Separate and combined applications of nontoxigenic Aspergillus flavus and A. parasiticus for biocontrol of aflatoxin in peanuts

A 2-year study was carried out to determine the effect of applying nontoxigenic strains of Aspergillus flavus and A. parasiticus to soil separately and in combination on preharvest aflatoxin contamination of peanuts. A naturally occurring, nontoxigenic strain of A. flavus and a UV-induced mutant of A. parasiticus were applied to peanut soils during the middle of each of two growing seasons using a formulation of conidia-coated hulled barley. In addition to an untreated control, treatments included soil inoculated with nontoxigenic A. flavus only, soil inoculated with nontoxigenic A. parasiticus only, and soil inoculated with a mixture of the two nontoxigenic strains. Plants were exposed to late-season drought conditions that were optimal for aflatoxin contamination. Results from year one showed that significant displacement (70%) of toxigenic A. flavus occurred only in peanuts from plots treated with nontoxigenic A. flavus alone; however, displacement did not result in a statistically significant reduction in the mean aflatoxin concentration in peanuts. In year two, soils were re-inoculated as in year one and all treatments resulted in significant reductions in aflatoxin, averaging 91.6%. Regression analyses showed strong correlations between the presence of nontoxigenic strains in peanuts and aflatoxin reduction. It is concluded that treatment with the nontoxigenic A. flavus strain alone is more effective than the A. parasiticus strain alone and equally as effective as the mixture. The U.S. Government’s right to retain a non-exclusive, royalty-free license in and to any copyright is acknowledged.     

Evaluation of different genotypes of nontoxigenic Aspergillus flavus for their ability to reduce aflatoxin contamination in peanuts

Aflatoxins produced by the fungus Aspergillus flavus are potent carcinogens and account for large monetary losses worldwide in peanuts, maize, and cottonseed. Biological control in which a nontoxigenic strain of A. flavus is applied to crops at high concentrations effectively reduces aflatoxins through competition with native aflatoxigenic populations. In this study, eight nontoxigenic strains of A. flavus belonging to different vegetative compatibility groups and differing in deletion patterns within the aflatoxin gene cluster were evaluated for their ability to reduce aflatoxin B₁ when paired with eight aflatoxigenic strains on individual peanut seeds. Inoculation of wounded viable peanut seeds with conidia demonstrated that nontoxigenic strains differed in their ability to reduce aflatoxin B₁. Reductions in aflatoxin B₁ often exceeded expected reductions based on a 50:50 mixture of the two A. flavus strains, although one nontoxigenic strain significantly increased aflatoxin B₁ when paired with an aflatoxigenic strain. Therefore, nontoxigenicity alone is insufficient for selecting a biocontrol agent and it is also necessary to test the effectiveness of a nontoxigenic strain against a variety of aflatoxigenic strains.     

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.     

Mycological assisted phytoremediation enhancement of bioenergy crops Zea mays and ‎Helianthus annuus in heavy metal contaminated lithospheric zone

Current investigation has for the first time utilized Trichocomaceae fungi i.e. Aspergillus niger, Aspergillus terreus, Aspergillus flavus and Pencillium i.e. Penicillium chrysogenum for augmenting the phytoremediation potential of bioenergy crops wheat (Zea mays) and ? sunflower (Helianthus annuus). Phytoremediation was done for mitigation of heavy metals i.e. Chromium (Cr), Copper (Cu), Lead (Pb) and Cadmium (Cd) from contaminated soils of agricultural significance. Phytoremediant crops were inoculated with fungal cultures by three methods i.e. mixing method, seed inoculation method and layering spreading method. Maize and sunflower plants after fungal inoculation were harvested after 60 days of germination. The estimation of % biomass and bioenergy of maize and sunflower plants was done. Results were indicative of the good phytoremediation potential of roots and shoots for uptake of heavy metals i.e. CrAspergillus niger, Aspergillus terreus and Aspergillus flavus by fungal inoculation methods. Sunflower and fungal inoculum of Aspergillus flavus and Penicillium chrysogenum extracted significant quantity of metals from the soil. By three fungal inoculation methods, range of % production of biomass was 84?87% and sunflower plants dry biomass 9.6 g yielded 0.16% of oil. Obtained results are have favored the use of fungal inoculation as an effective mode for phytoremediation augmentation of maize and sunflower. Furthermore, current work also signifies the sustainable conversion of bioenergy crops to biofuel production in a cost effective mode.     

Effect of nontoxigenic Aspergillus flavus and A. parasiticus on aflatoxin contamination of wounded peanut seeds inoculated with agricultural soil containing natural fungal populations

Peanuts and other seed and grain crops are commonly contaminated with carcinogenic aflatoxins, secondary metabolites produced by Aspergillus flavus and A. parasiticus. Aflatoxin contamination of peanuts in the field can be reduced by 77–98% with biological control through the application of nontoxigenic strains of these species, which competitively exclude native aflatoxin-producing strains from developing peanuts. In this study, viable peanut seeds were artificially wounded and inoculated with field soil containing natural fungal populations that were supplemented with conidia of nontoxigenic A. flavus NRRL 21882 (niaD nitrate-nonutilizing mutant) and A. parasiticus NRRL 21369 (conidial color mutant). Increasing soil densities of applied nontoxigenic strains generally resulted in an increase in the incidence of seed colonization by applied nontoxigenic strains, a decrease in seed colonization by native A. flavus and A. parasiticus, and a decrease in aflatoxin concentration in seeds. Reduction of aflatoxins in peanut seeds depended on both the density and the aflatoxin-producing potential of native populations and on the fungal strain used for biological control. Wild-type strain A. flavus NRRL 21882 and its niaD mutant were equally effective in reducing aflatoxins in peanuts, indicating that nitrate-nonutilizing mutants, which are easily monitored in the field, can be used for evaluating the efficacy of biocontrol strains.     

Metabolic precursor regulation of aflatoxin formation in toxigenic and non-toxigenic strains ofAspergillus flavus

Non-aflatoxin-producing isolates ofAspergillus flavus from nature and isolates ofA. flavus that had lost their toxigenic trait following laboratory transfer were compared biochemically. After the addition of aflatoxin B₁ precursors sterigmatocystin or O-methylsterigmatocystin to whole cell cultures, the non-toxin producing isolates from nature remained non-toxigenic while toxigenicity was restored in the nontoxigenic laboratory strains. Results imply a lack of enzymes needed for biochemical conversions of precursors to aflatoxin B₁ in natural non-producers and suppression of these enzymes in the nonproducing laboratory strains.     

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.     

Amino acid supplementation reveals differential regulation of aflatoxin biosynthesis in <Emphasis Type="Italic">Aspergillus flavus</Emphasis> NRRL 3357 and <Emphasis Type="Italic">Aspergillus parasiticus</Emphasis> SRRC 143

Aflatoxins are toxic and carcinogenic secondary metabolites produced by the fungi Aspergillus flavus and Aspergillus parasiticus. To better understand the molecular mechanisms that regulate aflatoxin production, the biosynthesis of the toxin in A. flavus and A. parasticus grown in yeast extract sucrose media supplemented with 50 mM tryptophan (Trp) were examined. Aspergillus flavus grown in the presence of 50 mM tryptophan was found to have significantly reduced aflatoxin B₁ and B₂ biosynthesis, while A. parasiticus cultures had significantly increased B₁ and G₁ biosynthesis. Microarray analysis of RNA extracted from fungi grown under these conditions revealed 77 genes that are expressed significantly different between A. flavus and A. parasiticus, including the aflatoxin biosynthetic genes aflD (nor-1), aflE (norA), and aflO (omtB). It is clear that the regulatory mechanisms of aflatoxin biosynthesis in response to Trp in A. flavus and A. parasiticus are different. These candidate genes may serve as regulatory factors of aflatoxin biosynthesis.     

Behaviour of Aspergillus flavus in presence of Aspergillus niger during biosynthesis of aflatoxin B1

Aspergillus niger or Aspergillus tamarii when grown as mixed cultures with toxigenic A. flavus inhibits biosynthesis of aflatoxin by A. flavus, owing primarily to its ability to produce inhibitors of aflatoxin biosynthesis and to their ability to degrade aflatoxin. Gluconic acid partly prevents aflatoxin production. The other factors such as changes in pH of the medium and the effect on the growth of A. flavus have no role in imparting capabilities to these cultures to inhibit aflatoxin production by A. flavus.     

Mycobiota and molecular detection of Aspergillus flavus and A. parasiticus aflatoxin contamination of Strawberry (Fragaria ananassa Duch.) fruits

Strawberry fungi were isolated from fresh fruits and juice on the two types of media (Sabouraud dextrose agar, SDA and potato-dextrose agar, PDA) at 28 °C. Nineteen fungal species belong to 12 genera were isolated from fruits and juice on both isolation media. The most common fungal genera and species were Aspergillus flavus, A. niger, Mucor racemosus, Neurospora crassa, Penicillium chrysogenum, Rhizopus stolonifer and Trichoderma harzianum. Twenty A. flavus and A. parasitics isolates were assayed for their abilities to produce aflatoxins. The concentration of aflatoxins ranged between 25.8–75.2 and 23.6–71.1 ng/ml at 350 and 365 nm, respectively. Among A. flavus and A. parasiticus strains tested, aflatoxin B contributed 30–60% of total isolates. However, G type contributed 85–90%. The R_f values of B₁, B₂, G₁ and G₂ were 0.79, 0.61, 0.44 and 0.32, respectively. High-performance liquid chromatography analysis of extracts revealed the presence of aflatoxins with variable levels.     

Studies on the mycoflora associated with sugarcane factory waste and pollution of River Nile in upper Egypt

Sixty-nine species and four varieties that belong to 28 genera of terrestial fungi were recovered from polluted and non-polluted water and mud samples on glucose and cellulose-Czapek's agar at 28°C. The most common species from the two substrates on the two types of media were Aspergillus flavus, A. fumigatus, A.niger, Cladosporium cladosporioides, Fusarium oxysporum, Mycosphaerella tassiana and Penicillium chrysogenum. Twenty-six species belonging to 14 genera were isolated from polluted (26 species and 14 genera) and non-polluted (17species and 10 genera) mud samples on Sabouraud's dextrose agar at 28°C. The most prevalent species were Acremonium retiulum, Alternaria alternata, Aspergillus flavus, Aphanoascus fulvescens, A. terreus, Aphanoascus sp., Penicillium funiculosum and Stachybotrys chartarum.     

Evaluation of the mycological status of luncheon meat with special reference to aflatoxigenic moulds and aflatoxin residues

The luncheon meat samples analyzed, which were produced locally by the two main luncheon meat producing companies in Egypt were relatively highly contaminated either by moulds and yeasts in general, aflatoxigenic species and aflatoxin residues in particular. The most frequently encountered fungi from the samples were yeasts, Aspergillus niger, A. flavus, Penicillium chrysogenum, Rhizopus stolonifer, Mucor circinelloides. Less common were Cladosporium sphaerospermum, Alternaria alternata, Mycosphaerella tassiana, P. aurantiogriseum and P. oxalicum. The most important aflatoxigenic species, A. flavus, was isolated frequently. It was 10% of the total fungal isolates from both samples of the two companies. Seven luncheon meat samples out of 50 analyzed were positive for aflatoxin B₁ or B₁ and G₁, while all samples were negative for aflatoxins B₂, G₂, M₁ and M₂. Aflatoxin B₁ was detected only in 4 and 3 samples out of 25 analyzed from each of company A and B, respectively. The highest detectable level, 11.1 ppb, was recorded in a sample from company B and the least, 0.5 ppb, in a sample from company A. Aflatoxin G₁, at concentration of 3.2 ppb, was detected in only one sample of the aflatoxin B₁ – contaminated 3 samples of company B: this sample also had the highest level of aflatoxin B₁. Some luncheon meat samples had higher numbers of aflatoxigenic A. flavus than others, however these samples were negative for aflatoxins. The hazardous potential of such contamination will be discussed. This revised version was published online in June 2006 with corrections to the Cover Date.     

A strain of Aspergillus flavus from China shows potential as a biocontrol agent for aflatoxin contamination

Aflatoxins are toxic and carcinogenic secondary metabolites produced by Aspergillus flavus and Aspergillus parasiticus. Strains of A. flavus that are non-aflatoxigenic (i.e., incapable of secreting aflatoxins) have proven effective in controlling contamination by these aflatoxin producing species in the field. In the present study, a non-aflatoxigenic A. flavus strain, GD-3, was isolated from a peanut field in Guangdong Province, China. Polymerase chain reaction (PCR) analysis showed that 12 aflatoxin biosynthesis genes (aflT, pksA, nor-1, fas-2, fas-1, aflR, aflJ, adhA, estA, norA, ver-1 and verA) were deleted in GD-3. Co-inoculation with a toxigenic strain, GD-15, at the ratio of 1:10, 1:1 or 10:1 (GD-3:GD-15), showed that GD-3 was capable of reducing detectable aflatoxin levels on three different substrates. This reduction ranged from 33% to 99% and correlated with competitor ratio. These results demonstrated that GD-3 was successful at reducing aflatoxin contamination and showed promise as a potential agent of biocontrol for local farmers.     

Morphological changes of conidiophores and mycelium of some hyphomycetes

The formation of enlarged cells and other abnormalities of mycelium and conidiophores occurring inAspergillus flavus Link,Penicillium chrysogenum Thom,Penicillium notatum Westling,Penicillium variabile Sopp.,Scopularipsis brevicaulis Bainier,Fusarium moniliforme Sheld.,Botrytis cinera Pers. ex Fr.,Botrytis allii Rud.,Humicola brunnea var.africana Fass. andTritirachium heimii var.grisea Fass. under the influence of uranium acetate, radioactive sediment from P?íbram mines (uranium mines in P?íbram, Bohemia), fungicidal compounds (fungizon, fungistatin, nitrofungin), antibiotic nystatin and antifungal bacteriumBacillus subtilis Cohn emend. Prazmowski were investigated. Enlarged phialides or phialides with extended orifices and collarettes and occasionally a conversion of phialides to spherical enlarged cells were observed inAspergillus flavus grown on a Sabouraud agar supplemented with uranium acetate. InPenicillium chrysogenum some conidiophores were completely reduced to enlarged cells. Less abnormalities were observed on the Czapek-Dox agar. The radioactive sediment as a component of malt agar caused either complete reduction of conidiophores inAspergillus flavus to dichotomously branched hyphae or sometimes only an enlargement of phialides. The antifungal effect ofBacillus subtilis was demonstrated in all studied strains. Enlarged cells instead of whole conidiophores were formed under the influence of the bacterium. This effect was observed inBotrytis allii, Botrytis cinerea and to a lesser extent inPenicillium notatum, Aspergillus flavus andFusarium moniliforme. The enlarged cells could still germinate and can be considered as reduced conidiophores. The used fungicidal compounds and the antibiotic nystatin inhibited to a certain degree the studied strains, however, their application did not bring about any abnormalities of mycelium and conidiophores.     

Reduction of aflatoxin B1 in chicken feed by using Saccharomyces cerevisiae, Rhizopus oligosporus and their combination

Aflatoxin B1 is a toxigenic and carcinogenic compound produced by Aspergillus flavus and Aspergillus parasiticus. An approach to prevent aflatoxin contamination in feed was carried out by using Saccharomyces cerevisiae (Sc) and Rhizopus oligosporus (Ro). Aspergillus flavus was cultured together with Sc, Ro and their combination (ScRo) in chicken feed. The aflatoxin B1 content was observed at day 0, 5, 10 and 15. The result showed that aflatoxin B1 contaminations in feed were reduced by Sc, Ro and ScRo addition. The highest reduction of aflatoxin B1 content was shown at day 5 for all treatments with Sc, Ro and ScRo. The best activity of reducing aflatoxin B1 was shown by Ro. Although the ability of reducing aflatoxin B1 of Sc, Ro or ScRo was not significantly different, Sc or Ro gave the better result than ScRo and they are better used individually.     

Aflatoxin and aflatoxicosis

A wide range of fungi, amounting to fifty-six species belonging to twenty-two genera, have been recovered from animal feeds and foodstuffs.The most frequent fungi were Aspergillus niger, and A. flavus, followed by Mucor racemosus, Alternaria alternata, Rhizopus stolonifer, Penicillium corylophilum and P. notatum. Three genera were found to be of moderate occurrence, namely, Mucor, Rhizopus and Alternaria. The three following genera were of low occurrence: Cladosporium, Fusarium, and Neurospora.The fluorescence method of detecting aflatoxin-producing strains demonstrated that only one isolate of A. flavus possesses this property. Certain species of Penicillium and Aspergillus produced fluorescent substances (metabolites) similar in color to B and G aflatoxin. These substances were subsequently proved not to be aflatoxin by (TLC) chromatography.The animal and public health significance from such toxins was also discussed.     

Heat-stability of peroxidase in mycelia of some toxigenic and nontoxigenic Aspergilli and Penicillia

Seven-day-old mycelia from 19 cultures of Aspergillus and 12 cultures of Penicillium were heated to 50, 60, 65, 70, 75, 80, 85, 90 or 95 C for no more than min, and tested for residual peroxidase. The peroxidase from all aspergilli survived heating at 50 through 80 C. Peroxidase from toxigenic strains of Aspergillus flavus, Aspergillus parasiticus and Aspergillus ochraceus survived heating at 85 C and often at 90 C, whereas peroxidase from nontoxigenic strains of A. flavus was inactivated at 90 C and markedly reduced in activity at 85 C. Peroxidase from all penicillia survived heating at all temperatures through 80 C, although the activity of several cultures was reduced at 80 C. Peroxidase activity in mycelia of two strains of Penicillium cyclopium and one of Penicillium puberulum failed to survive heating at 85 C. One strain each of Penicillium roqueforti and Penicillium viridicatum exhibited some peroxidase activity after heating at 90 C, whereas the peroxidase of all other penicillia was inactivated at this temperature.     

Isolation of endophytic fungi from Cannabis sativa and study their antifungal potential

A systemic study of fungal endophytes associated with different plant parts of Cannabis sativa and their antifungal activity was investigated in the present study. A total of 281 plant segments, including 91 leaves, 93 stem and 97 petioles samples, were screened for the isolation of endophytic fungi. Totally, 212 (77.65%) segments were found colonised by different fungi. Highest colonisation frequency were observed in stem parts (84.94%), then leaves (82.41%) and lowest 59.79% in petiole. Total eight fungal genera belonging to 12 species were isolated. Aspergillus is recorded as the most frequently occurring genera with three species Aspergillus niger, Aspergillus flavus and Aspergillus nidulans followed by Penicillium with two species Penicillium chrysogenum and Penicillium citrinum, while Phoma, Rhizopus, Colletotrichum, Cladosporium and Curvularia with single species. The antifungal potential of A. niger and A. flavus – two most frequently isolated endophytic fungi – was evaluated against two common plant pathogen, Colletotrichum gloeosporioides and Curvularia lunata. Different plant and fungal extracts individually and in combinations showed variations in antifungal activity against both the pathogens. The primary results obtained on antifungal activity of endophytes show their possible role in plant defence mechanism but it is a preliminary approach and more extensive research is still required.     

Effect of phytate on aflatoxin formation by Aspergillus parasiticus and Aspergillus flavus in synthetic media

The effect of phytate on the production of aflatoxins by Aspergillus parasiticus and Aspergillus flavus grown on synthetic media was examined. In the absence of pH control (initial pH 4.5–6.5) for A. parasiticus, phytate (14.3 mM) caused a six-fold decrease in aflatoxins in the medium and a ten-fold decrease in those retained by the mycelia. When the initial pH of the medium was adjusted to 4.5 no effect on aflatoxin production was observed. With A. flavus or A. parasiticus grown on media with a higher initial pH value (6 to 7), the presence of phytate in the media caused an increase in aflatoxin production. These results are inconsistent with previous studies which indicated that phytate depresses aflatoxin production by rendering zinc, a necessary co-factor for aflatoxin biosynthesis, unavailable to the mold.     

β-Carotene inhibition of aflatoxin biosynthesis amongAspergillus flavus genotypes from Illinois corn

Thirty-nineAspergillus flavus genotypes (DNA fingerprinting) isolated from corn grown in a field near Kilbourne, Illinois were evaluated for their sensitivity to β-carotene (50 μg/ml) inhibition of aflatoxin B₁ biosynthesis. Inhibition of aflatoxin was greater than 90% for 28 of the genotypes and >70% for 38 of the 39 genotypes. FiveA. flavus strains (4 fingerprint groups) isolated from molded raw peanuts, NRRL 3239, NRRL 3357, NRRL 6514, NRRL 6515 and NRRL 13135, produced greater quantities of aflatoxin than all 39 genotypes isolated from corn, and were less sensitive to β-carotene inhibition.Aspergillus flavus NRRL 3357 is commonly used as inoculum in variety trials for aflatoxin resistance. Isolate identity and sensitivity to potential inhibitors in corn can be critical in assessing corn resistance to aflatoxin.