Aflatoxin contamination of developing corn kernels

Preharvest of corn and its contamination with aflatoxin is a serious problem. Some environmental and cultural factors responsible for infection and subsequent aflatoxin production were investigated in this study. Stage of growth and location of kernels on corn ears were found to be one of the important factors in the process of kernel infection with A. flavus & A. parasiticus. The results showed positive correlation between the stage of growth and kernel infection. Treatment of corn with aflatoxin reduced germination, protein and total nitrogen contents. Total and reducing soluble sugar was increase in corn kernels as response to infection. Sucrose and protein content were reduced in case of both pathogens. Shoot system length, seeding fresh weigh and seedling dry weigh was also affected. Both pathogens induced reduction of starch content. Healthy corn seedlings treated with aflatoxin solution were badly affected. Their leaves became yellow then, turned brown with further incubation. Moreover, their total chlorophyll and protein contents showed pronounced decrease. On the other hand, total phenolic compounds were increased. Histopathological studies indicated that A. flavus & A. parasiticus could colonize corn silks and invade developing kernels. Germination of A. flavus spores was occurred and hyphae spread rapidly across the silk, producing extensive growth and lateral branching. Conidiophores and conidia had formed in and on the corn silk. Temperature and relative humidity greatly influenced the growth of A. flavus & A. parasiticus and aflatoxin production.     

Color mutants of Aspergillus flavus and Aspergillus parasiticus in a study of preharvest invasion of peanuts

A comparison of the invasion of flowers, aerial pegs, and kernels by wild-type and mutant strains of Aspergillus flavus or A. parasiticus along with aflatoxin analyses of kernels from different drought treatments have supported the hypothesis that preharvest contamination with aflatoxin originates mainly from the soil. Evidence in support of soil invasion as opposed to aerial invasion was the following. A greater percentage of invasion of kernels rather than flower or aerial pegs by either wild-type A. flavus or mutants. Significant invasion by an A. parasiticus color mutant occurred only in peanuts from soil supplemented with the mutant, whereas adjacent plants in close proximity but in untreated soil were only invaded by wild-type A. flavus or A. parasiticus. Aflatoxin data from drought-stressed, visibly undamaged peanut kernels showed that samples from soil not supplemented with a mutant strain contained a preponderance of aflatoxin B's (from wild-type A. flavus) whereas adjacent samples from mutant-supplemented soil contained a preponderance of B's plus G's (from wild-type and mutant A. parasiticus). Preliminary data from two air samplings showed an absence of propagules of A. flavus or A. parasiticus in air around the experimental facility.     

Color mutants of Aspergillus flavus and Aspergillus parasiticus in a study of preharvest invasion of peanuts.

A comparison of the invasion of flowers, aerial pegs, and kernels by wild-type and mutant strains of Aspergillus flavus or A. parasiticus along with aflatoxin analyses of kernels from different drought treatments have supported the hypothesis that preharvest contamination with aflatoxin originates mainly from the soil. Evidence in support of soil invasion as opposed to aerial invasion was the following. A greater percentage of invasion of kernels rather than flower or aerial pegs by either wild-type A. flavus or mutants. Significant invasion by an A. parasiticus color mutant occurred only in peanuts from soil supplemented with the mutant, whereas adjacent plants in close proximity but in untreated soil were only invaded by wild-type A. flavus or A. parasiticus. Aflatoxin data from drought-stressed, visibly undamaged peanut kernels showed that samples from soil not supplemented with a mutant strain contained a preponderance of aflatoxin B's (from wild-type A. flavus) whereas adjacent samples from mutant-supplemented soil contained a preponderance of B's plus G's (from wild-type and mutant A. parasiticus). Preliminary data from two air samplings showed an absence of propagules of A. flavus or A. parasiticus in air around the experimental facility.     

Detection of toxigenic isolates of Aspergillus flavus and related species on coconut cream agar

A new readily-prepared medium, coconut cream agar, was developed for the detection of aflatoxin production by isolates of Aspergillus flavus and related species. Coconut cream agar, which comprised coconut cream (50%) and agar (1.5%), detected isolates of A. flavus more effectively than the synthetic media tested and was as effective as media containing desiccated coconut. Fluorescence colouring of colonies grown on coconut cream agar could be used to differentiate A. flavus from A. parasiticus and A. nomius. In addition, conidial colour of A. flavus and A. nomius was quite distinct from that of A. parasiticus.     

Aflatoxin variability in pistachios.

Pistachio fruit components, including hulls (mesocarps and epicarps), seed coats (testas), and kernels (seeds), all contribute to variable aflatoxin content in pistachios. Fresh pistachio kernels were individually inoculated with Aspergillus flavus and incubated 7 or 10 days. Hulled, shelled kernels were either left intact or wounded prior to inoculation. Wounded kernels, with or without the seed coat, were readily colonized by A. flavus and after 10 days of incubation contained 37 times more aflatoxin than similarly treated unwounded kernels. The aflatoxin levels in the individual wounded pistachios were highly variable. Neither fungal colonization nor aflatoxin was detected in intact kernels without seed coats. Intact kernels with seed coats had limited fungal colonization and low aflatoxin concentrations compared with their wounded counterparts. Despite substantial fungal colonization of wounded hulls, aflatoxin was not detected in hulls. Aflatoxin levels were significantly lower in wounded kernels with hulls than in kernels of hulled pistachios. Both the seed coat and a water-soluble extract of hulls suppressed aflatoxin production by A. flavus.     

Influence of Fungicides and Irrigation Practice on Aflatoxin in Peanuts Before Digging

Peanuts grown under dryland conditions where drought stress occurred accumulated more aflatoxin before digging than peanuts grown under irrigation. Kernels became more susceptible to Aspergillus flavus and A. parasiticus invasion when the soil moisture in the pod zone approached levels at which moisture moved from the pod into the soil and the kernel moisture dropped below 31%. Isolation frequencies of these aspergilli from fresh-dug kernels were lowest in 1968 (maximum of 3%). In 1967 and 1969, maximum percentages of 100 and 74, respectively, were noted. Kernel infestation was correlated with degree of aflatoxin contamination. Dryland fresh-dug kernels contained a maximum of 35,800 parts per billion aflatoxin while a maximum of 50 parts per billion was detected in kernels from irrigated plots. In 1969 A. flavus infestation was as high as 59% in peanuts from irrigated plots; however, no aflatoxin was detected. Absence of aflatoxin in these samples is attributed to the higher kernel moisture content which reduced the aflatoxin-producing potential of A. flavus. Statistical analysis of the data revealed no significant differences in degree of fungal infestation, production levels, and grade factors between any fungicide treatments.     

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.     

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.     

Channeling of vanadium in two species of fungi.

Aspergillus flavus and Penicillium italicum were cultivated on Dox medium amended with different concentrations of a vanadium salt; vanadium pentoxide (V2O5). The fungal isolates were found to absorb considerable quantities of vanadium. Fungal contents of carbohydrates and proteins were decreased by the presence of vanadium in the growth environment. Vanadium was incorporated into several types of low and high molecular weight protein(s) and peptides. In case of P. italicum, lysine, tyrosine, glycine, methionine, valine, alanine, proline, glutamic acid and histidine were detected in the fungal cell free extract. While in case of A. flavus, the following amino acids were detected: cystine, methionine, leucine, phenylalanine, glycine, alanine, tyrosine, lysine, histidine, arginine and valine.     

Characterization and population analysis of the mating-type genes in Aspergillus flavus and Aspergillus parasiticus

We characterize the mating-type genes in Aspergillus flavus,Aspergillus parasiticus and Petromyces alliaceus. A single MAT1-1 or MAT1-2 gene was detected in the genomes of A. flavus and A. parasiticus, which is consistent with a potential heterothallic organization of MAT genes in these species. In contrast, the only known, functionally homothallic species in Aspergillus section Flavi, P. alliaceus, has tightly linked (<2kb) MAT1-1 and MAT1-2 genes, typical of other self-fertile homothallic euascomycetes. This is the first example of linked MAT genes within a homothallic species of Aspergillus. We tested the null hypothesis of no significant difference in the frequency of MAT1-1 and MAT1-2 in A. flavus and A. parasiticus sampled from a single peanut field in Georgia. For each species, mating-type frequencies were determined for the total population samples and for samples that were clone-corrected based on vegetative compatibility groups (VCGs) and aflatoxin gene cluster haplotypes. There was no significant difference in the frequency of the two mating types for A. flavus and A. parasiticus in either VCG or haplotype clone-corrected samples. The existence of both mating-type genes in equal proportions in A. flavus and A. parasiticus populations, coupled with their expression at the mRNA level and the high amino acid sequence identity of MAT1-1 (77%) and MAT1-2 (83%) with corresponding homologs in P. alliaceus, indicates the potential functionality of these genes and the possible existence of a sexual state in these agriculturally important species.     

Interrelation of growth media and water activity in sclerotia characteristics of Aspergillus section Flavi

AIMS: To examine sclerotium characteristics of two Aspergillus flavus and two A. parasiticus strains at different growth media and water stress. METHODS AND RESULTS: The effects of growth media and water activity (0.999, 0.971, 0.955 and 0.937) on characteristics of sclerotia production (number, size and volume) of four isolates of Aspergillus section Flavi were examined. There was total inhibition under the driest conditions (0.955 and 0.937). When an osmotic potential of 0.971 was generated in Czapek agar (CD) and maize meal extract agar with sucrose and sodium nitrate (MMEA S/N), an increase in sclerotial size and volume was observed. The amount of sclerotia produced by cultures at 0.999 a(w) value was higher on CD. CONCLUSIONS: The data show that the sclerotia characteristics of A. flavus and A. parasiticus have been influenced by water availability and growth media composition. SIGNIFICANCE AND IMPACT OF THE STUDY: The information obtained shows that if we know the nutritional and water stress requirements for sclerotia production, it could be possible to develop effective prevention strategies to inhibit the survival of these fungi in grain.     

Intracellular hydrolases of Aspergillus parasiticus and Aspergillus flavus

When the distribution profile of hydrolases in mycelial homogenates and culture filtrates of A. parasiticus and A. flavus was examined, six hydrolytic enzymes viz. N-acetyl-beta-glucosaminidase, aryl sulfatase, alkaline proteinase, cathepsin B, cathepsin D and aminopeptidase were detected in homogenate. The culture filtrates were devoid of any activity of these enzymes. The enzyme levels varied with the stage of incubation. The most abundant fungal exopeptidase showing preference for basic amino acid naphthylamides seems to be an aminopeptidase B. Incorporation of CEPA, an ethylene generating compound, stimulated the amino peptidase activity in the mycelium but inhibited the enzyme in vitro. The enzyme was also inhibited by different aflatoxins to varying degree. While aminopeptidase B was located intracellularly, a non-dialysable, heat-stable inhibitor of the enzyme was found to be secreted in the culture filtrate. This peptide inhibitor was however ineffective on the other enzymes.     

Sporogenic effect of polyunsaturated fatty acids on development of Aspergillus spp.

Aspergillus spp. are frequently occurring seed-colonizing fungi that complete their disease cycles through the development of asexual spores, which function as inocula, and through the formation of cleistothecia and sclerotia. We found that development of all three of these structures in Aspergillus nidulans, Aspergillus flavus, and Aspergillus parasiticus is affected by linoleic acid and light. The specific morphological effects of linoleic acid include induction of precocious and increased asexual spore development in A. flavus and A. parasiticus strains and altered sclerotium production in some A. flavus strains in which sclerotium production decreases in the light but increases in the dark. In A. nidulans, both asexual spore production and sexual spore production were altered by linoleic acid. Spore development was induced in all three species by hydroperoxylinoleic acids, which are linoleic acid derivatives that are produced during fungal colonization of seeds. The sporogenic effects of these linoleic compounds on A. nidulans are similar to the sporogenic effects of A. nidulans psi factor, an endogenous mixture of hydroxylinoleic acid moieties. Light treatments also significantly increased asexual spore production in all three species. The sporogenic effects of light, linoleic acid, and linoleic acid derivatives on A. nidulans required an intact veA gene. The sporogenic effects of light and linoleic acid on Aspergillus spp., as well as members of other fungal genera, suggest that these factors may be significant environmental signals for fungal development.     

Effect of corn and peanut cultivation on soil populations of Aspergillus flavus and A. parasiticus in southwestern Georgia.

The effect of corn and peanut cultivation on the proportion of Aspergillus flavus to A. parasiticus in soil was examined. Soil populations were monitored in three fields during three different years in southwestern Georgia. Each field was planted in both peanuts and corn, and soil was sampled within plots for each crop. A. flavus and A. parasiticus were present in similar proportions in plots from all fields at the beginning of the growing season. A. terreus, A. niger, and A. fumigatus were the other dominant aspergilli in soil. Fields A and B did not show drought stress in peanut or corn plants, and soil populations of A. flavus and A. parasiticus remained stable during the course of the year. In field C, drought stress in corn plants with associated A. flavus infection and aflatoxin contamination greatly increased soil populations of A. flavus relative to A. parasiticus upon dispersal of corn debris to the soil surface by a combine harvester. Colonization of organic debris after it has been added to the soil may maintain soil populations of A. parasiticus despite lower crop infection.     

Intrafungal distribution of aflatoxins among conidia and sclerotia of Aspergillus flavus and Aspergillus parasiticus

This research examines the distribution of aflatoxins among conidia and sclerotia of toxigenic strains of Aspergillus flavus Link and Aspergillus parasiticus Speare cultured on Czapek agar (21 days, 28 degrees C). Total aflatoxin levels in conidia and sclerotia varied considerably both within (intrafungal) and among strains. Aspergillus flavus NRRL 6554 accumulated the highest levels of aflatoxin (conidia: B1, 84000 ppb; G1, 566000 ppb; sclerotia: B1, 135000 ppb; G1, 968000 ppb). Substantial aflatoxin levels in conidia could place at risk those agricultural workers exposed to dust containing large numbers of A. flavus conidia. Cellular ratios of aflatoxin B1 to aflatoxin G1 were nearly identical in conidia and sclerotia even though levels of total aflatoxins in these propagule types may have differed greatly. Aflatoxin G1 was detected in sclerotia of all A. flavus strains but in the conidia of only one strain. Each of the A. parasiticus strains examined accumulated aflatoxin G1 in both sclerotia and conidia. These results are examined in the context of current evolutionary theory predicting an increase in the chemical defense systems of fungal sclerotia, propagules critical to the survival of these organisms.     

Quantitative analysis of free amino acids and carbohydrates in spring barley anthers at different stages of maturity

The content of the carbohydrates glucose, fructose and sucrose was determined in spring barley anthers at different stages of maturity. During maturation the sucrose content of the anthers increased markedly. The following 17 free amino acids were detected in anthers of different stages of maturity: aspartic acid, glutamic acid, serine, alanine, arginine, leucine, isoleucine, lysine, α-aminobutyric acid, glutamine, proline, tyrosine, phenylalanine, valine, threonine, cystine and glycine. Quantitative analysis was only carried out in amino acids present in higher concentrations in the analysed samples. These were: aspartic acid, glutamic acid, α-aminobutyric acid, proline, serine, valine and glutamine, and a mixture of amino acids (leucine, isoleucine, valine and phenylalanine). The total content of free amino acids increased with increasing maturity of the anthers. However, not all amino acids followed contributed to this increase, but only proline, glutamic acid, aspartic acid and glutamine. A small difference was found in the variety Gopal in which the aspartic acid content did not increase significantly, but the content of the mixture of amino acids and serine did. With the exception of green anthers of the variety Firlbecks Union, proline was present in the highest concentration in all samples analysed.     

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.     

Preharvest aflatoxin contamination: effect of moisture and substrate variation in developing cottonseed and corn kernels

Variations in moisture and substrate in preharvest corn kernels and cottonseed were linked with the ability of Aspergillus parasiticus to infect the seed and produce aflatoxin. Osmotic pressures and moisture content (MC) levels of developing starch-rich corn kernels and lipid-rich cottonseed were determined. For in vivo studies, corn kernels and cottonseed were inoculated with A. parasiticus conidia and retained on plants through maturation. For in vitro studies, samples of corn kernels and cottonseed were collected at various stages, sterilized, inoculated, incubated for 2 weeks, and assayed for toxin. Aflatoxin levels were highest in corn kernels inoculated at 28 days postflowering (52% MC) in both the in vivo and in vitro tests. Toxin concentrations in cottonseed were greatest with inoculation at 35 days postflowering (70% MC) in seed retained on the plant, but toxin accumulation continued to increase with the maturity of the seed inoculated in cottonseed used in the in vitro trials. Moisture and substrate conditions in the midrange of seed development provided optimum conditions for fungal development and toxin production in seed retained on the plant.     

Preharvest aflatoxin contamination: effect of moisture and substrate variation in developing cottonseed and corn kernels.

Variations in moisture and substrate in preharvest corn kernels and cottonseed were linked with the ability of Aspergillus parasiticus to infect the seed and produce aflatoxin. Osmotic pressures and moisture content (MC) levels of developing starch-rich corn kernels and lipid-rich cottonseed were determined. For in vivo studies, corn kernels and cottonseed were inoculated with A. parasiticus conidia and retained on plants through maturation. For in vitro studies, samples of corn kernels and cottonseed were collected at various stages, sterilized, inoculated, incubated for 2 weeks, and assayed for toxin. Aflatoxin levels were highest in corn kernels inoculated at 28 days postflowering (52% MC) in both the in vivo and in vitro tests. Toxin concentrations in cottonseed were greatest with inoculation at 35 days postflowering (70% MC) in seed retained on the plant, but toxin accumulation continued to increase with the maturity of the seed inoculated in cottonseed used in the in vitro trials. Moisture and substrate conditions in the midrange of seed development provided optimum conditions for fungal development and toxin production in seed retained on the plant.     

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

S im , T.S., T eo , T heresa & S im , T.F. 1985. A note on the screening of dried shrimps, shrimp paste and raw groundnut kernels for aflatoxin-producing Aspergillus flavus. Journal of Applied Bacteriology 59 , 29–34.
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.