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1.
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.  相似文献   

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3.
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.  相似文献   

4.
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.  相似文献   

5.
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.  相似文献   

6.
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.  相似文献   

7.
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.  相似文献   

8.
ABSTRACT Russin, J. S., Guo, B. Z., Tubajika, K. M., Brown, R. L., Cleveland, T. E., and Widstrom, N. W. 1997. Comparison of kernel wax from corn genotypes resistant or susceptible to Aspergillus flavus. Phytopathology 87: 529-533.Kernels of corn genotype GT-MAS: gk are resistant to Aspergillus flavus. Earlier studies showed that this resistance is due in part to kernel pericarp wax. Experiments were conducted to compare wax from GTMAS: gk kernels with that from kernels of several susceptible commercial hybrids. GT-MAS: gk had more pericarp wax than did the susceptible hybrids. Scanning electron microscopy revealed that GT-MAS: gk kernels appeared rough and showed abundant wax deposits on kernel surfaces. Susceptible kernels appeared much more smooth and lacked the abundant surface deposits observed in GT-MAS: gk. In vitro bioassays showed that kernel wax from GT-MAS: gk reduced A. flavus colony diameter by 35%. Colony diameters on a medium amended with wax from susceptible kernels did not differ from those of controls. Thin-layer chromatography and analyses of chromatograms using NIH Image software showed a distinctive composition for GT-MAS: gk kernel wax. Chromatograms of wax from GT-MAS: gk contained a peak unique to this genotype, but also lacked a peak common to all susceptible hybrids. This is the first report of specific kernel factors involved in resistance to A. flavus in corn.  相似文献   

9.
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.  相似文献   

10.
A Chitinase from Tex6 Maize Kernels Inhibits Growth of Aspergillus flavus   总被引:2,自引:0,他引:2  
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.  相似文献   

11.
Evaluating commercial maize hybrids for resistance to gibberella ear rot   总被引:2,自引:0,他引:2  
An integral component of breeding maize for resistance to Fusarium graminearum ear rot is the identification of resistant genotypes. Since natural infection is not consistent from year to year, maize researchers must use manual techniques to inoculate the plant material with fungal spores. Information is presented here on site resistance of commercial maize hybrids to F. graminearum over three years and at two locations. Additionally, results of an investigation on the two predominant techniques of inoculating maize, the silk channel and kernel inoculation methods, are reported. Of 61 commercial hybrids tested, only two were ranked as moderately resistant to the fungus by both inoculation methods. These two hybrids also had a stable response to the F. graminearum infection across seven environments when the silk channel inoculation method was used. The majority of the hybrids were ranked as either susceptible or highly susceptible and less than 10% of the hybrids had a stable response to fungal infection. In the investigation of methodology, it was concluded that silk browning would be the least laborious way to identify the ideal time to complete silk channel inoculations. It was found that kernel inoculations using the pin inoculation method should take place between 11 and 15 days after 50% silking to achieve proper hybrid discrimination. Mist irrigation increased mold severity ratings and resulted in greater discrimination between hybrids with varying levels of resistance to F. graminearum infection.  相似文献   

12.
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.  相似文献   

13.
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.  相似文献   

14.
Almonds can be contaminated with aflatoxins, produced mainly by Aspergillus flavus and A. parasiticus. Infection can be facilitated by insect injuries during hull split, which begins four to six weeks before harvest. Within this period, it is unknown which kernel stages are most susceptible to aflatoxin contamination. Developing almonds of the Nonpareil cultivar were inoculated weekly with a spore suspension of A. flavus or A. parasiticus for five weeks after hull split in 2013. The almonds were infested with eggs of the lepidopteron navel orangeworm (NOW) (Amyelois transitella) before each spore inoculation. Aflatoxin levels were quantified at harvest using HPLC. Aflatoxin contamination was consistently higher in NOW-damaged kernels, although aflatoxins were also detected in undamaged kernels at each inoculation date. Insect injury is not required for kernel infection but it is a key risk factor for high aflatoxin contamination. Laboratory inoculations were also performed on Nonpareil almond kernels collected during the summers of 2013 and 2015. Aflatoxin levels were significantly lower on dried almonds but the ability to produce aflatoxins was restored when almonds were incubated with high humidity or when the Aspergillus species were inoculated on almond meal agar amended with ground kernels. Therefore, aflatoxins can accumulate in kernels with low aw, should sufficient moisture favors aflatoxin production. In our field experiment, the orchard micro-climate had sufficient humidity to enable aflatoxin production in both damaged and undamaged dried kernels.  相似文献   

15.
Huang Z  White DG  Payne GA 《Phytopathology》1997,87(6):622-627
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.  相似文献   

16.
Aspergillus flavus inoculation techniques were compared on aflatoxin-resistant and -susceptible corn hybrids for inducing aflatoxin contamination andA. flavus kernel infection. A dry carrier technique was comparable to the standard inoculation techniques (the side-needle and a spray technique) in differentiating between the resistant and the susceptible hybrids in the first year of the study. However, only hybrids inoculated with the side-needle technique had statistically different levels of aflatoxin andA. flavus kernel infection in the second year of the study. In a second study, a modified pinbar technique with inoculations near the tip or base of the ear was compared with the side-needle technique. When developing ears were inoculated near the base with the modified pinbar, adequate levels of aflatoxin were induced both years to distinguish between the resistant and susceptible corn hybrids. The modified pinbar technique has the potential of being a useful tool in evaluating corn germplasm for aflatoxin resistance. http://www.phytoparasitica.org posting May 4, 2007.  相似文献   

17.
ABSTRACT The relative importance of several infection pathways (silks, stalks, and seed) leading to kernel infection of maize hybrids by Fusarium moniliforme was investigated in field experiments in 1993 and 1994. Systemic movement of specific fungal strains within plants was detected by using vegetative compatibility as a marker. Transmission of F. moniliforme from inoculated seed to stalks and developing kernels was detected in two of three field experiments; the seed-inoculated strain was detected in kernels on approximately 10% of ears. The percentage of kernels infected with the seed-inoculated strain ranged from 0 to 70%, with a mean of 0 to 2.5% (0 to 8.3% of F. moniliforme-infected kernels). Other pathways to kernel infection were more effective than seed transmission and systemic infection. F. moniliforme strains inoculated into the crowns and stalks of plants were found throughout the stalks and in up to 95% of the kernels in individual plants. Infection through the silks was clearly the most effective pathway to kernel infection. This was the only inoculation method that significantly increased overall incidence of F. moniliforme infection in kernels; the silk-inoculated strain infected up to 100% of the kernels in individual ears, with a treatment mean as high as 83.7% of kernels. When plants were silk-inoculated, the percentage of kernels infected by other F. moniliforme strains from the seed or stalk was reduced, apparently due to competition among strains. This study provides evidence that systemic development of F. moniliforme from maize seed and stalk infections can contribute to kernel infection, but silk infection is a more important pathway for this fungus to reach the kernels.  相似文献   

18.
ABSTRACT Infection of peanut (Arachis hypogaea) seed by Aspergillus flavus and A. parasiticus is a serious problem that can result in aflatoxin contamination in the seed. Breeding resistant cultivars would be an effective approach to reduce aflatoxin accumulation. The objective of this study was to investigate the expression of the pathogenesis-related (PR) protein beta-1,3-glucanase and the isoform patterns in peanut seed inoculated with A. flavus. Peanut genotypes GT-YY9 and GT-YY20 (both resistant to A. flavus infection) and Georgia Green and A100 (both susceptible to A. flavus infection) were used in this study. The activities of beta-1,3-glucanase were similar in the uninfected seed of all genotypes, but increased significantly in the resistant genotypes after inoculation in comparison with the susceptible genotypes. An in-gel (native polyacrylamide gel electrophoresis [PAGE]) enzymatic activity assay of beta-1,3-glucanase revealed that there were more protein bands corresponding to beta-1,3-glucanase isoforms in the infected seed of resistant genotypes than in the infected seed of susceptible genotypes. Both acidic and basic beta-1,3-glucanase isoforms were detected in the isoelectric focusing gels. Thin-layer chromatography analysis of the hydrolytic products from the reaction mixtures of the substrate with the total protein extract or individual band of native PAGE revealed the presence of enzymatic hydrolytic oligomer products. The individual bands corresponding to the bands of beta-1,3-glucanase isoforms Glu 1 to 5 were separated on the sodium dodecyl sulfate-PAGE, resulting in two bands of 10 and 13 kDa, respectively. The sequences of fragments of the 13-kDa major protein band showed a high degree of homology to conglutin, a storage protein in peanut seed. Conglutin is reported as a peanut allergen, Ara h2. Our data provide the first evidences for peanut having beta-1,3-glucanase activities and the association with the resistance to A. flavus colonization in peanut seed. We have not directly demonstrated that conglutin has beta-1,3-glucanase activity.  相似文献   

19.
A two-year field study was conducted to determine the effects of artificial inoculation techniques on the pathogenicity and virulence of Aspergillus niger kernel infection on two maize hybrids. Test plants included in the study were hybrids resistant and susceptible to Aspergillus flavus to determine if the host resistance mechanisms that limited A. flavus infection would also suppress A. niger infection. Ears were inoculated with the silk-channel, side-needle, and spray techniques 7?days after midsilk (50% of the plants in a plot had silk emerging). Ears were also inoculated with a modified-pinbar technique 21?days after midsilk. Kernel infection in 2008 in inoculated plants ranged from 2% to 11% and from 2% to 45% in the resistant and susceptible hybrids, respectively. In 2009, kernel infection in inoculated plants ranged from 13% to 32% and from 10% to 67% in the resistant and susceptible, respectively. The silk-channel, side-needle, and modified-pinbar techniques produced significantly higher levels of kernel infection in the susceptible hybrid in both years than the spray technique. When hybrids were compared, the silk-channel, side-needle, and modified-pinbar techniques induced significantly higher levels of infections in the susceptible hybrid than in the resistant hybrid in 2008 and 2009. The level of A. niger pathogenicity and virulence increased when conidia were placed inside the husks of developing ears by wounding (modified-pinbar and side-needle techniques) or non-wounding (silk-channel technique) inoculation methods. Although A. niger kernel infection was significantly lower in the A. flavus resistant hybrid compared to the A. flavus susceptible hybrid, A. niger infection levels were much higher than A. flavus infection levels typically observed in both of these hybrids in past studies.  相似文献   

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