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.     

Aflatoxin production by entomopathogenic isolates of Aspergillus parasiticus and Aspergillus flavus

Fourteen isolates of Aspergillus parasiticus and 2 isolates of Aspergillus flavus isolated from the mealybug Saccharicoccus sacchari were analyzed for production of aflatoxins B1, B2, G1, and G2 in liquid culture over a 20-day period. Twelve Aspergillus isolates including 11 A. parasiticus and 1 A. flavus produced aflatoxins which were extracted from both the mycelium and culture filtrate. Aflatoxin production was detected at day 3 and was detected continually for up to day 20. Aflatoxin B1 production was greatest between 7 and 10 days and significantly higher quantities were produced by A. flavus compared to A. parasiticus. Aflatoxin production was not a stable trait in 1 A. parasiticus isolate passaged 50 times on agar. In addition to loss of aflatoxin production, an associated loss in sporulation ability was also observed in this passaged isolate, although it did maintain pathogenicity against S. sacchari. An aflatoxin B1 concentration of 0.16 micrograms/mealybug (14.2 micrograms/g wet wt) was detected within the tissues of infected mealybugs 7 days after inoculation. In conclusion, the ability of Aspergillus isolates to produce aflatoxins was not essential to the entomopathogenic activity of this fungus against its host S. sacchari.     

Factors influencing the inhibition of aflatoxin production in corn by Aspergillus niger

Aspergillus niger, a mold commonly associated with Aspergillus flavus in damaged corn, interferes with the production of aflatoxin when grown with A. flavus on autoclaved corn. The pH of corn-meal disks was adjusted using NaOH-HCl, citric acid-sodium citrate, or a water extract of A. niger fermented corn. Aflatoxin formation was completely inhibited below pH 2.8-3.0, irrespective of the system used for pH adjustment. When grown in association with A. flavus NRRL 6432 on autoclaved corn kernels, A. niger NRRL 6411 lowered substrate pH sufficiently to suppress aflatoxin production. The biodegradation of aflatoxin B1 or its conversion to aflatoxin B2a were eliminated as potential mechanisms by which A. niger reduces aflatoxin contamination. A water extract of corn kernels fermented with A. niger caused an additional inhibition of aflatoxin formation apart from the effects of pH.     

Southwestern corn borer (Lepidoptera: Crambidae) damage and aflatoxin accumulation in maize

Aflatoxin, a potent carcinogen, is produced by the fungus Aspergillus flavus Link: Fr. Drought, high temperatures, and insect damage contribute to increased levels of aflatoxin contamination in corn, Zea mays L. Plant resistance is widely considered a desirable method of reducing aflatoxin contamination. Germplasm lines with aflatoxin resistance have been developed. This investigation was undertaken to determine whether crosses among these lines exhibited resistance to southwestern corn borer, Diatraea grandiosella Dyar, and to assess the effects of southwestern corn borer feeding on aflatoxin accumulation. Differences in ear damage among southwestern corn borer infested hybrids were significant. Estimates of general combining ability effects indicated that the lines Mp80:04, Mp420, and Mp488 contributed to reduced ear damage, and SC213 and T165 contributed to greater damage when used in hybrids. Mean aflatoxin levels were 254 ng/g for hybrids infested with southwestern corn borer larvae and 164 ng/g for noninfested hybrids in 2000 when environmental conditions were conducive to aflatoxin production. In contrast, the overall mean aflatoxin level for southwestern corn borer infested hybrids was only 5 ng/g in 1999 when environmental conditions did not favor aflatoxin accumulation. Crosses that included lines selected for aflatoxin resistance as parents (Mp80:04 and Mp313E) exhibited lower levels of aflatoxin contamination both with and without southwestern corn borer infestation in 2000. Only the experimental line Mp80:04 contributed significantly to both reduced southwestern corn borer damage and reduced aflatoxin contamination.     

Corn earworm,Helicoverpa zea (Lepidoptera: Noctuidae) and other insect associated resistance in the maize inbred Tex6

A 2-yr field and laboratory study investigated insect resistance of the maize, Zea mays L., inbred Tex6, which has previously demonstrated resistance to Aspergillus ear rot and aflatoxin production, relative to susceptible inbred B73. Field studies indicated significantly greater resistance to insect feeding of V4-V8 growth stage Tex6 plants compared with B73 plants in both years, primarily to flea beetles (Chaetonema spp.). Field studies of natural (1999) and artificial (2000) infestations of corn earworms, Helicoverpa zea (Boddie), indicated much lower levels of kernel damage at milk stage (approximately three-fold) and smaller surviving larvae (approximately three-fold) in Tex6 compared with B73 ears. At harvest similar trends in reduction of numbers of damaged kernels per ear, as well as incidence and numbers of kernels per ear symptomatically infected by Fusarium spp. were noted. Laboratory studies indicated little difference in mortality or survivor weight of caterpillars or sap beetle adults caged with milk stage kernels of the two inbreds. However, assays with silks indicated significantly greater mortality of H. zea in both 1999 and 2000, and European corn borer, Ostrinia nubilalis (Hübner) in 1999 (only year tested) when fed Tex6 silks compared with B73 silks. Pollinated Tex6 silks were generally darker colored and more toxic than unpollinated silks. Thus, it is possible that commercially usable inbreds with resistance to insects, which also contribute to the mycotoxin problem through vectoring and damage, could be produced using Tex6 as a source.     

Partial characterization of the mode of action of benzoic acid on aflatoxin biosynthesis.

Aflatoxin production by a toxigenic strain of Aspergillus flavus was greatly reduced by benzoic acid and sodium benzoate in synthetic media. The reduction was accompanied by the appearance of a yellow pigment. Spectral analyses partially characterized this pigment as closely related to an acetyl derivative of a versiconal-type compound. A cell-free extract prepared from A. flavus grown in synthetic media was active in converting this yellow compound into aflatoxin B1 in the presence of reduced nicotinamide adenine dinucleotide phosphate at 25 degrees C (pH 7.4). In the presence of benzoic acid and its salt or autoclaved cell-free extract, conversion of yellow compound to aflatoxin B1 was prevented. These results suggest that the yellow compound is an intermediate in the secondary metabolic cycle involved in aflatoxin B1 production. Benzoic acid, sodium benzoate, or autoclaving the cell-free extract appear to have respectively blocked or denatured an enzymatic step late in the biosynthetic pathway of aflatoxin B1.     

Stimulation by Hyphopichia burtonii and Bacillus amyloliquefaciens of aflatoxin production by Aspergillus flavus in irradiated maize and rice grains

Aspergillus flavus was grown on maize and rice extract agars and on irradiated viable cracked maize and rice grains, either in pure culture or in dual culture with wild strains of either Hyphopichia burtonii or Bacillus amyloliquefaciens. Aflatoxin production by A. flavus and its growth and interactions with the other microorganisms were studied at three water activities (aw) (0.98, 0.95, and 0.90) and two temperatures (25 and 16 degrees C). Both H. burtonii and B. amyloliquefaciens markedly stimulated growth and aflatoxin production by A. flavus on cracked maize, especially at 25 degrees C and 0.95 and 0.98 aw. No aflatoxin was detected in pure cultures of A. flavus on cracked rice after 12 days of incubation at 25 degrees C, but some was produced by mixed cultures at 16 degrees C and 0.98 aw. The morphological interactions among A. flavus, H. burtonii, and B. amyloliquefaciens were also examined on maize and rice extract agars under similar controlled conditions.     

Dusky Sap Beetle Mediated Dispersal of Bacillus subtilis to Inhibit Aspergillus flavus and Aflatoxin Production in Maize Zea mays L.

Laboratory assays were performed with detached milk stage maize ( Zea mays L.) ears and dusky sap beetles ( Carpophilus lugubris Murray) carrying the Kodiak Concentrate formulation of the bacterium, Bacillus subtilis (Ehrenberg) Cohn. After 1 day of exposure to the B. subtilis- contaminated C. lugubris , the colonization of mechanically damaged kernels by Aspergillus flavus Link ex. Fries was reduced from 82% (if the A. flavus was inoculated first) to 41% (if B. subtilis was added by C. lugubris before the A. flavus ). Field cage studies were performed with an autoinoculative device containing B. subtilis into which C. lugubris beetles were introduced. C. lugubris -dispersed B. subtilis reduced visible A. flavus colonization by 97% when the A. flavus was added to purposely damaged maize ears 4 days after C. lugubris were released from the autoinoculator. In 1993 field studies, none of the purposely damaged ears that allowed access to C. lugubris beetles emerging from autoinoculators containing B. subtilis had visible sporulating A. flavus compared with 92% of ears that did not allow access of C. lugubris but that subsequently had the A. flavus inoculum added. In 1994 field studies, 70% of the ears that excluded C. lugubris had aflatoxin levels greater than 200 ppb in purposely damaged kernels, as opposed to less than 10% of kernels that permitted access by natural populations of C. lugubris that probably acquired B. subtilis from a single autoinoculator. Aflatoxin levels in these ears were negatively correlated with the presence of both B. subtilis and C. lugubris . The B. subtilis was widely dispersed over a 16-ha area as indicated by maize ear and C. lugubris trap sampling. These studies indicate that autoinoculative dispersal of B. subtilis by natural populations of C. lugubris is a potentially useful means for reducing A. flavus and aflatoxin in maize.     

Stimulation by Hyphopichia burtonii and Bacillus amyloliquefaciens of aflatoxin production by Aspergillus flavus in irradiated maize and rice grains.

Aspergillus flavus was grown on maize and rice extract agars and on irradiated viable cracked maize and rice grains, either in pure culture or in dual culture with wild strains of either Hyphopichia burtonii or Bacillus amyloliquefaciens. Aflatoxin production by A. flavus and its growth and interactions with the other microorganisms were studied at three water activities (aw) (0.98, 0.95, and 0.90) and two temperatures (25 and 16 degrees C). Both H. burtonii and B. amyloliquefaciens markedly stimulated growth and aflatoxin production by A. flavus on cracked maize, especially at 25 degrees C and 0.95 and 0.98 aw. No aflatoxin was detected in pure cultures of A. flavus on cracked rice after 12 days of incubation at 25 degrees C, but some was produced by mixed cultures at 16 degrees C and 0.98 aw. The morphological interactions among A. flavus, H. burtonii, and B. amyloliquefaciens were also examined on maize and rice extract agars under similar controlled conditions.     

Fungicidal and anti-aflatoxigenic effects of the essential oil of Cymbopogon citratus (DC.) Stapf. (lemongrass) against Aspergillus flavus Link. isolated from stored rice

AIMS: To develop a natural fungicide against aflatoxigenic fungi, to protect stored rice, using the essential oil of lemongrass. METHODS AND RESULTS: Aspergillus flavus Link. was isolated from stored rice and identified as an aflatoxigenic strain. Lemongrass oil was tested against A. flavus and the test oil was fungistatic and fungicidal against the test pathogen at 0.6 and 1.0 mg ml(-1), respectively. Aflatoxin production was completely inhibited at 0.1 mg ml(-1). The results obtained from the thin layer chromatographic bioassay and gas chromatography indicated citral a and b as the fungicidal constituents in lemongrass oil. During the fumigant toxicity assay of lemongrass oil, the sporulation and the mycelial growth of the test pathogen were inhibited at the concentrations of 2.80 and 3.46 mg ml(-1), respectively. CONCLUSION: Lemongrass oil could be used to manage aflatoxin formation and fungal growth of A. flavus in stored rice. SIGNIFICANCE AND IMPACT OF THE STUDY: Currently, fungicides are not used to control fungal pests or mycotoxin production on stored rice. Rice treated with the essential oil of lemongrass could be used to manage fungal pests as well as the insect pests in stored rice. The essential oil is chemically safe and acceptable to consumers, as synthetic chemical fungicides can cause adverse health effects to consumers.     

Effects of various acids and salts on growth and aflatoxin production by Aspergillus flavus NRRL 3145.

The effects of sodium chloride, sodium acetate, benzoic acid, sodium benzoate, malonic acid, and sodium malonate on growth and aflatoxin production by Aspergillus flavus were investigated in synthetic media. Sodium chloride at concentrations equivalent to or greater than 12 g/100 ml inhibited growth and aflatoxin production, while at 8 g or less/100 ml, growth and aflatoxin production were stimulated. At 2 g or less/100 ml, sodium acetate also stimulated growth and aflatoxin production, but reduction occurred with 4 g or more/100 ml. Malonic acid at 10, 20, 40, and 50 mM reduced growth and aflatoxin production (over 50%) while sodium malonate at similar concentrations but different pH values had the opposite effect. Benzoic acid (pH 3.9) and sodium benzoate (pH 5.0) at 0.4 g/100 ml completely inhibited growth and aflatoxin production. Examination of the effect of initial pH indicated that the extent of inhibitory action of malonic acid and sodium acetate was a function of initial pH. The inhibitory action of benzoic acid and sodium benzoate appeared to be a function of undissociated benzoic acid molecules. Aflatoxin reduction was usually accompanied by an unidentified orange pigment, while aflatoxin stimulation was accompanied by unidentified blue and green fluorescent spots but with lower Rf values that aflatoxins B1, G1, B2, and G2 standards.     

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.     

黄曲霉毒素B1生物脱毒的研究进展

黄曲霉毒素是一组由黄曲霉、寄生曲霉等多种真茵产生的次级代谢产物,具有强烈的毒性,可以引起动物肝脏肿大、病变甚至癌变,对人和家畜的健康产生极大的威胁。本文简介了黄曲霉毒素B_1的分子结构、理化性质、污染现状,综述了黄曲霉毒素B_1生物脱毒方面及其应用的研究进展,重点讲述通过微生物降解黄曲霉毒素B_1的研究近况。     

Occurrence of aflatoxin in some liver curative herbal medicines

Fifty herbal medicine samples of seven different taxa known to cure liver disorders were analysed for aflatoxin contamination. Twenty-three samples, out of 50, were contaminated with various levels of aflatoxins. Amongst the 23 contaminated samples the maximum level of aflatoxin B¹ recorded was 2.23 μg g^-1 in Asparagus racemosus and the minimum 0.28 μg g^-1 in Emblica officinalis . Aflatoxin G¹ was only found in one species, Terminalia belarica . Aflatoxin production of the isolates of Aspergillus flavus was also examined and the highest levels were produced by isolates from A. racemosus (1.07-2.47 μg ml^-1). Aflatoxin contamination of herbal drugs may be a risk for patients because the level of aflatoxins is much higher than the tolerance level fixed by the WHO for foods.     

Effects of soil moisture and temperature on preharvest invasion of peanuts by the Aspergillus flavus group and subsequent aflatoxin development

Four soil temperature and moisture treatment regimens were imposed on Florunner peanuts 94 days after planting in experimental plots in 1980. At harvest (145 days after planting), the incidence of the Aspergillus flavus group and the aflatoxin concentration were greatest in damaged kernels. Extensive colonization of sound mature kernels (SMK) by the A. flavus group occurred with the drought stress treatment (56% kernels colonized); colonization was less in the irrigated plot (7%) and the drought stress plot with cooled soil (11%) and was intermediate in the irrigated plot with heated soil (26%). Aflatoxin was virtually absent from SMK with the last three treatments, but it was found at an average concentration of 244 ppb (ng/g) in drought-stressed SMK. Colonization of SMK by the A. flavus group and aflatoxin production were greater with hot dry conditions. Neither elevated temperature alone nor drought stress alone caused aflatoxin contamination in SMK. When the ratio of SMK colonized by A. flavus compared with A. niger was greater than 19:1, there was aflatoxin contamination, but there was none if this ratio was less than 9:1. Irrigation caused a higher incidence of A. niger than drought did. This may have prevented the aflatoxin contamination of undamaged peanuts.     

Effects of soil moisture and temperature on preharvest invasion of peanuts by the Aspergillus flavus group and subsequent aflatoxin development.

Four soil temperature and moisture treatment regimens were imposed on Florunner peanuts 94 days after planting in experimental plots in 1980. At harvest (145 days after planting), the incidence of the Aspergillus flavus group and the aflatoxin concentration were greatest in damaged kernels. Extensive colonization of sound mature kernels (SMK) by the A. flavus group occurred with the drought stress treatment (56% kernels colonized); colonization was less in the irrigated plot (7%) and the drought stress plot with cooled soil (11%) and was intermediate in the irrigated plot with heated soil (26%). Aflatoxin was virtually absent from SMK with the last three treatments, but it was found at an average concentration of 244 ppb (ng/g) in drought-stressed SMK. Colonization of SMK by the A. flavus group and aflatoxin production were greater with hot dry conditions. Neither elevated temperature alone nor drought stress alone caused aflatoxin contamination in SMK. When the ratio of SMK colonized by A. flavus compared with A. niger was greater than 19:1, there was aflatoxin contamination, but there was none if this ratio was less than 9:1. Irrigation caused a higher incidence of A. niger than drought did. This may have prevented the aflatoxin contamination of undamaged peanuts.     

Inhibition of aflatoxin B production of Aspergillus flavus, isolated from soybean seeds by certain natural plant products

AIMS: The inhibitory effect of cowdung fumes, Captan, leaf powder of Withania somnifera, Hyptis suaveolens, Eucalyptus citriodora, peel powder of Citrus sinensis, Citrus medica and Punica granatum, neem cake and pongamia cake and spore suspension of Trichoderma harzianum and Aspergillus niger on aflatoxin B(1) production by toxigenic strain of Aspergillus flavus isolated from soybean seeds was investigated. METHODS AND RESULTS: Soybean seed was treated with different natural products and fungicide captan and was inoculated with toxigenic strain of A. flavus and incubated for different periods. The results showed that all the treatments were effective in controlling aflatoxin B(1) production. Captan, neem cake, spore suspension of T. harzianum, A. niger and combination of both reduced the level of aflatoxin B(1) to a great extent. Leaf powder of W. somnifera, H. suaveolens, peel powder of C. sinensis, C. medica and pongamia cake also controlled the aflatoxin B(1) production. CONCLUSIONS: All the natural product treatments applied were significantly effective in inhibiting aflatoxin B(1) production on soybean seeds by A. flavus. SIGNIFICANCE AND IMPACT OF THE STUDY: These natural plant products may successfully replace chemical fungicides and provide an alternative method to protect soybean and other agricultural commodities from aflatoxin B(1) production by A. flavus.     

Sequence comparison of aflR from different Aspergillus species provides evidence for variability in regulation of aflatoxin production

Aflatoxin contamination of foods and feeds is a world-wide agricultural problem. Aflatoxin production requires expression of the biosynthetic pathway regulatory gene, aflR, which encodes a Cys6Zn2-type DNA-binding protein. Homologs of aflR from Aspergillus nomius, bombycis, parasiticus, flavus, and pseudotamarii were compared to investigate the molecular basis for variation among aflatoxin-producing taxa in the regulation of aflatoxin production. Variability was found in putative promoter consensus elements and coding region motifs, including motifs involved in developmental regulation (AbaA, BrlA), regulation of nitrogen source utilization (AreA), and pH regulation (PacC), and in coding region PEST domains. Some of these elements may affect expression of aflJ, a gene divergently transcribed from aflR, that also is required for aflatoxin accumulation. Comparisons of phylogenetic trees obtained with either aligned aflR intergenic region sequence or coding region sequence and the observed divergence in regulatory features among the taxa provide evidence that regulatory signals for aflatoxin production evolved to respond to a variety of environmental stimuli under differential selective pressures. Phylogenetic analyses also suggest that isolates currently assigned to the A. flavus morphotype SBG represent a distinct species and that A. nomius is a diverse paraphyletic assemblage likely to contain several species.     

Degradation of aflatoxin by Aspergillus flavus

Aflatoxin degradative activity was demonstrated in 6- to 12-d-old intact mycelium and cell-free extracts of Aspergillus flavus. The addition of cycloheximide, SKF 525-A or metyrapone to cultures of A. flavus prevented subsequent degradation of the aflatoxins, while in cell-free extracts degradation was inhibited by SKF 525-A, metyrapone and cytochrome c but not by KCN. In cell-free extracts, aflatoxin degradation was enhanced by NADPH and NaIO4. The results suggest the involvement of cytochrome P-450 monooxygenases in the aflatoxin degradative activity of A. flavus.