Resistance to aflatoxin contamination in corn as influenced by relative humidity and kernel germination.

Kernels of corn population GT-MAS:gk, resistant to aflatoxin B1 production by Aspergillus flavus, and susceptible Pioneer hybrid 3154 were tested for aflatoxin when incubated under different relative humidities (RH). High aflatoxin levels were not detected in either genotype at RH < 91%. Resistance in GT-MAS:gk was consistent across all RH levels (91 to 100%) at which significant aflatoxin accumulation was detected. Aflatoxin levels in GT-MAS:gk averaged about 98% less than those in susceptible Pioneer 3154, which suggests that storage of this or other genotypes with similar resistance mechanisms may be possible under moisture conditions less exacting than are required with susceptible hybrids. Results for fungus growth and sporulation ratings on kernel surfaces were similar to those for aflatoxin levels. When kernels of both genotypes were preincubated 3 days at 100% RH prior to inoculation with A. flavus, germination percentages increased to very high levels compared to those of kernels that were not preincubated. In preincubated kernels aflatoxin levels remained consistently low in GT-MAS:gk but decreased markedly (61%) in Pioneer 3154. When eight susceptible hybrids were evaluated for aflatoxin accumulation in preincubated kernels, seven of these supported significantly lower toxin levels than kernels not subjected to preincubation. Average reduction across hybrids was 83%, and reductions within hybrids ranged from 68 to 96%. Preincubated kernels of one susceptible hybrid (Deltapine G-4666) supported aflatoxin levels comparable to those in resistant GT-MAS: gk. Data suggest that an inhibitor of aflatoxin biosynthesis may be induced during kernel germination. Possible mechanisms for embryo effects on resistance to aflatoxin accumulation are discussed.     

Resistance to aflatoxin accumulation in kernels of maize inbreds selected for ear rot resistance in West and Central Africa

Thirty-six inbred lines selected in West and Central Africa for moderate to high resistance to maize ear rot under conditions of severe natural infection were screened for resistance to aflatoxin contamination using the previously established kernel screening assay. Results showed that more than half the inbreds accumulated aflatoxins at levels as low as or lower than the resistant U.S. lines GT-MAS:gk or MI82. In 10 selected aflatoxin-resistant or aflatoxin-susceptible inbreds, Aspergillus flavus growth, which was quantified using an A. flavus transformant containing a GUS-beta-tubulin reporter gene construct, was, in general, positively related to aflatoxin accumulation. However, one aflatoxin-resistant inbred supported a relatively high level of fungal infection, whereas two susceptibles supported relatively low fungal infection. When kernels of the 10 tested lines were profiled for proteins using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, significant variations from protein profiles of U.S. lines were observed. Confirmation of resistance in promising African lines in field trials may significantly broaden the resistant germplasm base available for managing aflatoxin contamination through breeding approaches. Biochemical resistance markers different from those being identified and characterized in U.S. genotypes, such as ones inhibitory to aflatoxin biosynthesis rather than to fungal infection, may also be identified in African lines. These discoveries could significantly enhance the host resistance strategy of pyramiding different traits into agronomically useful maize germplasm to control aflatoxin contamination.     

Mycotoxins in cereal grain Part 14. Histochemical examination of fusarium-damaged wheat kernels

Wheat kernels with visible Fusarium-damage, naturally infected, have been examined with histochemical techniques to observe mycelium growth inside kernels and change in kernels cells. Kernels infected with F. culmorum were found to be damaged more extensively, comparatively to those infected with F. graminearum, F. avenaceum and F. nivale/(Microdochium nivale). Growth of Fusarium mycelium was most abundant between pericarp and aleurone layer and alongside of scutellum. Mycelium colonized also endosperm and embryo — both plumula and radicula.     

Mycotoxins in cereal grain. Part 14. Histochemical examination of Fusarium-damaged wheat kernels

Wheat kernels with visible Fusarium-damage, naturally infected, have been examined with histochemical techniques to observe mycelium growth inside kernels and change in kernels cells. Kernels infected with F. culmorum were found to be damaged more extensively, comparatively to those infected with F. graminearum, F. avenaceum and F. nivale/(Microdochium nivale). Growth of Fusarium mycelium was most abundant between pericarp and aleurone layer and alongside of scutellum. Mycelium colonized also endosperm and embryo--both plumula and radicula.     

Construction and preliminary evaluation of an Aspergillus flavus reporter gene construct as a potential tool for screening aflatoxin resistance

Effective preharvest strategies to eliminate aflatoxin accumulation in crops are not presently available. The molecular biology of aflatoxin biosynthesis has been extensively studied, and genetic and molecular tools such as reporter gene systems for the measurement of fungal growth have been developed. A reporter construct containing the Aspergillus flavus beta-tubulin gene promoter fused to Escherichia coli beta-glucuronidase (GUS) has been shown to be a reliable tool for the indirect measurement of fungal growth in maize kernels. Since cost-saving alternative methods for the direct measurement of aflatoxin levels are needed to facilitate more widespread field and laboratory screening of maize lines, a new reporter gene construct involving the promoter region of the omtA gene of the aflatoxin biosynthetic pathway was constructed and tested. Expression of GUS activity by this construct (omtA::GUS) was correlated with aflatoxin accumulation in culture. In the fungal transformant GAP26-1, which harbors this construct, aflatoxin production and GUS expression on sucrose-containing medium showed the same temporal pattern of toxin induction. Furthermore, GUS expression by GAP26-1 was shown to be associated with aflatoxin accumulation in maize kernels inoculated with this strain. Our results suggest that this and other reporter gene pathway promoter constructs may provide superior alternatives to direct aflatoxin quantification with respect to time, labor, and materials for the screening of maize lines for resistance to aflatoxin accumulation.     

Scanning electron microscopy of Fusarium damaged kernels of spring wheat

Kernels of five wheat cultivars (Triticum aestivum) of different bread-making quality were examined. Grown under field conditions, heads of wheat were inoculated in the flowering stage with an aqueous suspension of Fusarium culmorum conidia. Wheat heads were collected from the control and inoculated plots at full maturity. Control (non-inoculated) kernels without any symptoms of disease and Fusarium damaged kernels (FDK) were examined under scanning electron microscopy (SEM). Examination of the FDK fraction confirmed localisation of Fusarium hyphae on the surface and inside the tissues of kernels. Observations of the endosperm from Fusarium infected kernels revealed presence of fungal hyphae in the endosperm and some characteristic structural changes in many of its regions, such as partial or complete lack of the protein matrix, damage to large and small starch granules caused by fungal amylolytic enzymes, disappearance of small starch granules as the colonisation progressed, complete disappearance of the starchy endosperm under severe infection. Fungal colonisation of the endosperm and structural changes in its area were highly variable traits within the FDK fraction of a given cultivar.     

Comparison of constitutive and inducible maize kernel proteins of genotypes resistant or susceptible to aflatoxin production.

Maize genotypes resistant or susceptible to aflatoxin production or contamination were compared for differences in both constitutive and inducible proteins. Five additional constitutive proteins were found to be associated with resistance in over 8 of the 10 genotypes examined. Among these, the 58- and 46-kDa proteins were identified as globulin-1 and globulin-2, respectively. Differences in the ability to induce specific antifungal proteins, such as the higher synthesis of the 22-kDa zeamatin in resistant genotypes, were also observed between resistant and susceptible kernels incubated under germinating conditions (31 degrees C, 100% humidity). Both constitutive and inducible proteins appear to be necessary for kernel resistance. Embryo-killed kernels (unable to synthesize new proteins) supported the highest level of aflatoxins, whereas imbibed kernels (to hasten protein induction) supported the lowest among all treatments. This suggests that the synthesis of new proteins by the embryo plays an important role in conferring resistance. However, significantly lower levels of aflatoxin production in embryo-killed resistant kernels than in susceptible ones suggest that, in reality, high levels of constitutive antifungal proteins are indispensable to kernel resistance.     

Comparison of Aspergillus ear rot and aflatoxin contamination in grain of high-oil and normal-oil corn hybrids

High-oil corn (Zea mays L.) grain is a valuable component of feed for monogastric livestock. One method of increasing the concentration of oil in corn grain is the TopCross method. With TopCross, ears of a cytoplasmic male-sterile, normal-oil hybrid are pollinated by a male-fertile, high-oil synthetic hybrid. The concentration of oil in the resulting grain is increased because of xenia effects. Kernels of high-oil corn typically have a larger germ and a smaller endosperm than kernels of comparable normal hybrids. The growth of Aspergillus flavus Link:Fr within germ tissue has been reported to be more extensive than that on the whole corn kernel; therefore, the severity of Aspergillus ear rot could be more extensive and aflatoxin concentrations could be higher in high-oil grain produced by TopCross than in grain with a lower concentration of oil. The objective of this study was to compare Aspergillus ear rot severity levels and aflatoxin concentrations in the grains of hybrids crossed with high-oil or normal-oil pollinators. Fifteen hybrids were evaluated in 1998 and 1999 in Urbana, Ill. Primary ears were inoculated with A. flavus and evaluated for susceptibility to Aspergillus ear rot and aflatoxin production in grain. Concentrations of aflatoxin and oil in corn kernels were significantly higher for high-oil hybrids than for normal-oil hybrids; however, ear rot severity was unaffected by the type of pollinator. These results suggest that grain from high-oil hybrids is at greater risk for aflatoxin contamination during some growing seasons.     

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

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

No effect of soybean lipoxygenase on aflatoxin production in Aspergillus flavus-inoculated seeds

Soybean lines lacking lipoxygenase (LOX) activity were compared with soybean lines having LOX activity for the ability to support growth and aflatoxin B1 production by the fungal seed pathogen Aspergillus flavus. Whole seeds, broken seeds, and heat-treated (autoclaved) whole seeds were compared. Broken seeds, irrespective of LOX presence, supported excellent fungal growth and the highest aflatoxin levels. Autoclaved whole seeds, with or without LOX, produced good fungal growth and aflatoxin levels approaching those of broken seeds. Whole soybean seeds supported sparse fungal growth and relatively low aflatoxin levels. There was no significant difference in aflatoxin production between whole soybean seeds either with or without LOX, although there did seem to be differences among the cultivars tested. The heat treatment eliminated LOX activity (in LOX+ lines), yet aflatoxin levels did not change substantially from the broken seed treatment. Broken soybean seeds possessed LOX activity (in LOX+ lines) and yet yielded the highest aflatoxin levels. The presence of active LOX did not seem to play the determinant role in the susceptibility of soybean seeds to fungal pathogens. Seed coat integrity and seed viability seem to be more important characteristics in soybean seed resistance to aflatoxin contamination. Soybean seeds lacking LOX seem safe from the threat of increased seed pathogen susceptibility.     

Mycotoxins in cereal grain. Part III. Production of ochratoxin A in different varieties of wheat,rye and barley

Kernels of several varieties of wheat, rye and barley were found to have different resistance to fungi attack and ochratoxin A production, particularly in first step of fungus development on kernels. Zinc was stated to be a limiting factor of ochratoxin production. Viable sound kernels were very resistant against fungi. Dead (e.g. autoclaved) kernels were attacked by fungus very quickly. Varieties with stronger resistance to fungi invasion during storage were selected.     

Distribution of antifungal proteins in maize kernel tissues using immunochemistry

This study examined the distribution of two antifungal proteins, ribosome-inactivating protein (RIP) and zeamatin, in maize kernel tissues. Proteins were extracted from endosperm (including aleurone layer) and embryo tissues of imbibed maize kernels. Western blot analyses revealed that RIP-like protein was present at higher levels in endosperm than in embryo tissues, whereas zeamatin-like protein was more concentrated in embryo tissues than in endosperm tissues. However, there were three protein bands in the endosperm and two bands in the embryo that reacted to anti-RIP antibody in Western blot analyses. Tissue prints were conducted to localize the antifungal proteins. Imbibed kernels were cut longitudinally and transversely and blotted onto nitrocellulose membranes. Using antibodies against maize RIP and zeamatin, RIP was found primarily in the aleurone layer of the endosperm and glandular layer of scutellum, whereas zeamatin was located mainly in the kernel embryo. These results provide insight into the potential functions of these antifungal proteins, especially since the presence of RIP and zeamatin within maize kernels uniquely protects kernels from pathogens.     

Restriction fragment length polymorphism assessment of the heterogeneous nature of maize population GT-MAS:gk and field evaluation of resistance to aflatoxin production by Aspergillus flavus.

Aflatoxin, produced by Aspergillus flavus, is one of the most toxic and carcinogenic substances known and contaminates many agricultural commodities such as corn, peanuts, cottonseed, and tree nuts. The challenge to breeders/plant pathologists is to identify lines that have resistance to aflatoxin production. Maize population GT-MAS:gk has been identified and released as a germplasm with resistance to aflatoxin contamination. In the present study, we assessed genetic divergence in the GT-MAS:gk population using restriction fragment length polymorphism (RFLP) DNA markers to survey 11 selfed inbred lines and conducted field evaluations for the dissimilarities in aflatoxin production among these inbred lines in comparison with a sister population, GT-MAS:pw.nf. The 11 selfed inbred lines were assayed for DNA polymorphism using 113 RFLP markers in 10 linkage groups covering 1,518.2 centimorgans (cM; unit of gene or chromosome size). Considerable variation among the inbreds was detected with RFLP markers, of which 42 probe-enzyme combinations gave 102 polymorphic bands. Cluster analysis based on genetic similarities revealed associations and variations among the tested lines. Three polymorphic groups were distinguished by cluster analysis. Two years of field evaluation data showed that aflatoxin concentrations among the lines were significantly different in both years (P < 0.001). Maturity data were also different. Thus, this study demonstrates that the maize population GT-MAS:gk is heterogeneous and that individuals may be different in resistance to A. flavus infection and aflatoxin production. Therefore, the most resistant lines should be inbred to increase homogeneity, and resistance should be confirmed through progeny testing.     

Mycotoxins in cereal grain. Part X. Invasion of fungi mycelium into wheat and barley kernels

Histochemical examination of wheat kernels invaded by Aspergilli and Penicillia proved penetration of fungal mycelium into subaleurone and endosperm cells through aleurone layer cells. Walls of aleurone layer cells in invaded kernels were found to be thicker and split. Formation of spores in cleistothecia's in endosperm cells was found. The observation of kernels under a stereoscopic microscope was found to be a sensitive way to detect fungi invasion in cereal kernels.     

Influence of mechanical damage and repeated infestation of sorghum on its resistance to Sitophilus oryzae (L.) (Coleoptera: Curculionidae)

Abrading the pericarp rendered resistant sorghum cultivars as susceptible as abraded susceptible cultivars to attack by Sitophilus oryzae (L.). A previous exposure of selected sorghum cultivars to S. oryzae broke down pericarp resistance factor(s). Damage to sorghum kernels during harvesting and handling could therefore destroy resistance to rice weevil attack.     

Mould infection and aflatoxin contamination of the peanut kernels harvested from spring and fall crops as affected by artificial inoculation of the seeded kernels with Aspergillus flavus and Aspergillus niger

Spring and fall crops of peanut are grown each year in Taiwan. Mould infection and aflatoxin contamination of crops as affected by artificial inoculation of the seeded kernels with conidia of Aspergillus flavus, A niger and a combination of A flavus and A niger (inocula > 10⁵ CFU kernel⁻¹) were determined. Three cultivars, ie Tainan 9, Tainan 11 and Tainan 12, were consecutively grown for fall 1996, spring 1997, fall 1997 and spring 1998 crops with green vegetable pea for rotation. In crops from uninoculated kernels (control), percentages of germination and harvested plants were higher in spring crops than in fall crops. Inoculation with A niger alone or with A flavus resulted in various levels of seed and seedling mortality and lower yields of peanut pods than yields of the other inoculation treatments. When harvested and sized (US No 1), kernels were subjected to examination for mould colonisation and analysis of aflatoxin content. Fairly low percentages were colonised and aflatoxin contents were low or non-detectable. Colonisation and aflatoxin content were independent of artificial inoculation. Average aflatoxin contents in the kernels harvested from all cultivars and crops ranged from 0 to 6.1 µg kg⁻¹. However, the highest levels of aflatoxin content among samples of the four crops were 4.0, 18.2, 9.6 and 36.7 µg kg⁻¹, respectively. © 1999 Society of Chemical Industry     

Correlation and classification of single kernel fluorescence hyperspectral data with aflatoxin concentration in corn kernels inoculated with Aspergillus flavus spores

The objective of this study was to examine the relationship between fluorescence emissions of corn kernels inoculated with Aspergillus flavus and aflatoxin contamination levels within the kernels. Aflatoxin contamination in corn has been a long-standing problem plaguing the grain industry with potentially devastating consequences to corn growers. In this study, aflatoxin-contaminated corn kernels were produced through artificial inoculation of corn ears in the field with toxigenic A. flavus spores. The kernel fluorescence emission data were taken with a fluorescence hyperspectral imaging system when corn kernels were excited with ultraviolet light. Raw fluorescence image data were preprocessed and regions of interest in each image were created for all kernels. The regions of interest were used to extract spectral signatures and statistical information. The aflatoxin contamination level of single corn kernels was then chemically measured using affinity column chromatography. A fluorescence peak shift phenomenon was noted among different groups of kernels with different aflatoxin contamination levels. The fluorescence peak shift was found to move more toward the longer wavelength in the blue region for the highly contaminated kernels and toward the shorter wavelengths for the clean kernels. Highly contaminated kernels were also found to have a lower fluorescence peak magnitude compared with the less contaminated kernels. It was also noted that a general negative correlation exists between measured aflatoxin and the fluorescence image bands in the blue and green regions. The correlation coefficients of determination, r ², was 0.72 for the multiple linear regression model. The multivariate analysis of variance found that the fluorescence means of four aflatoxin groups, <1, 1–20, 20–100, and ≥100 ng g^?1 (parts per billion), were significantly different from each other at the 0.01 level of alpha. Classification accuracy under a two-class schema ranged from 0.84 to 0.91 when a threshold of either 20 or 100 ng g^?1 was used. Overall, the results indicate that fluorescence hyperspectral imaging may be applicable in estimating aflatoxin content in individual corn kernels.     

Assessing Purdue Improved Crop Storage (PICS) bags to mitigate fungal growth and aflatoxin contamination

Storing maize in regions of the world without sufficient drying and storage capacity is challenging due to the potential risk of aflatoxin contamination produced by Aspergillus flavus. This study sought to determine if storage of maize in Purdue Improved Crop Storage (PICS) bags prevents mold growth and aflatoxin accumulation. PICS bags are a three-layer, hermitic bag-system that forms a barrier against the influx of oxygen and the escape of carbon dioxide. Maize conditioned at 12, 15, 18, and 21% grain moisture was inoculated with 50 g of maize kernels infected with fluorescent-marked strain of A. flavus. The grain was stored in either PICS or woven bags at 26 °C, and percent oxygen/carbon dioxide levels, fungal growth, aflatoxin, moisture content, and kernel germination were assessed after 1 and 2 months incubation. Maize stored in woven bags was found to equilibrate with the ambient moisture environment over both storage periods, while PICS bags retained their original moisture levels. Aspergillus flavus growth and aflatoxin accumulation were not observed in maize stored in any PICS bags. No aflatoxin B1 was detected in woven bags containing low-moisture maize (12 and 15%), but detectable levels of aflatoxin were observed in high moisture maize (18 and 21%). The percentage of oxygen and carbon dioxide within PICS bags were dependent on initial grain moisture. Higher carbon dioxide levels were observed in the bags stored for 1 month than for 2 months. High initial moisture and carbon dioxide levels correlated with low kernel germination, with the 18 and 21% treatment groups having no seeds germinate. The results of the study demonstrate that storage of maize in PICS bags is a viable management tool for preventing aflatoxin accumulation in storage.     

Efficacy of ozone as a fungicidal and detoxifying agent of aflatoxins in peanuts

BACKGROUND: Peanut contamination by fungi is a concern of processors and consumers owing to the association of these micro‐organisms with quality deterioration and aflatoxin production. In this study the fungicidal and detoxifying effects of ozone on aflatoxins in peanuts was investigated. Peanut kernels were ozonated at concentrations of 13 and 21 mg L^?1 for periods of 0, 24, 48, 72 and 96 h. RESULTS: Ozone was effective in controlling total fungi and potentially aflatoxigenic species in peanuts, with a reduction in colony‐forming units per gram greater than 3 log cycles at the concentration of 21 mg L^?1 after 96 h of exposure. A reduction in the percentage of peanuts with internal fungal populations was also observed, particularly after exposure to ozone at 21 mg L^?1. A reduction in the concentrations of total aflatoxins and aflatoxin B1 of approximately 30 and 25% respectively was observed for kernels exposed to ozone at 21 mg L^?1 for 96 h. CONCLUSION: It was concluded that ozone is an important alternative for peanut detoxification because it is effective in controlling potentially aflatoxigenic fungi and also acts in the reduction of aflatoxin levels in kernels. Copyright © 2011 Society of Chemical Industry     

Aspergillus flavus infection and aflatoxin production in mixtures of high-moisture and dry maize

High-moisture (26·6–27·9% m.c.) and dry (9·8% m.c.) fractions of white and yellow maize were examined for fungal development and aflatoxin production during an 8-week incubation at 25°C. Treatment procedures included blending of either high-moisture white with dry yellow or high-moisture yellow with dry white maize fractions (average moisture in blend, 14%) and inoculation of some test maizes with A. flavus spores. At sampling time white and yellow components of maize blends were manually separated and all of the maize samples were analyzed for levels of moisture, fungal infection and aflatoxin. Moisture levels in maize blends equilibrated rapidly during the initial 2–4 days of incubation; neither dry yellow nor dry white exceeded 13% moisture during the trial period. Only a limited incidence of A. flavus was observed on uninoculated maize. but in samples treated with A. flavus spores a high infection rate developed; from 58 to 98% of the kernels in dry fractions of inoculated blends were infected with A. flavus during the trial. Aflatoxin was detected in high-moisture maize and in both high-moisture and dry fractions of inoculated maize blends. Up to 500 μg aflatoxin B₁/kg of corn was found after the 8-week incubation in a dry fraction of inoculated maize blends.