Amy1, the alpha-Amylase Gene of Aspergillus flavus: Involvement in Aflatoxin Biosynthesis in Maize Kernels

ABSTRACT Aspergillus flavus is the causal agent of an ear and kernel rot in maize. In this study, we characterized an alpha-amylase-deficient mutant and assessed its ability to infect and produce aflatoxin in wounded maize kernels. The alpha-amylase gene Amy1 was isolated from A. flavus, and its DNA sequence was determined to be nearly identical to Amy3 of A. oryzae. When Amy1 was disrupted in an aflatoxigenic strain of A. flavus, the mutant failed to produce extracellular alpha-amylase and grew 45% the rate of the wild-type strain on starch medium. The mutant produced aflatoxin in medium containing glucose but not in a medium containing starch. The alpha-amylase-deficient mutant produced aflatoxin in maize kernels with wounded embryos and occasionally produced aflatoxin only in embryos of kernels with wounded endosperm. The mutant strain failed to produce aflatoxin when inoculated onto degermed kernels. In contrast, the wild-type strain produced aflatoxin in both the endosperm and embryo. These results suggest that alpha-amylase facilitates aflatoxin production and growth of A. flavus from a wound in the endosperm to the embryo. A 14-kDa trypsin inhibitor associated with resistance to A. flavus and aflatoxin in maize also inhibited the alpha-amylase from A. flavus, indicating that it is a bifunctional inhibitor. The inhibitor may have a role in resistance, limiting the growth of the fungus in the endosperm tissue by inhibiting the degradation of starch.     

Identification of a maize kernel stress-related protein and its effect on aflatoxin accumulation

ABSTRACT Aflatoxins are carcinogens produced mainly by Aspergillus flavus during infection of susceptible crops such as maize. Through proteomic comparisons of maize kernel embryo proteins of resistant and susceptible genotypes, several protein spots previously were found to be unique or upregulated in resistant embryos. In the present study, one of these protein spots was sequenced and identified as glyoxalase I (GLX-I; EC 4.4.1.5). The full-length cDNA of the glyoxalase I gene (glx-I) was cloned. GLX-I constitutive activity was found to be significantly higher in the resistant maize lines compared with susceptible ones. After kernel infection by A. flavus, GLX-I activity remained lower in susceptible genotypes than in resistant genotypes. However, fungal infection significantly increased methylglyoxal (MG) levels in two of three susceptible genotypes. Further, MG was found to induce aflatoxin production in A. flavus culture at a concentration as low as 5.0 muM. The mode of action of MG may be to stimulate the expression of aflR, an aflatoxin biosynthesis regulatory gene, which was found to be significantly upregulated in the presence of 5 to 20 muM MG. These data suggest that GLX-I may play an important role in controlling MG levels inside kernels, thereby contributing to the lower levels of aflatoxins found in resistant maize genotypes.     

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.     

A Corn Trypsin Inhibitor with Antifungal Activity Inhibits Aspergillus flavus alpha-Amylase

ABSTRACT In this study, we found that the inhibition of fungal growth in potato dextrose broth (PDB) medium by the 14-kDa corn trypsin inhibitor (TI) protein, previously found to be associated with host resistance to aflatoxin production and active against various fungi, was relieved when exogenous alpha-amylase was added along with TI. No inhibitory effect of TI on fungal growth was observed when Aspergillus flavus was grown on a medium containing either 5% glucose or 1% gelatin as a carbon source. Further investigation found that TI not only inhibited fungal production of extracellular alpha-amylase when A. flavus was grown in PDB medium containing TI at 100 mug ml(-1) but also reduced the enzymatic activity of A. flavus alpha-amylase by 27%. At a higher concentration, however, TI stimulated the production of alpha-amylase. The effect of TI on the production of amyloglucosidase, another enzyme involved in starch metabolism by the fungus, was quite different. It stimulated the production of this enzyme during the first 10 h at all concentrations studied. These studies suggest that the resistance of certain corn genotypes to A. flavus infection may be partially due to the ability of TI to reduce the production of extracellular fungal alpha-amylase and its activity, thereby limiting the availability of simple sugars for fungal growth. However, further investigation of the relationship between TI levels and fungal alpha-amylase expression in vivo is needed.     

Germination induces accumulation of specific proteins and antifungal activities in corn kernels

ABSTRACT This study examined protein induction and accumulation during imbibition and germination of corn kernels, as well as antifungal activities of extracts from germinating kernels against Aspergillus flavus and Fusarium moniliforme. Genotypes studied included GT-MAS:gk and Mp420, which are resistant to A. flavus infection and aflatoxin accumulation, and Pioneer 3154 and Deltapine G-4666, which are susceptible to A. flavus infection and aflatoxin accumulation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis resolved five protein bands that were present at higher concentrations in germinated kernels than in nongerminated kernels. Western blot analyses revealed that one of these proteins reacted with the 22-kDa zeamatin antiserum, and a zeamatin-like protein accumulated to a higher concentration in germinated kernels. Two protein bands from dry kernels that reacted with ribosome-inactivating protein (RIP) antiserum were identified as the 32-kDa proRIP-like form and an 18-kDa peptide of the two peptides that form active RIP. However, in germinated kernels, two protein bands that reacted with RIP antiserum were identified as two RIP-like peptides with a molecular mass of approximately 18 and 9 kDa. Purified RIP and zeamatin from corn inhibited growth of A. flavus. Bioassays of germinated kernel extracts from all four genotypes exhibited antifungal activity against A. flavus and F. moniliforme, with extracts from the susceptible genotypes showing greater inhibition zones. This study provides evidence of protein induction in corn kernels during imbibition or the early stages of germination, and the induced proteins may be related to our previous findings of germination-associated resistance in the corn kernel, especially in the susceptible kernels.     

Tissue-specific components of resistance to Aspergillus ear rot of maize

Aspergillus flavus and other Aspergillus spp. infect maize and produce aflatoxins. An important control measure is the use of resistant maize hybrids. There are several reports of maize lines that are resistant to aflatoxin accumulation but the mechanisms of resistance remain unknown. To gain a better understanding of resistance, we dissected the phenotype into 10 components: 4 pertaining to the response of silk, 4 pertaining to the response of developing kernels, and 2 pertaining to the response of mature kernels to inoculation with A. flavus. In order to challenge different tissues and to evaluate multiple components of resistance, various inoculation methods were used in experiments in vitro and under field conditions on a panel of diverse maize inbred lines over 3 years. As is typical for this trait, significant genotype-environment interactions were found for all the components of resistance studied. There was, however, significant variation in maize germplasm for susceptibility to silk and kernel colonization by A. flavus as measured in field assays. Resistance to silk colonization has not previously been reported. A significant correlation of resistance to aflatoxin accumulation with flowering time and kernel composition traits (fiber, ash, carbohydrate, and seed weight) was detected. In addition, correlation analyses with data available in the literature indicated that lines that flower later in the season tend to be more resistant. We were not able to demonstrate that components identified in vitro were associated with reduced aflatoxin accumulation in the field.     

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.     

Infection and colonization of peanut pods by Aspergillus parasiticus and the expression of the aflatoxin biosynthetic gene,nor-1, in infection hyphae

The aflatoxigenic fungi, Aspergillus flavus and A. parasiticus infect a wide variety of crops, all of which produce oil-rich seed. A histological study of the host–pathogen interaction between peanut,Arachis hyphogea , and A. parasiticus was performed in a system where peanuts remained attached to the plant and were inoculated without wounding. For infection studies, a genetically-tagged strain of A. parasiticus, G5, was engineered to harbor the β-glucuronidase (GUS) reporter gene under control of the nor-1 promoter from the aflatoxin biosynthetic pathway. There was a similar temporal pattern of aflatoxin B1 production and appearance of GUS activity in cultures ofA. parasiticus G5. This strain was used to follow infection and aflatoxin production during colonization of undamaged, drought-stressed peanuts. The fungus colonized all tissues of the peanut pod and appeared to gain ingress through the corky layer of the pericarp. Both intra- and inter-cellular colonization were observed. Fungal colonization of the cotyledons resulted in visible depletion of storage bodies within cells. Two morphologically distinct types of hyphae, wider hyphae and narrower hyphae, were seen throughout the pod tissues. Statistical analysis revealed that the narrower hyphae were significantly more likely to produce GUS activity than wider ones. GUS activity was found in hyphae infecting the pericarp, embryo and cotyledons indicating expression of aflatoxin biosynthetic genes in these tissues. Interestingly, GUS activity was not observed in the hyphae colonizing the testa.     

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.     

Influence of Bacillus spp. isolated from maize agroecosystem on growth and aflatoxin B(1) production by Aspergillus section Flavi

A total of 59 bacteria of the Bacillus genus were isolated from different components of a maize agroecosystem and their antifungal activity against Aspergillus section Flavi was evaluated. Thirty-three and 46% of these bacteria were able to inhibit Aspergillus flavus Link and A. parasiticus Speare respectively at water activity (a(w)) 0.982; however, when a(w) was 0.955, these percentages were decreased and only three isolates were able to inhibit Aspergillus section Flavi. The majority of bacilli acted as contact antagonists, while a small number of isolates were able to form inhibition zones. In maize meal extract agar, Aspergillus section Flavi growth rate and aflatoxin B(1) (AFB(1)) production were significantly reduced when these strains were paired at a(w) 0.982 with bacilli at all inoculum levels studied. However, two bacilli isolated were able to reduce growth rate and aflatoxin production when a(w) was 0.955. Lag phase increase followed the same general pattern as growth rate reduction. When Aspergillus section Flavi was grown in sterile maize in the presence of three Bacillus strains at a(w) 0.982, the reduction in count (colony-forming units (cfu) g(-1) maize) was less than 30%, except when Aspergillus section Flavi grew with Bacillus amyloliquefaciens UNRCLR. However, levels of detectable AFB(1) were significantly reduced in these interactions at a(w) 0.982.     

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.     

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.     

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.     

储存玉米中黄曲霉毒素主要产生菌的检测及污染预防研究

通过对储存玉米霉变初期的感官症状观察、分离菌的PCR检测及在不同环境条件下的生长预测模型建立,探讨了识别、预防储存玉米发生黄曲霉毒素及其主要产生菌污染的实用方法。结果表明:籽粒色泽及致密性改变、表面有潮湿感、粮堆内局部发热等症状的出现可表征储存玉米有可能发生真菌污染。以毒素合成相关的全局性调控因子veA基因为靶标,对污染玉米样品分离菌进行PCR检测,扩增出约1.9kb的条带,与预期大小相符,证明污染菌是黄曲霉或寄生曲霉。污染曲霉在不同玉米水分活度和环境温度下的生长数据,经Baranyi函数拟合、估测其最大生长速度,并建立了生长速度随玉米水分活度和环境温度变化的多项式回归模型;模型显示玉米水分活度和环境温度对污染曲霉的生长影响具有协同性;要确保储存玉米安全,储存参数的限值选择应远离适合污染菌生长的区域。本研究为储存玉米安全管理决策、玉米水分活度和环境温度限值的选择及调控提供支持,利于降低储存玉米的黄曲霉毒素及其主要产生曲霉(黄曲霉或寄生曲霉)的污染风险。     

Control of Aspergillus growth and aflatoxin production using natural maize phytochemicals under different conditions of water activity

The effects of the natural phytochemicals trans-cinnamic acid (CA) and ferulic acid (FA) alone at concentrations of 1-25 mM and in 16 combinations (M: mixtures) on growth and aflatoxin B(1) production by Aspergillus flavus Link and A. parasiticus Speare were evaluated. Studies on growth rate and aflatoxin B(1) production were carried out in vitro in relation to a water activity a(w) of 0.999, 0.971, 0.955 and 0.937. Overall, CA at concentrations of 10 and 20 mM and FA-CA mixtures M3 (20 + 5 mM respectively), M8 (25 + 5 mM), M9 (1 + 10 mM), M10 (10 + 10 mM), M11 (20 + 10 mM), M12 (25 + 10 mM), M13 (1 + 20 mM), M14 (10 + 20 mM), M15 (20 + 20 mM) and M16 (25 + 20 mM) were the treatments most effective at inhibiting growth of the four species assayed. All strains were much more sensitive to all natural phytochemicals tested on growth rate at a(w) = 0.937. CA and the FA-CA mixtures M1 (1 + 1 mM respectively), M4 (25 + 1 mM), M5 (1 + 5 mM), M6 (10 + 1 mM), M7 (20 + 1 mM), M8 (25 + 5 mM), M9 (1 + 10 mM), M10 (10 + 10 mM), M11 (20 + 10 mM), M12 (25 + 10 mM), M13 (1 + 20 mM), M14 (10 + 20 mM), M15 (20 + 20 mM) and M16 (25 + 20 mM) completely inhibited aflatoxin B(1) production by all strains at a(w) = 0.999, 0.971, 0.955 and 0.937. Decreased aflatoxin B(1) levels in comparison with the control were observed with FA at 1, 10, 20 and 25 mM with the strains RCM89, RCM108 and RCM38 at a(w) = 0.971, 0.955 and 0.999 respectively. The data show that CA and FA can be considered as effective fungitoxicants for A. flavus and A. parasiticus in in vitro assay. The information obtained is part of an ongoing study to determine their application at the storage level.     

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.     

Relationships among resistances to fusarium and Aspergillus ear rots and contamination by fumonisin and aflatoxin in maize

ABSTRACT Fusarium verticillioides, F. proliferatum, and Aspergillus flavus cause ear rots of maize and contaminate the grain with mycotoxins (fumonisin or aflatoxin). The objective of this study was to investigate the relationships between resistance to Fusarium and Aspergillus ear rots and fumonisin and aflatoxin contamination. Based on a previous study of 143 recombinant inbred lines from the cross NC300 x B104, 24 lines with the highest and 24 lines with the lowest mean fumonisin concentration were selected for further evaluation. Paired plots of each line were inoculated with F. verticillioides and F. proliferatum or with A. flavus in replicated trials in 2004 and 2005 in Clayton, NC, and College Station, TX. The low-fumonisin group had significantly lower levels of fumonisin, aflatoxin, and Fusarium and Aspergillus ear rots. Across year-location environments, all four traits were significantly correlated; the genotypic correlation (r(G)) ranged from r(G) = 0.88 (aflatoxin and Aspergillus ear rot) to r(G) = 0.99 (Fusarium and Aspergillus ear rots). Quantitative trait loci (QTLs) were identified and their effects estimated. Two QTLs affected both toxin concentrations, one QTL affected both ear rots, and one QTL affected Aspergillus and Fusarium rots and fumonisin. These results suggest that at least some of the genes involved in resistance to ear rots and mycotoxin contamination are identical or genetically linked.