Comparison of some screening methods for aflatoxigenic moulds

Thirty seven strains of the Aspergillus flavus group isolated from animal mixed feeds have been screened for their ability to produce aflatoxins in yeast extract and sucrose (YES), aflatoxin producing ability (APA), and coconut agar medium (CAM) media. The concentration and detection of the aflatoxins by different methods is compared. Five known aflatoxin-positive and one aflatoxin-negative strains have been used as controls. Only 5 out of the 37 strains (13.5%) were aflatoxin-producers in YES medium. Of these five strains and the five known aflatoxin-positive strains, only three showed blue fluorescence in APA medium and four in CAM medium. Generally, the aflatoxin concentration in CAM medium was higher than in YES and APA media. Using the agar-plug method and by direct spotting of the YES broth on TLC plates, some aflatoxin-producing strains were not detected.     

Considerations on the distribution of aflatoxigenic Aspergillus flavus in feeds

The distribution of aflatoxin producing isolates of the Aspergillus flavus group in feeds was studied. Aflatoxin production was investigated by a sequential method previously reported (fluorescence in Coconut Agar Medium, rapid extraction from a wheat medium, and total extraction from the same wheat medium). Twenty-seven of 32 samples contained A. flavus, and 21 of them had at least one aflatoxicogenic isolate of A. flavus. Of the 115 isolates analysed, 65 produced aflatoxins, mainly B aflatoxins.     

Aspergillus flavus colonization and aflatoxin B1 formation in barley grain during interaction with other fungi

Colonization of barley grain by Aspergillus flavus and formation of aflatoxin B₁ in the presence of Penicillium verrucosum, Fusarium sporotrichioides, and Hyphopichia burtonii were studied over a three-week period in all combinations of 20 or 30 °C and 0.97, 0.95 or 0.90 a_w. Grain colonization was assessed initially by observing hyphal extension on the grain surface, using scanning electron microscopy, and then from the proportion of seeds infected and numbers of colony forming units (cfu) formed. Aflatoxin b¹ concentrations were determined by enzyme linked immunosorbent assay using a monoclonal antibody. These studies showed that interaction between A. flavus and other fungi in paired culture had different effects on both colonization and aflatoxin formation depending on the species involved and environmental conditions. Germination of A. flavus spores was unaffected by the presence of other species on the grain surface. Subsequently, three principal patterns of A. flavus colonization of barley grain were observed through the incubation period in the presence of other fungal species: (a) colonization unaffected by the presence of other species; (b) colonization initially slower in the presence of other species but later differing little from pure cultures; and (c) colonization adversely affected by the presence of other species. Five main patterns of aflatoxin B₁ production were observed relative to pure culture but with no consistent relationship with species, a_w, temperature or incubation period; (a) little changed; (b) increased slowly; (c) decreased; (d) enhanced; and (e, f) increased initially but later decreased to (e) the same level as in pure culture or (f) to less than in pure culture. Generally, production of aflatoxin B₁ by A. flavus was less than in pure culture but sometimes was changed only slightly by the presence of P. verrucosum, F. sporotrichioides or H. burtonii or was temporarily enhanced.     

Mycotoxins in Australia: biocontrol of aflatoxin in peanuts

The major mycotoxin problem in Australia is the formation of aflatoxins in peanuts by Aspergillus flavus and A. parasiticus. This is controlled by good farm management practice, segregation into grades on aflatoxin content at intake to shelling facilities, colour sorting and aflatoxin assays. A second problem is the potential presence of ochratoxin A in grapes and grape products, resulting from infection by Aspergillus carbonarius. Good quality control before and during wine making ensures ochratoxin A is kept to very low levels, but in dried vine fruit, ochratoxin A levels may be higher. Biocontrol by competitive exclusion has been developed as the most promising means of controlling aflatoxins in peanuts. Some details of the process are given, including some basic laboratory experiments.     

Substrate depletion during formation of aflatoxin and kojic acid on corn inoculated with Aspergillus flavus

During a period of 8 years 300 cases of dermatophytoses involving both hairy areas and the glabrous skin were found to be caused by M. canis. There was scalp involvement in 60%, including 8 infants and 27 adults; most of the adults presented Kerion-like lesions and presented various clinical aspects such as seborrhea capitis, folliculitis and discois lupus erythematosus. In the 21 patients showing invasion of the beard the clinical manifestations included superficial erythematosquamous patches with hyperemic slightly elevated margins, folliculitis or abscess-like lesions and Kerion-like lesions. Among the lesions found on the glabrous skin there were unusual aspects of tinea faciei in 19 adults, mimicking lymphocytic infiltration, granuloma faciale or discoid lupus erythematosus. Some of the cases of tinea corporis found in 70 patients also had lesions simulating various other dermatological entities, including erythema multiforme, psoriasiform eruption, pityriasis rosea and seborrheic dermatitis. The hands were invaded in 5 adults patients, with involvement of the finger nails in one. Repeated mycologic examinations were necessary to establish the true etiology in many of these cases.     

Isolation of aflatoxigenic strains of Aspergillus and detection of aflatoxin B1 from feeds in India

In a preliminary study, 256 feed samples collected from different parts of Northern India were examined for the presence of aflatoxigenic strains of Aspergillus flavus/parasiticus and for detection of Aflatoxin B₁ (AFB₁). Out of 198 A. flavus and 15 A. parasiticus strains isolated, 76% and 86% respectively, were found to be toxigenic. Aflatoxin B₁ content of these feeds, as estimated by thin layer chromatography (TLC) and enzyme linked immunosorbent assay (ELISA) were very high (average 0.412 ± 0.154 ppm) in comparison to the permissible Indian regulation level (0.03 ppm). Seasonal variation of incidence and level of toxin in feed was recorded and it was high during monsoon/post monsoon period.This revised version was published online in October 2005 with corrections to the Cover Date.     

Different media and methodologies for the detection of aflatoxin production by Aspergillus flavus strains isolated from trout feed

We have studied the aflatoxin producing capacity of 41 Aspergillus flavus strains isolated from the mycoflora present of natural media (wheat, rice and mixed feed) synthetic medium (Aflatoxin Producing Ability Medium) and semisynthetic media (Coconut Agar Medium and Glucose Yeast Extract Agar) were compared. Aflatoxins were analysed on days 4 and 8 post-inoculation under an incubation temperature of 28 °C. A total of 30 strains (75.7%) were producers on natural media as detected by Thin Layer Chromatography: 23 strains on wheat, 27 on rice and 12 on mixed feed. The results by qualitative flourescence tests on synthetic and semisynthetic media were: 3 strains positive on Coconut Agar Medium (CAM) 1 on Glucose Yeast Extract Agar (GY + Agar) and none on Aflatoxin Producing Ability Medium (APA).     

Influence of formic acid on fungal flora of barley and on aflatoxin production inAspergillus flavus link

In recent yearsAspergillus flavus and aflatoxin production have been noted on several occasions in grain preserved with formic acid. Samples of mouldy barley treated with formic acid and stored in an open bin were investigated for the presence of fungi. In the lower part of the bin there was a clear dominance ofFusarium sporotrichioides, and deoxynivalenol and neosolaniol were detected.A. flavus andA. fumigatus were also present.Paecilomyces variotii occurred, almost as a pure culture, in the upper part of the bin, but no patulin was found. Cultivation of four fungal isolates from these genera on laboratory substrates containing formic acid showedP. variotii to be the most tolerant to formic acid, withstanding 150 mM, but still without patulin production.F. sporotrichioides andA. fumigatus tolerated only 6 mM formic acid. The growth ofA. flavus was reduced and atypical at 60 mM formic acid. Pretreatment ofA. flavus spores with formic acid increased aflatoxin production about 800 times.     

Occurrence of Aspergillus section Flavi and aflatoxin B1 in corn genotypes and corn meal in Argentina

A study has been carried out in Argentina on samples of corn genotypes from a breeding station as well as in commercially available corn meal. All samples were analyzed for fungal infection and aflatoxin B₁.Mycological analysis of corn genotypes showed the presence of three principal genera of filamentous fungi Fusarium (100%), Penicillium (67%) and Aspergillus (60%). In the genus Fusarium three species were identified, F. moniliforme (42%), F. nygamai (56%) andF. proliferatum (1.8%). Eight species ofPenicillium were identified, the predominant species isolated were P. minioluteum, P. funiculosum and P. variabile. In the genus ranked third in isolation frequency, two species were identified, A. flavus and A. parasiticus, the percentage of infection was 78% and 21%, respectively. Only one corn genotype was contaminated with aflatoxin B₁ at a level of 5 ppb. The cornmeal samples showed great differences in fungal contamination, the values ranging from 1 × 10¹ to 7 × 10⁵ cfu g^–1. Fusarium (68%), Aspergillus (35%) and Penicillium (21%) were the most frequent genera isolated. Among the genus, Aspergillus, A. parasiticus (38%) was the most frequent species isolated. All the samples of corn meal were negative to aflatoxin B₁. These results indicate a low degree of human exposure to aflatoxins in Argentina through the ingestion of maize or corn meal.This revised version was published online in October 2005 with corrections to the Cover Date.     

Evaluation of biological control formulations to reduce aflatoxin contamination in peanuts

A two-year study was conducted to evaluate the efficacy of three formulations of nontoxigenic strains of Aspergillus flavus and Aspergillus parasiticus to reduce preharvest aflatoxin contamination of peanuts. Formulations included: (1) solid-state fermented rice; (2) fungal conidia encapsulated in an extrusion product termed Pesta; (3) conidia encapsulated in pregelatinized corn flour granules. Formulations were applied to peanut plots in 1996 and reapplied to the same plots in 1997 in a randomized design with four replications, including untreated controls. Analysis of soils for A. flavus and A. parasiticus showed that a large soil population of the nontoxigenic strains resulted from all formulations. In the first year, the percentage of kernels infected by wild-type A. flavus and A. parasiticus was significantly reduced in plots treated with rice and corn flour granules, but it was reduced only in the rice-treated plots in year two. There were no significant differences in total infection of kernels by all strains of A. flavus and A. parasiticus in either year. Aflatoxin concentrations in peanuts were significantly reduced in year two by all formulation treatments with an average reduction of 92%. Reductions were also noted for all formulation treatments in year one (average 86%), but they were not statistically significant because of wide variation in the aflatoxin concentrations in the untreated controls. Each of the formulations tested, therefore, was effective in delivering competitive levels of nontoxigenic strains of A. flavus and A. parasiticus to soil and in reducing subsequent aflatoxin contamination of peanuts.     

The occurrence of Aspergillus flavus in vegetative tissue of cotton plants and its relation to seed infection

Twenty-seven mature cotton bolls with Aspergillus flavus Link colonies naturally occurring on the surface of the boll or lint were collected in the field in Arizona along with their subtending stems and peduncles. Bolls inoculated through the carpel wall 30 days after anthesis were allowed to mature in the field and were collected in the same manner. The seed and stem and peduncle sections of each boll were surface-sterilized, plated on agar media and observed for A. flavus. Seventy-eight percent of the naturally contaminated bolls with A. flavus in the seed also had the fungus in the stem and peduncle, whereas only 31% of the naturally contaminated bolls with no A. flavus in the seed had the fungus in the stem or peduncle. This difference was significant (P=0.0125), indicating a positive relationship between seed infection and stem and peduncle infection. All of the bolls inoculated through the carpel wall had A. flavus in the seed, but only 11% of the stem and peduncle sections were infected, indicating that the fungus does not readily grow downward from the boll into the supporting stem or peduncle.This unidirectional pattern of movement (upward) was further substantiated in greenhouse experiments where cotton seedlings were inoculated at the cotyledonary leaf scar with A. flavus and plants were sequentially harvested, surface sterilized and plated. Aspergillus flavus was isolated from the cotyledonary leaf scar, flower buds, developing bolls, and stem sections in the upper portion of the plant. It was never isolated from roots or stem sections below the cotyledonary node, again indicating that the fungus does not readily move downward through the plant.     

Divergent regulation of aflatoxin production at acidic pH by two <Emphasis Type="Italic">Aspergillus</Emphasis> strains

Production of aflatoxins (AF) by Aspergillus flavus and A. parasiticus is known to occur only at acidic pH. Although typical A. flavus isolates produced more AF as the external pH became increasingly acidic, an atypical strain from West Africa produced less. The lower AF production was not well correlated with decreases in expression of the aflatoxin pathway regulatory gene, aflR, or of two other biosynthesis genes.     

Time ofAspergillus flavus infection and aflatoxin formation in ripening of figs

Figs in an orchard were inoculated with an aflatoxigenicAspergillus flavus strain in two ways by spore injection or by dusting at three maturation stages: firm ripe, shrivelled, and dried. Fruits were individually examined for fungal development and analyzed for aflatoxin B₁ (AF B₁) after 2, 4, 6, 8 and 10 days. Fruit injected at the first stage showed fungal development and AF B₁ contamination within two days. The toxin level increased sharply to 1 ppm after 10 days. The mean level of AF B₁ (284.75 ng/g) was significantly higher than those observed in other conditions. Figs dusted at the first stage showed only a tiny fungal growth even after 10 days. AF B₁ appeared after 6 days with a low frequency (35%), mean level (7.6 ng/g) and a great variation among figs (0.22–15 ng/g). Among fruits inoculated during the shrivelled fig and dried fruit stages, no fungal growth was observed and AF B₁ was detected with a lower incidence in association with low mean levels (less than 1.25 ng/g). Methods of prevention of aflatoxin contamination at the critical step, the firm ripe stage, are discussed.     

A mycological and bacteriological survey on feed ingredients and mixed poultry feeds in Reunion island

A survey was carried out in Reunion island to obtain data on the occurrence of fungi, aflatoxigenic strains of Aspergillus flavus, aflatoxins, total aerobic bacteria and salmonellae of 150 samples of mixed poultry feeds and raw materials. These were collected at five farms over a 3-month period during the warm rainy season.White corn and Brazilian soybean meal seemed to present a better microbiological quality than yellow corn and US soybean meal.Mixed poultry feeds presented a high total mold count reflecting the mold flora of raw materials. The most frequent and abundant fungi were Aspergillus flavus, A. glaucus group, Fusarium spp., Penicillium spp., A. candidus, Mucor spp., A. restrictus, Scopulariopsis spp., Cladosporium spp. and A. versicolor. Of the 118 A. flavus strains screened, 42 (35.6%) were aflatoxigenic. Yellow corn samples were the most frequently contamined with aflatoxigenic strains (54.5%), followed by mixed feeds (44%).Of the 66 samples tested, 24 (36%) contained aflatoxins (traces to 22 ng/g). A good correlation seemed to exist between presence of at least one aflatoxigenic strain per sample and presence of aflatoxins.     

Fluorometric analysis of iodinated aflatoxin in minicultures ofAspergillus flavus andAspergillus parasiticus

Summary A convenient miniassay for aflatoxin has been developed for cultures ofAspergillus flavus andA. parasiticus grown for 3–10 days in 10 ml of a coconut extract medium. The sensitivity of the assay, as measured by photofluorometry (365 nm maximum excitation; 445 nm maximum emission), is of the order of 0.01 M (3.12 ng/ml) for aflatoxin B₁ dissolved in aqueous iodine (0.26 mM). High performance liquid chromatography, monitored by fluorometric analysis of both an aflatoxin B₁ standard and selected culture filtrates, confirmed the sensitivity of the assay and indicated specificity for iodine-enhanced fluorescence of aflatoxin in the coconut extract medium. Thin layer chromatography further confirmed the aflatoxin titers and the specificity for enhancement of aflatoxins B₁ and G₁ in culture filtrates.Alabama Agricultural Experiment Station Journal No. 6-871297.     

Incidence of aflatoxin producing strains and aflatoxin contamination in dry fruit slices of quinces (Cydonia oblonga Mill.) from the Indian State of Jammu and Kashmir

An investigation was undertaken to obtain data on the occurrence of aflatoxins and the aflatoxin producing potential of Aspergillus flavus strains isolated from dry fruit slices of quinces produced in jammu and Kashmir, India. A total of 147 A. flavus isolates recovered from dr fruit slices were grown in liquid rice flour medium and screened for the production of various aflatoxins by thin layer chromatography. The results showed that 23.14% of the tested isolates were aflatoxigenic, producing aflatoxins B₁and B₂ in varying amounts. Aflatoxins G₁ and G₂ were not detected. All 25 of the investigated market samples were also found to be aflatoxin B₁ positive and the level of contamination ranged from 96 to 8164 g/kg of the dry fruit which is quite high in comparison to the permissible level of 30 ppb. As per these results biochemical composition of dry fruit slices of quinces, along with climatic conditions seem to be very favourable for aflatoxin production by the toxigenic A. flavus strains. Therefore,monitoring of aflatoxins in dry fruit slices of quincesis recommended for this region.This revised version was published online in October 2005 with corrections to the Cover Date.     

Assessment of aflatoxin and cyclopiazonic acid production by Aspergillus flavus isolates from Hungary

Thirty-two isolates of Aspergillus flavus were obtained from various sources in Hungary. All isolates were morphologically identified as A. flavus and three atypical variants were confirmed as A. flavus by comparing their DNA with an ex type culture of A. flavus. None of these isolates produced aflatoxins when tested on coconut agar or grown on rice medium and culture extracts examined by thin layer chromatography. Also, none of the isolates converted sterigmatocystin, O-methyl sterigmatocystin, norsolorinic acid, or sodium acetate to aflatoxin. However, 59% of the isolates produced cyclopiazonic acid based on thin layer chromatographic analysis of culture extracts. The isolates that lack the ability to produce both aflatoxin and cyclopiazonic acid are potential candidates for use in bicontrol studies.     

Research on the aflatoxin problem in groundnut at ICRISAT

Summary Aflatoxin contamination of groundnut is a serious problem in most groundnut producing countries and as such is given high research priority by the Groundnut Improvement Program of ICRISAT. Since 1979 we have concentrated on selecting cultivars resistant to seed invasion and colonization by toxigenicAspergillus flavus, and/or to aflatoxin production following invasion by the fungus. Resistance to invasion and colonization byA. flavus of rehydrated, mature seed has been found, and confirmed, in some cultivars. We have also screened several groundnut cultivars for seed resistance in the field, both under natural conditions and with the inoculum of the fungus added to the soil in the pod zone. Some cultivars with resistance to seed colonization also showed resistance to seed invasion byA. flavus. None of the cultivars tested has shown complete resistance to aflatoxin production but significant cultivar differences occurred in the amounts of aflatoxin produced in seeds inoculated with a toxigenic strain ofA. flavus.ICRISAT Journal Article No. JA-316     

Conidial movement of nontoxigenic Aspergillus flavus and A. parasiticus in peanut fields following application to soil

The use of nontoxigenic strains of Aspergillus flavus and A. parasiticus in biological control effectively reduces aflatoxin in peanuts when conidium-producing inoculum is applied to the soil surface. In this study, the movement of conidia in soil was examined following natural rainfall and controlled precipitation from a sprinkler irrigation system. Conidia of nontoxigenic A. flavus and A. parasiticus remained near the soil surface despite repeated rainfall and varying amounts of applied water from irrigation. In addition, rainfall washed the conidia along the peanut furrows for up to 100 meters downstream from the experimental plot boundary. The dispersal gradient was otherwise very steep upstream along the furrows and in directions perpendicular to the peanut rows. The retention of biocontrol conidia in the upper soil layers is likely important in reducing aflatoxin contamination of peanuts and aerial crops such as corn and cottonseed. This revised version was published online in August 2006 with corrections to the Cover Date.