groundnuts from aflatoxin contamination, through use of proper postharvest handling practices. Specifically the study determined the effects of harvesting dates and drying methods on aflatoxin contamination. Field experiments were carried out both at Chitedze and Chitala Agricultural Research Stations in Malawi during 2017/2018 growing season. A randomized complete block design in a split plot arrangement with three harvesting dates as the main plot and four drying methods as the sub-plots replicated three times was used. Groundnut was assessed for kernel infection by Aspergillus flavus, and level of aflatoxin contamination. Significantly low levels of about 0.5μg/ Kg of A. flavus infection and aflatoxin contamination were observed at 90 days after sowing (DAS). Higher aflatoxin contamination of up to 5μg/ Kg was observed at 80 DAS, and 10 days late after physiological maturity (100 DAS). This study also identified Mandela cock, aframe drying rack as effective drying method that can reduce aflatoxin contamination in groundnuts by 75 %. Moreover, Mandela cock drying method was shown as the most effective compared to A-frame and drying rack drying method. Current study therefore recommends for adoption of timely harvesting at physiological maturity, and drying using either Mandela cock or A-frame and drying rack. Further studies need to be carried on biological control of aflatoxin contamination.

Investigation of Pseudomonas fluorescens strain 3JW1 on preventing and reducing aflatoxin contaminations in peanuts.

Pseudomonas fluorescens strain 3JW1, which has a broad-spectrum antimicrobial activity, was studied to investigate whether it affects the amounts of aflatoxin B1 (AFB1) produced by Aspergillus flavus. It was found that the bacterium reduced the amounts of AFB1 in potato dextrose broth (PDB) and peanut medium by 97.8% and 99.4%, respectively. It also reduced AFB1 by ~183 μg/kg (55.8%) when applied onto peanut kernels. This strain reduced AFB1 via three mechanisms. First, it significantly inhibited A. flavus growth; second, our data showed that strain 3JW1 inhibits aflatoxin biosynthesis by A. flavus; and third, P. fluorescens strain 3JW1 is capable of degrading AFB1 at a rate as high as 88.3% in 96 hours. This is the first report demonstrating that Pseudomonas fluorescens can reduce toxin contamination caused by A. flavus on peanut kernels. Our findings indicate that P. fluorescens strain 3JW1 had multiple effects including reducing A. flavus infection and aflatoxin contamination. And the results also highlight the potential applications of the strain 3JW1 for the biological control of aflatoxin contamination in peanuts and other susceptible crops.

AtoxigenicAspergillus flavus biological control of aflatoxin contamination: what is the mechanism?

The term ‘competitive exclusion’ involving physical blockage of growth or access of the toxigenic strain to the seed target has been used to describe the mechanism of biological control of aflatoxin contamination. However, recent evidence suggests that a form of intraspecific aflatoxin inhibition requiring growth of the competing strains together during the infection process in such a way that hyphae physically interact or touch is the trigger for preventing induction of aflatoxin synthesis. This direct touch-based inhibition of aflatoxin synthesis is posited to be the mechanistic basis of biological control in this system. Evidence for this idea comes from the published observations that co-culture of toxigenic and atoxigenic strains in a suspended disc system, in which the hyphae physically interact, prevents aflatoxin production. However, growth of the same strains in the same medium in the two compartments of a filter insert plate well system, separating the atoxigenic and toxigenic strains with a 0.4 μm or 3.0 μm filter, allows aflatoxin production approaching that of the toxigenic strain alone. When the strains are mixed and placed in both the insert and the well compartments, the intraspecific aflatoxin inhibition occurs as it did in the suspended disc culture system. This further suggests that neither nutrient competition nor soluble signal molecules, which should pass through the filter, are involved in intraspecific aflatoxin inhibition. When the two strains are separated by a 12 μm filter that would allow some passage of conidia or hyphae between the compartments the aflatoxin synthesis is approximately half that of the toxigenic strain alone. This phenomenon could be termed ‘competitive inclusion’ or ‘competitive phenotype conversion’. Work of others that relates to understanding the phenomenon is discussed, as well as anAspergillus flavus population biology study from the Louisiana maize agro-ecosystem which has biological control implications.

Inhibition of Aspergillus parasiticus and cancer cells by marine actinomycete strains

Ten actinomycete strains isolated from the Yellow Sea off China’s coasts were identified as belonging to two genera by 16S rDNA phylogenetic analysis: Streptomyces and Nocardiopsis. Six Streptomyces strains (MA10, 2SHXF01-3, MA35, MA05-2, MA05-2-1 and MA08-1) and one Nocardiopsis strain (MA03) were predicted to have the potential to produce aromatic polyketides based on the analysis of the KSα (ketoacyl-synthase) gene in the type II PKS (polyketides synthase) gene cluster. Four strains (MA03, MA01, MA10 and MA05-2) exhibited significant inhibitory effects on mycelia growth (inhibition rate >50%) and subsequent aflatoxin production (inhibition rate >75%) of the mutant aflatoxigenic Aspergillus parasiticus NFRI-95. The ethyl acetate extracts of the broth of these four strains displayed significant inhibitory effects on mycelia growth, and the IC50 values were calculated (MA03: 0.275 mg mL−1, MA01: 0.106 mg mL−1, MA10: 1.345 mg mL−1 and MA05-2: 1.362 mg mL−1). Five strains (2SHXF01-3, MA03, MA05-2, MA01 and MA08-1) were selected based on their high cytotoxic activities. The ethyl acetate extract of the Nocardiopsis strain MA03 was particularly noted for its high antitumor activity against human carcinomas of the cervix (HeLa), lung (A549), kidney (Caki-1) and liver (HepG2) (IC50: 2.890, 1.981, 3.032 and 2.603 μg mL−1, respectively). The extract also remarkably inhibited colony formation of HeLa cells at an extremely low concentration (0.5 μg mL−1). This study highlights that marine-derived actinomycetes are a huge resource of compounds for the biological control of aflatoxin contamination and the development of novel drugs for human carcinomas.

The Role of Essential Oils and the Biological Detoxification in the Prevention of Aflatoxin Borne Diseases

Fungi are an important group of microorganisms. They are studied due to their significant impact on the environment, industry and human health. In relation to biological aspects, biogeochemical cycling of elements, the world of nature would not be possible without the contribution of fungi as the primary decomposers of organic material. On the other hand, food decay by the fungi spoilage causes considerable economic losses and constitutes a health risk for consumers due to the potential of fungi to produce mycotoxins. Mycotoxins are chemically diverse secondary metabolites that can be harmful both to animal and human health. Aflatoxins, a widely studied group of toxins among mycotoxins, are mainly biosynthesized by Aspergillus flavus and Aspergillus parasiticus. Therefore, the control of fungi and the production of aflatoxins, especially the most toxic aflatoxins B1 and G1, is essential and decisive. Over the last few decades, numerous studies have demonstrated that plants, especially their essential oils, contain diverse bioactive components that can protect crops from becoming contaminated by different mold species, Aspergillus in particular, with an emphasis on A. flavus and A. parasiticus; as well as that, they can prevent the contamination of food produce during the processing and storage and prevent the production of aflatoxins. Plant essential oils are expected to be more advantageous than synthetic compounds because of their relatively safe status, easy decomposition, environmentally friendly and non-phytotoxic properties. This paper also presents the recent research in biological control of aflatoxin contamination.

Structure of an Aspergillus flavus population from maize kernels in northern Italy

In order to gain insight into the causal agents of aflatoxin contamination of maize in Italy, populations of Aspergillus flavus on maize produced in the most affected area were characterized. Forty-six percent of A. flavus, isolated from maize kernels collected in 5 districts of northern Italy between 2003 and 2010, were unable to produce detectable levels of aflatoxins. The genetic diversity of the population was assessed by analysis of vegetative compatibility groups (VCGs) and presence or absence of several aflatoxin biosynthesis genes. Forty-eight VCGs were identified through complementation between nitrate non-utilizing mutants. Twenty-five VCGs contained only atoxigenic isolates, and the remaining 23 only aflatoxin producers. Members of the largest atoxigenic VCG (IT6) were found in 4 of the 5 districts sampled. Six deletion patterns of genes in the aflatoxin biosynthesis gene cluster were detected. No deletions in the cluster were detected for twelve atoxigenic isolates and 10 had the entire cluster deleted. One isolate had a deletion pattern only seen once before in Nigeria. The basis for initial selection of endemic atoxigenic strains of A. flavus for biological control of aflatoxin contamination of maize in Italy is provided.

Intraspecific aflatoxin inhibition in Aspergillus flavus is thigmoregulated, independent of vegetative compatibility group and is strain dependent.

Biological control of preharvest aflatoxin contamination by atoxigenic stains of Aspergillus flavus has been demonstrated in several crops. The assumption is that some form of competition suppresses the fungus's ability to infect or produce aflatoxin when challenged. Intraspecific aflatoxin inhibition was demonstrated by others. This work investigates the mechanistic basis of that phenomenon. A toxigenic and atoxigenic isolate of A. flavus which exhibited intraspecific aflatoxin inhibition when grown together in suspended disc culture were not inhibited when grown in a filter insert-plate well system separated by a .4 or 3 µm membrane. Toxigenic and atoxigenic conidial mixtures (50∶50) placed on both sides of these filters restored inhibition. There was ∼50% inhibition when a 12 µm pore size filter was used. Conidial and mycelial diameters were in the 3.5–7.0 µm range and could pass through the 12 µm filter. Larger pore sizes in the initially separated system restored aflatoxin inhibition. This suggests isolates must come into physical contact with one another. This negates a role for nutrient competition or for soluble diffusible signals or antibiotics in aflatoxin inhibition. The toxigenic isolate was maximally sensitive to inhibition during the first 24 hrs of growth while the atoxigenic isolate was always inhibition competent. The atoxigenic isolate when grown with a green fluorescent protein (GFP) toxigenic isolate failed to inhibit aflatoxin indicating that there is specificity in the touch inhibiton. Several atoxigenic isolates were found which inhibited the GFP isolate. These results suggest that an unknown signaling pathway is initiated in the toxigenic isolate by physical interaction with an appropriate atoxigenic isolate in the first 24 hrs which prevents or down-regulates normal expression of aflatoxin after 3–5 days growth. We suspect thigmo-downregulation of aflatoxin synthesis is the mechanistic basis of intraspecific aflatoxin inhibition and the major contributor to biological control of aflatoxin contamination.

Reduction of aflatoxin B1 in stored peanuts (Arachis hypogaea L.) using Saccharomyces cerevisiae.

Aflatoxin B(1) is a toxigenic and carcinogenic compound produced by Aspergillus flavus and Aspergillus parasiticus. To inhibit aflatoxin contamination of peanuts, seeds of two peanut breeds, IAC Caiapó and IAC Runner 886, were inoculated with A. parasiticus (1.0 × 10(6) spores per ml) and the yeast Saccharomyces cerevisiae (3.2 × 10(7) cells per ml) and incubated at 25°C for 7 and 15 days. Two experiments were conducted for each incubation period separately. The treatments were completely randomized, with three replications per treatment. Treatments included the two cultivars and three types of inoculation (pathogen alone, yeast and pathogen, and yeast 3 h before pathogen). Aflatoxin B(1) was quantified with a densitometer at 366 nm after thin layer chromatography. Aflatoxin B(1) contamination in peanuts was reduced after the addition of S. cerevisiae. The concentration of aflatoxin B(1) decreased by 74.4 and 55.9% after 7 and 15 days, respectively. The greatest aflatoxin reduction was observed when S. cerevisiae was inoculated 3 h before the pathogen in IAC Caiapó seeds and incubated for 7 days at 25°C. The use of S. cerevisiae is a promising strategy for biological control of aflatoxin contamination in peanuts.

Influence of the host contact sequence on the outcome of competition among aspergillus flavus isolates during host tissue invasion.

Biological control of aflatoxin contamination by Aspergillus flavus is achieved through competitive exclusion of aflatoxin producers by atoxigenic strains. Factors dictating the extent to which competitive displacement occurs during host infection are unknown. The role of initial host contact in competition between pairs of A. flavus isolates coinfecting maize kernels was examined. Isolate success during tissue invasion and reproduction was assessed by quantification of isolate-specific single nucleotide polymorphisms using pyrosequencing. Isolates were inoculated either simultaneously or 1 h apart. Increased success during competition was conferred to the first isolate to contact the host independent of that isolate's innate competitive ability. The first-isolate advantage decreased with the conidial concentration, suggesting capture of limited resources on kernel surfaces contributes to competitive exclusion. Attempts to modify access to putative attachment sites by either coating kernels with dead conidia or washing kernels with solvents did not influence the success of the first isolate, suggesting competition for limited attachment sites on kernel surfaces does not mediate first-isolate advantage. The current study is the first to demonstrate an immediate competitive advantage conferred to A. flavus isolates upon host contact and prior to either germ tube emergence or host colonization. This suggests the timing of host contact is as important to competition during disease cycles as innate competitive ability. Early dispersal to susceptible crop components may allow maintenance within A. flavus populations of genetic types with low competitive ability during host tissue invasion.

Biological control of aflatoxin contamination in corn using a nontoxigenic strain of Aspergillus flavus.

A 2-year study was conducted to determine the efficacy of different applications of a nontoxigenic strain of Aspergillus flavus for reducing aflatoxin contamination in corn. Treatments consisted of the nontoxigenic strain in the form of (i) conidia-coated hulled barley applied to soil when corn was about 0.8 m tall, (ii) conidia-coated hulled barley applied in plant whorls prior to tasseling, (iii) multiple applications of a spray formulation of conidia during silking, and (iv) untreated control. Treatments were replicated eight times in individual plots consisting of four rows of 18 m each. In year 1, no significant differences were associated with treatments for aflatoxin, total A. flavus colonization, or incidence of nontoxigenic isolates of A. flavus in corn, which were all relatively high, ranging from 83.8 to 93.1%. In year 2, the whorl application produced a significantly lower mean aflatoxin concentration of 49.5 ppb compared with all other treatments, while both the soil (108.3 ppb) and spray applications (173.7 ppb) were significantly reduced compared with the control (191.6 ppb). The whorl application was the only treatment that had a significantly higher incidence (86.5%) of nontoxigenic isolates of A. flavus than the control had, which was still relatively high at 69.1%. Data indicated that applications of the nontoxigenic strain influenced untreated corn, thus reducing the apparent effect of the biocontrol treatments. Larger-scale studies with greater separation between treated and untreated fields are warranted.

Development of Biocontrol Technology to Manage Aflatoxin Contamination in Peanuts

Abstract Aflatoxin contamination of peanuts results from invasion and growth of the fungi, Aspergillus flavus and A. parasiticus. Peanut pods develop in the soil where they are in contact with propagules of these ubiquitous fungi. When peanuts are subjected to drought conditions as pods are maturing, they become susceptible to contamination. A method of biological control of aflatoxin contamination was developed in which a competitive, nontoxigenic strain of A. flavus is applied to the soil to competitively exclude the toxigenic strains in the invasion of peanuts. The biocontrol product is comprised of conidia of the nontoxigenic strain coated onto the surface of hulled barley, which is applied to peanut fields during the middle of the growing season. After uptake of moisture the conidia germinate, grow, and sporulate, yielding a dominant population of the nontoxigenic strain in the soil. Several plot and field studies showed that aflatoxin in farmers' stock peanuts was reduced by 80 to 90% with this technique. The patented technology was licensed by a company that markets the biocontrol product under the trade name, afla-guard®. In 2004, the U. S. Environmental Protection Agency issued a Section 3 registration for use of afla-guard® to control aflatoxin contamination in peanuts. Analyses of peanuts from the first commercial use of afla-guard® in various locations in Georgia and Alabama showed aflatoxin reductions averaging 85% in farmers' stock peanuts and as high as 98% in shelled stock.

Biological control of aflatoxin contamination of crops.

Aflatoxins produced primarily by two closely related fungi, Aspergillus flavus and Aspergillus parasiticus, are mutagenic and carcinogenic in animals and humans. Of many approaches investigated to manage aflatoxin contamination, biological control method has shown great promise. Numerous organisms, including bacteria, yeasts and nontoxigenic fungal strains of A. flavus and A. parasiticus, have been tested for their ability in controlling aflatoxin contamination. Great successes in reducing aflatoxin contamination have been achieved by application of nontoxigenic strains of A. flavus and A. parasiticus in fields of cotton, peanut, maize and pistachio. The nontoxigenic strains applied to soil occupy the same niches as the natural occurring toxigenic strains. They, therefore, are capable of competing and displacing toxigenic strains. In this paper, we review recent development in biological control of aflatoxin contamination.

Biological Control of Aflatoxin Contamination of Crops

Aflatoxin contamination of crops compromises the safety of food and feed supplies and causes significant economic losses each year. Of the many research approaches being studied to reduce and, ultimately, eliminate aflatoxin contamination, biological control is one of the more promising, particularly for the near‐term. Numerous organisms have been tested for biological control of aflatoxin contamination including bacteria, yeasts, and nontoxigenic strains of the causal organisms, Aspergillus flavus and A. parasiticus. Most of the field successes to date have been achieved by applying certain nontoxigenic strains of A. flavus and A. parasiticus to soil of susceptible crops, such as peanuts, cotton, and corn. The applied strains occupy the same niche as the naturally occurring toxigenic strains and competitively exclude them when crops are susceptible to infection. Various formulations have been used to apply the nontoxigenic strains to soil, but the most effective methods have been to combine the desired s...

Effect of application of nontoxigenic strains of Aspergillus flavus and A. parasiticus on subsequent aflatoxin contamination of peanuts in storage

Experiments were conducted to determine the potential for biological control of aflatoxin contamination of peanuts during storage. Florunner peanuts were treated in field plots by applying competitive, nontoxigenic strains of Aspergillus flavus and A. parasiticus, at 76 and 67 days after planting in 1998 and 1999, respectively. After harvest, half the peanuts from both treated and control plots were sprayed with an aqueous conidial suspension containing the nontoxigenic strains; the other half of the peanuts from each group were not sprayed. The peanuts were then placed in separate compartments of a miniature warehouse. Therefore, storage treatments consisted of peanuts that were (1) not treated at all; (2) treated prior to storage only; (3) field-treated only; (4) treated both in the field and prior to storage. Peanuts were stored for 3–5 months under high temperature and relative humidity conditions designed to promote aflatoxin contamination. In 1998, peanuts were not contaminated with aflatoxins prior to storage. After storage, peanuts that were never treated with the competitive fungi contained an average of 78.0 ppb of aflatoxins. Peanuts not treated in the field but receiving the spray treatment before storage contained 48.8 ppb. Peanuts treated in the field only averaged 1.4 ppb, and peanuts treated both in the field and prior to storage contained 0.8 ppb. In 1999, peanuts suffered from late-season drought and were contaminated with aflatoxins at harvest, with controls averaging 516.8 ppb compared with 54.1 ppb in treated peanuts. After storage, non-field-treated peanuts averaged 9145.1 ppb compared with 374.2 ppb for peanuts that had been field-treated, a 95.9% reduction. Spraying of pods with the nontoxigenic strains postharvest but prior to storage provided no additional protection against aflatoxin contamination. Results demonstrated that field application of the nontoxigenic strains had a carry-over effect and reduced aflatoxin contamination that occurred in storage.

Inhibition of Aflatoxin B1 Production by Aspergillus parasiticus Using Nonaflatoxigenic Strains: Role of Vegetative Compatibility

The effect of vegetative compatibility on the inhibition of aflatoxin B 1 production by Aspergillus parasiticus was examined using nonaflatoxigenic strains. Nonaflatoxigenic white-conidial mutants were paired in different proportions on an agar medium with aflatoxigenic yellow-conidial mutants belonging to the same isolate, to the same vegetative compatibility group but with the original wild types differing in phenotype, and to different vegetative compatibility groups. Heterokaryosis as a result of hyphal anastomosis was detected by the presence of conidiogenous structures with a mixture of green and parental (white and/or yellow) chains of conidia. Sclerotium production (number and dry weight) was significantly greater in pairings of compatible strains that formed heterokaryons than in pairings of strains from different vegetative compatibility groups. In contrast, there were no consistent differences in aflatoxin B 1 inhibition by nonaflatoxigenic strains in pairings from the same vegetative compatibility group and pairings from different groups. Therefore, the composition of vegetative compatibility groups within a population may be of minor importance in predicting the efficacy of a particular nonaflatoxigenic strain for the biological control of aflatoxin contamination of crops.

INFLUENCE OF OTHER FUNGI ON AFLATOXIN PRODUCTION BY ASPERGILLUS FLAVUS IN MAIZE KERNELS

Experiments were designed to determine whether certain nontoxigenic fungi commonly isolated from maize kernels can affect aflatoxin B1 development when inoculated with A. flavus onto individual unsterilized, and autoclaved maize kernels. Trichoderma viride and Aspergillus niger were found to be strongly antagonist inhibiting the growth of A. flavus by 87 and 66% respectively, whereas Aspergillus versicolor, Fusarium moniliforme, Paecilomyces variotii and Emericella quadrillineata inhibited the growth of A. flavus by less than 51%. Less aflatoxin B1 was detected when A. flavus was paired with A. niger or T. viride than with the other test fungi. When A. niger or T. viride was introduced onto the kernels 72 h before inoculation with A. flavus, no aflatoxin B1 was detected in unsterilized kernels and the levels of aflatoxin B1 were greatly reduced from 700 ppb to 160 and 140 ppb in autoclaved kernels, respectively. When inoculation of A. flavus followed 72 h of incubation of either A. niger and T. viride, no aflatoxin B1 was detected. However, when both A. niger and T. viride were introduced 72 h after inoculation with A. flavus, the levels of aflatoxin B1 were reduced to 385 and 560 ppb, respectively in unsterilized and autoclaved maize kernels. Trichoderma viride and Aspergillus niger may be useful in biological control of aflatoxin contamination of maize kernels.; Accepted for Publication June 11, 1997