Inducers of Aflatoxin Biosynthesis from Colonized Maize Kernels Are Generated by an Amylase Activity from Aspergillus flavus

ABSTRACT Aflatoxin biosynthesis was induced by compounds in filtrates (EF) obtained from cultures consisting of ground maize kernels colonized by Aspergillus flavus. The inducing activity increased to a maximum at 4 days of incubation and then decreased. Amylase activity was detected in the EF, suggesting that the inducers are products of starch degradation (glucose, maltose, and maltotriose). Analysis of the enzyme by isoelectric focusing electrophoresis indicated a single alpha-amylase with a pI of 4.3. No maltase or amyloglucosidase was detected in the EF. High-pressure liquid chromatography analysis of the EF indicated the presence of glucose, maltose, and maltotriose in near-equal molar concentrations (about 15 mM). With a beta-glucuronidase (GUS) reporter assay consisting of A. flavus transformed with an aflatoxin gene promoter-GUS reporter gene fusion to monitor induction of aflatoxin biosynthesis, the minimum concentration of glucose, maltose, or maltotriose that induced measurable GUS activity was determined to be 1 mM. These results support the hypothesis that the best inducers of aflatoxin biosynthesis are carbon sources readily metabolized via glycolysis. They also suggest that alpha-amylase produced by A. flavus has a role in the induction of aflatoxin biosynthesis in infected maize kernels.     

Corn Seed Proteins Inhibitory to Aspergillus flavus and Aflatoxin Biosynthesis

ABSTRACT This study reports the presence of two fractions from corn seeds inhibitory to aflatoxin formation. Using a sensitive laboratory assay that can measure both inhibition of fungal growth and inhibition of aflatoxin biosynthesis, we examined aqueous extracts from seeds of Tex6, a corn inbred shown to be highly resistant to aflatoxin accumulation in field and laboratory evaluations. In these extracts, we identified two biologically active fractions. One inhibited growth of Aspergillus flavus and, thus, aflatoxin accumulation, and the other inhibited aflatoxin formation with little effect on fungal growth. The compounds responsible for these activities appear to be proteaceous, as they are water soluble, heat labile, and sensitive to proteinase K treatment. The compounds were partially purified by ultrafiltration and chromatography. The estimated molecular mass of the growth inhibitor is approximately 28 kDa, and that of the aflatoxin biosynthesis inhibitor appears to be greater than 100 kDa. Partially purified preparations of the growth inhibitor and aflatoxin biosynthesis inhibitor cause 50% inhibition at 26 and 75 mug of protein/ml, respectively. The presence of these compounds in Tex6 may explain its resistance to aflatoxin accumulation.     

A Chitinase from Tex6 Maize Kernels Inhibits Growth of Aspergillus flavus

ABSTRACT The maize inbred Tex6 has resistance to colonization and aflatoxin accumulation by Aspergillus flavus. A protein inhibitory to growth of A. flavus has been identified from aqueous extracts of mature Tex6 seeds. This study reports the purification of a chitinase associated with this inhibitory activity to electrophoretic homogeneity and the further characterization of its properties. The inhibitory protein, which has an M(r) of 29,000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, is an endochitinase that is also capable of exochitinase activity. The enzyme has an optimal pH of 5.5 and a temperature optimum of 45 degrees C. Chitinase activity in maize kernels peaked approximately 36 days after pollination. The Tex6 chitinase purified in this study is capable of inhibiting the growth of A. flavus by 50% at a concentration of 20 mug/ml. Our data indicate that chitinase activity in Tex6 kernels makes a major contribution to the antifungal activity in this maize genotype. Partial peptide sequence of the chitinase showed it to differ from previously reported chitinases.     

Identification of a Maize Kernel Pathogenesis-Related Protein and Evidence for Its Involvement in Resistance to Aspergillus flavus Infection and Aflatoxin Production

ABSTRACT Aflatoxins are carcinogens produced by Aspergillus flavus and A. parasiticus during infection of susceptible crops such as maize. Several aflatoxin-resistant maize genotypes have been identified and kernel proteins have been suggested to play an important role in resistance. In the present study, one protein (#717), which was expressed fivefold higher in three resistant lines compared with three susceptible ones, was identified using proteomics. This protein was sequenced and identified as a pathogenesis-related protein (PR-10) based on its sequence homology. To assess the involvement of this PR-10 protein (ZmPR-10) in host resistance of maize against fungal infection and aflatoxin production, the corresponding cDNA (pr-10) was cloned. It encodes a protein of 160 amino acids with a predicted molecular mass of 16.9 kDa and an iso-electric point of 5.38. The expression of pr-10 during kernel development increased fivefold between 7 and 22 days after pollination, and was induced upon A. flavus infection in the resistant but not in the susceptible genotype. The ZmPR-10 overexpressed in Escherichia coli exhibited a ribonucleolytic and antifungal activities. Leaf extracts of transgenic tobacco plants expressing maize pr-10 also demonstrated RNase activity and inhibited the growth of A. flavus. This evidence suggests that ZmPR-10 plays a role in kernel resistance by inhibiting fungal growth of A. flavus.     

Identification of Maize Kernel Endosperm Proteins Associated with Resistance to Aflatoxin Contamination by Aspergillus flavus

ABSTRACT Aflatoxins are carcinogens produced mainly by Aspergillus flavus during infection of susceptible crops such as maize (Zea mays). Previously, embryo proteins from maize genotypes resistant or susceptible to A. flavus infection were compared using proteomics, and resistance-associated proteins were identified. Here, we report the comparison of maize endosperm proteins from five resistant and five susceptible genotypes, and the identification of additional resistance-associated proteins using the same approach. Ten protein spots were upregulated twofold or higher in resistant lines compared with susceptible ones. Peptide sequencing of these proteins identified them as a globulin-2 protein, late embryogenesis abundant proteins (LEA3 and LEA14), a stress-related peroxiredoxin antioxidant (PER1), heat-shock proteins (HSP17.2), a cold-regulated protein (COR), and an antifungal trypsin-inhibitor protein (TI). The gene encoding one such upregulated protein, PER1, was cloned and overexpressed in Escherichia coli. The overexpressed PER1 protein demonstrated peroxidase activity in vitro. In addition, per1 expression was significantly higher in the resistant genotype Mp420 than in the susceptible genotype B73 during the late stage of kernel development, and was significantly induced upon A. flavus infection, suggesting that it may play an important role in enhancing kernel stress tolerance and aflatoxin resistance. The significance of other identified proteins to host resistance and stress tolerance also is discussed.     

Effect of Carotenoids on Aflatoxin B(1) Synthesis by Aspergillus flavus

ABSTRACT Carotenes and xanthophylls occurring in yellow corn and related terpenoids were tested for their effect on growth and aflatoxin B(1) production by Aspergillus flavus NRRL 3357, using the suspended disc culture method. Aflatoxin synthesis was inhibited at concentrations of beta-carotene, lutein, and zeaxanthin comparable to those found in the horny endosperm of mature corn. Usually growth was not significantly affected. Inhibition of aflatoxin biosynthesis was greater for compounds with an alpha-ionone-type ring (alpha-carotene, lutein, or alpha-ionone) compared with compounds with a beta-ionone ring. The presence of hydroxy groups on the rings tended to decrease inhibition, but did not override the effect of the ring type; lutein was similar to alpha-carotene and zeaxanthin was similar to beta-carotene in inhibition. A mutant accumulating norsolorinic acid (NA), A. parasiticus SRRC 162, incubated with alpha-carotene produced reduced levels of both NA and aflatoxin, indicating that inhibition occurred before NA. Additional A. flavus strains tested against 50 mug/ml of beta-carotene had 89 to 96% inhibition, which was significantly more sensitive than NRRL 3357. A. parasiticus strains were less sensitive and generally had similar or lower inhibition than NRRL 3357. The results indicate that the presence of carotenoids in endosperm may decrease the amount of aflatoxin produced by A. flavus.     

Aspergillus flavus and Aflatoxin Contamination of Leguminous Trees of the Sonoran Desert in Arizona

ABSTRACT Aspergillus spp. in section Flavi were frequently associated with desert tree legumes in uncultivated areas of the Sonoran Desert. Of 270 samples of debris and fruits of mesquite (Prosopis spp.), ironwood (Olneya tesota), acacia (Acacia spp.), and palo verde (Cercidium and Parkinsonia spp.), 87% were positive for A. flavus (S and L strains) and A. tamarii. A. flavus was the most common species (87%) among the 3,763 isolates examined. Mesquite pods were both the substrate from which A. flavus was recovered most frequently and the substrate from native habitats with the greatest aflatoxin content. In vitro, most desert legumes supported significant growth, reproduction, and aflatoxin production by A. flavus, with mesquite pods yielding 1 x 10(10) propagules/g and 5,000 mug/kg of aflatoxin B(1). Twenty percent of legume pods collected in the desert contained measurable quantities of aflatoxin, ranging from 1 to >2,500 mug/kg. Insect-damaged mesquite pods had significantly higher aflatoxin than intact pods. Legumes are apparently important reservoirs of aflatoxin-producing fungi and significant sources of aflatoxin contamination in the native Sonoran Desert habitats of Arizona.     

Advances in the Development of Host Resistance in Corn to Aflatoxin Contamination by Aspergillus flavus

ABSTRACT Aflatoxins are toxic, highly carcinogenic secondary metabolites of Aspergillus flavus and A. parasiticus, which when produced during fungal infection of a susceptible crop in the field or after harvest contaminate food and feed and threaten human and animal health. Although there are several management strategies that may reduce aflatoxin contamination of corn, the preeminent strategy for elimination of aflatoxin is to develop preharvest host resistance to aflatoxin accumulation. This strategy has gained even greater prominence due to recent discoveries of natural resistance in corn that can be exploited in plant-breeding strategies. The ability to identify resistant corn genotypes has been enhanced by the development of a laboratory kernel-screening assay and by a strain of A. flavus genetically engineered to produce beta-glucuronidase, an enzyme whose activity can be monitored to assess the degree of fungal infection in kernels. Investigations of resistant corn genotypes have associated kernel pericarp wax characteristics with resistance, identified kernel proteins associated with resistance to and inhibition of fungal growth or aflatoxin biosynthesis, and identified chromosome regions associated with resistance to Aspergillus ear rot and aflatoxin production. Such research advances could lead, in the near future, to commercially available, agronomically acceptable corn lines with multiple preharvest resistances to aflatoxin contamination.     

Resistance to Aspergillus flavus in Corn Kernels Is Associated with a 14-kDa Protein

ABSTRACT Corn genotypes resistant or susceptible to Aspergillus flavus were extracted for protein analysis using a pH 2.8 buffer. The profile of protein extracts revealed that a 14-kDa protein is present in relatively high concentration in kernels of seven resistant corn genotypes, but is absent or present only in low concentration in kernels of six susceptible ones. The N-terminal sequence of this 14-kDa protein showed 100% homology to a corn trypsin inhibitor. The 14-kDa protein purified from resistant varieties also demonstrated in vitro inhibition of both trypsin activity and the growth of A. flavus. This is the first demonstration of antifungal activity of a corn 14-kDa trypsin inhibitor protein. The expression of this protein among tested genotypes may be related to their difference in resistance to A. flavus infection and subsequent aflatoxin contamination.     

Interactions Between Fusarium verticillioides, Aspergillus flavus, and Insect Infestation in Four Maize Genotypes in Lowland Africa

ABSTRACT An experiment was designed to compare cycles of selection of four maize genotypes for ear- and grain-quality characteristics, interactions with Aspergillus flavus and Fusarium verticillioides infection, and insect ear infestation in two seasons. Mean infection levels by A. flavus and F. verticillioides were significantly higher in inoculated rows than in the controls. The F. verticillioides-inoculated rows had significantly more coleopteran beetles and lepidopteran borers per ear than the controls and A. flavus-inoculated rows. Genotypes and cycles of selection within genotype were not different with respect to number of insects or percent fungal incidence in the ear, but they were different for husk extension, field weight, 100-grain weight, and grain density. Inoculation with either fungus resulted in significantly higher percentage of floaters (i.e., loss of grain density) and lower grain weight than the controls. Aflatoxin (B1 and B2) in A. flavus-inoculated rows averaged 327 ppb in the first season and 589 ppb in the second (dryer) season. Fumonisin levels in F. verticillioides-inoculated rows did not differ between seasons, with an average of 6.2 ppm across seasons. In the noninoculated control rows, fumonisin was significantly higher in the first (5.3 ppm) than in the second (3.1 ppm) season. For all genotypes, husk extension and yield parameters decreased in the fungal-inoculated treatments. General ear-rot scoring was significantly correlated with incidence of F. verticillioides in kernels and grain-weight loss but not with A. flavus in the grain.     

Inhibition of aflatoxin production in Aspergillus flavus by pyrazolines and pyrazoles

A new series of 1,3,5-triarylpyrazolines, obtained from 3, 4-dichloroacetophenone, were readily dehydrogenated by tetrachloro-o-benzoquinone at room temperature to the pyrazoles. The spectra and fluorescence of the compounds are discussed. The pyrazolines and pyrazoles did not significantly affect the mycelial growth of 19 fungal isolates, but at low concentrations, they inhibited aflatoxin production by Aspergillus flavus.     

Biocontrol of Aspergillus flavus in groundnut using Trichoderma harzianum stain kd

Journal of Plant Diseases and Protection - Pre-harvest infection of groundnut (Arachis hypogea) during drought stress by strains of Aspergillus flavus and Aspergillus parasiticus is a major health...     

Field release of a non-toxigenic Aspergillus flavus L strain in central Benin

Journal of Plant Diseases and Protection - Non-toxigenic strains of Aspergillus flavus (Link) can be used as biological control agents to reduce the contamination of grains such as maize with...     

Comparison of soil and corn kernel Aspergillus flavus populations: evidence for niche specialization

Aspergillus flavus is considered a generalist-opportunistic pathogen, but studies are beginning to show that A. flavus populations have strains specific to various hosts. The research objective was to determine whether A. flavus soil populations consist of solely saprophytic strains and strains which can be facultatively parasitic on corn. A. flavus was isolated from both corn kernels and soil within 11 Louisiana fields. Sixteen vegetative compatibility groups (VCGs) were identified among 255 soil isolates. Only 6 of the 16 VCGs were identified in the 612 corn isolates and 88% of corn isolates were in two VCGs, whereas only 5% of soil isolates belonged to the same two VCGs. Isolates were characterized for aflatoxin B1 production and sclerotial size. A random subset of the isolates (99 from corn and 91 from soil) were further characterized for simple-sequence repeat (SSR) haplotype and mating type. SSR polymorphisms revealed 26 haplotypes in the corn isolates and 78 in the soil isolates, and only 1 haplotype was shared between soil and corn isolates. Corn and soil populations were highly significantly different for all variables. Differences between corn and soil populations indicate that some soil isolates are not found in corn and some isolates have become specialized to infect corn. Further understanding of A. flavus virulence is important for development of resistant hybrids and for better biological control against toxigenic A. flavus.     

Potential involvement of Aspergillus flavus laccases in peanut invasion at low water potential

Aspergillus flavus accumulates carcinogenic aflatoxins in peanuts, mainly in immature kernels during drought. Aspergillus flavus invasion induces accumulation of phytoalexins, mostly stilbenoids in peanut, as a plant defence mechanism. Because fungal laccases are often related to pathogenicity and can degrade stilbenoids, this study reports for the first time the expression of A. flavus laccases in the presence of kernels, hulls and low water potential in relation to the accumulation of phytoalexins in peanut kernels. Packed‐cell volume (PCV) of A. flavus biomass was significantly higher (P ≤ 0·01) in the presence of mature kernels, dead kernels, and mature and immature peanut hulls than the control. The presence of kernels and hulls lowered the level of expression of three A. flavus laccases by 4–6‐fold (P < 0·01), whereas 3% sucrose up‐regulated them by 35–304‐fold, and low water potential (?1·1 MPa) up‐regulated them by 85–248‐fold (P < 0·01). Phytoalexins that accumulated in peanut kernels in the presence of A. flavus and were quantified by HPLC‐DAD‐MS were primarily the stilbenoids: 3′‐isopentadienyl‐3,5,4′‐trihydroxystilbene (IPD), chiricanine‐A, arachidin‐2, arachidin‐3 and arahypin‐1. Apparent degradation of phytoalexins was observed when using a priori induction of phytoalexins in seeds in combination with a priori induction of laccases in A. flavus. The up‐regulation of laccase expression observed at ?1·1 MPa and at high sucrose concentration could be contributing to peanut invasion in immature kernels under drought conditions.     

Adult Plant Phenotype of the Rp1-DJ Compound Rust Resistance Gene in Maize

ABSTRACT The complex structure of the rp1 rust resistance locus of maize allows two or more resistance genes to be recombined together in coupling phase. The phenotypic effects of the Rp1-DJ compound gene, which carries both Rp1-D and Rp1-J, were analyzed. The Rp1-DJ compound gene was associated with a chlorotic spotting phenotype in some genetic backgrounds. At the seedling stage, lines carrying Rp1-DJ are fully susceptible to Puccinia sorghi biotype HI1, which is virulent on lines with the two genes singly. At later stages of growth, however, Rp1-DJ lines show partial resistance when compared with sibling lines not carrying the compound gene. The Rp1-DJ gene also confers partial resistance to P. polysora in adult plants.