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1.
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
2.
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
3.
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
4.
Guo BZ Chen ZY Brown RL Lax AR Cleveland TE Russin JS Mehta AD Selitrennikoff CP Widstrom NW 《Phytopathology》1997,87(11):1174-1178
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
5.
ABSTRACT Aflatoxins are carcinogens produced by Aspergillus flavus and A. parasiticus during infection of susceptible crops such as maize (Zea mays L.). Resistant maize genotypes have been identified, but the incorporation of resistance into commercial lines has been slow due to the lack of selectable markers. Here we report the identification of potential markers in resistant maize lines using a proteomics approach. Kernel embryo proteins from each of two resistant genotypes have been compared with those from a composite of five susceptible genotypes using large format two-dimensional gel electrophoresis. Through these comparisons, both quantitative and qualitative differences have been identified. Protein spots have been sequenced, and based on peptide sequence homology analysis, are categorized as follows: storage proteins (globulin 1 and globulin 2), late embryogenesis abundant (LEA) proteins related to drought or desiccation (LEA3 and LEA14), water- or osmo-stress related proteins (WSI18 and aldose reductase), and heat-stress related proteins (HSP16.9). Aldose reductase activity measured in resistant and susceptible genotypes before and after infection suggests the importance of constitutive levels of this enzyme to resistance. Results of this study point to a correlation between host resistance and stress tolerance. The putative function of each identified protein is discussed. 相似文献
6.
7.
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
8.
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. 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.W. Schaafsma R.W. Nicol L.M. Reid 《European journal of plant pathology / European Foundation for Plant Pathology》1997,103(8):737-746
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
11.
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
12.
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
13.
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
14.
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
15.
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. 相似文献
16.
ABSTRACT The inheritance of resistance to aflatoxin production in corn (Zea mays) caused by the fungus Aspergillus flavus was studied following inoculation in progeny derived from the cross between the susceptible inbred B73 and the resistant inbred LB31. In 1993, the susceptible parent B73 (P(1)), resistant parent LB31 (P(2)), F(1), F(3), and BCP(1)-selfed generations were evaluated. In 1994, the study was expanded to include the F(2), BCP(1), and BCP(2) generations. Aflatoxin concentrations were higher in 1993 than 1994. Generation mean analysis showed that additive and dominant gene action were important for resistance to aflatoxin production. Potence ratios indicated dominance for resistance in both years. In 1993, aflatoxin values of the F(1) generation were significantly lower than the calculated mid-parent values, indicating dominant gene action favoring resistance. In 1994, values of the F(1) generation were not significantly lower than the calculated midparent value. The frequency distributions of aflatoxin values for families of the F(3) and BCP(1)-selfed generations were skewed toward the resistant parent, also indicating dominance. Heritability based on a progeny mean basis of F(3) families representing the additive variance plus one-fourth of the dominance variance was estimated at 66% over both years combined. Based on these results, selection for resistance to aflatoxin production in progeny derived from the cross between B73 and LB31 should be effective. 相似文献
17.
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. 相似文献
18.
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
19.
Claudia Patricia Bolívar Forero Maria del Pilar Moncada 《European journal of plant pathology / European Foundation for Plant Pathology》2014,138(2):307-321
Coffee berry disease (CBD) is caused by the fungus Colletotrichum kahawae and is restricted to the African continent, where it generates losses of up to 80 % of coffee production. Weather conditions in certain growing areas at high altitudes in Colombia appear to be very favourable for the development of this disease. Certain genotypes of Coffee arabica are resistant to this pathogen, such as the Timor Hybrid and some Ethiopian accessions. It is important to identify the proteins in these coffee genotypes that are associated with resistance to this fungus. Therefore, we compared the proteomes of two genotypes that are resistant to different isolates of C. kahawae with the proteome of the susceptible coffee genotype Caturra. We optimized the methodology applied for the extraction, cleaning and purification of proteins from the green fruit pericarp at 150 to 170 days after flowering. Through two-dimensional differential gel electrophoresis, proteomic map images were obtained for the resistant and susceptible genotypes. Fifty-two protein spots that were significantly different between the resistant and susceptible genotypes were detected. These protein spots were isolated and sequenced via mass spectrometry. The sequence analysis identified 14 proteins in the Timor Hybrid and 14 in CCC1147 that were associated with resistance and pathogen defence. 相似文献
20.
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. 相似文献