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.     

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.     

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.     

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.     

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.     

柠檬醛对中药材黄曲霉菌的抑制研究

[目的]从霉变中药材(枳壳、泽泻)中分离鉴定黄曲霉菌(Aspergillus flavus),以中药材源黄曲霉菌为研究对象,分析柠檬醛对其抑制效果,为后期开发绿色、高效、安全的植物源防霉剂提供数据支撑.[方法]采用点样法从霉变中药材上分离黄曲霉菌,通过形态学和分子生物学手段鉴定,并测定了其产毒能力;利用孢子计数法定量研究柠檬醛对中药材源黄曲霉菌的体外抑制;通过人为接种的方式研究柠檬醛对实际中药材样本霉变的防治效果.[结果]从霉变中药材上分离了两株菌(ZK和ZX),形态学和分子学鉴定其为黄曲霉菌,该两株中药材源黄曲霉菌的产毒量分别为3.01μg/kg和345μg/kg,危害不容忽视.体外抑菌试验结果表明柠檬醛对ZK和ZX的半数有效抑制浓度分别为719.42 mg/L和689.19 mg/L,具有良好的体外抑制效果;柠檬醛对中药材实际样本具有良好的霉变防控作用,能有效防治泽泻和枳壳霉变的浓度分别是4μL/g和8μL/g.[结论]霉变中药材中分离得到的黄曲霉菌具有产生黄曲霉毒素的能力;柠檬醛对中药材黄曲霉菌有良好抑制作用,具有开发成植物源防霉剂的潜能.     

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.     

Comparison of Kernel Wax from Corn Genotypes Resistant or Susceptible to Aspergillus flavus

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.     

Methods to Sample Air Borne Propagules of Aspergillus flavus

Three techniques (cyclone samplers, filter samplers and rotorods) were evaluated for sampling airborne propagules of Aspergillus flavus in a cultivated region of southwest Arizona. Analysis of variance indicated no significant difference between cyclone and filter samplers in quantity of colony forming units caught, but there was a positive correlation between catches of the three separate impaction samplers (r = 0.84–0.99, P>0.05). There was no detectable correlation between impaction sampler catches and cyclone catches (r=0.12–0.33; P>0.05). Cyclone samplers collected a dry sample that was easy to process for quantification of fungal propagules. Size of conidia of A. flavus (combined with filter retention studies) suggests that the predominant propagules caught by the cyclone sampler were conidia, rather than sclerotia or infected vegetative matter. Using a water-soluble coating, rotorods collected viable conidia of A. flavus under controlled environment conditions, but not in the field, although viable propagules of other fungi were caught, including other Aspergilli. In the desert environment the rotorods became overloaded with particles of dust if operated for more than 2 h. Where isolate culture is required, cyclone samplers are ideal for collecting airborne A. flavus propagules in dry climates.     

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.     

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...     

Identification of hydrolytic activities expressed by Aspergillus flavus grown on cotton carpel tissue

To identify proteins important for invasion into host plant tissue, Aspergillus flavus was cultured on medium containing cotton carpel tissue as the sole carbon source. We identified several hydrolases suggesting they are important as A. flavus virulence factors for plant colonization. Specifically, Aspergillus flavus AF13 secreted at least two endoxylanase activities and a pectolytic activity when grown on the cotton carpel tissue medium. A concentrated sample derived from the A. flavus growth medium (6-day) was subjected to gel filtration chromatography on a BioGel P-30 column. A major endoxylanase activity was separated from the other fungal-secreted proteins. Additional fungal secreted proteins were partially resolved by gel filtration chromatography on a BioGel P-60 column. Multiple proteins with molecular weights in the 20 to 70 kD range were present in the harvested fungal growth medium. Analysis of these fungal-secreted proteins by liquid chromatography/tandem mass spectrometry identified both endo- and exo-glucanase proteins, α-l-arabinofuranosidase, glucoamylase, α-amylase A, pectate lyase A, xylanase F1, acetylxylan esterase, glutaminase A, as well as conserved hypothetical proteins of unknown function. These proteins likely assist A. flavus in the maceration of plant cell walls, allowing for pathogenic entry and accession of host nutrient resources. Pectolytic and xylanolytic hydrolases, as well as glucanases, appear to be important A. flavus virulence factors.     

Influence of plant host species on intraspecific competition during infection by Aspergillus flavus

Compositions of Aspergillus flavus populations determine the extent to which crops become contaminated with aflatoxins. In the current study, influences of diverse crop hosts on competition among A. flavus isolates were quantified with pyrosequencing. Maize, cotton, soyabean and sorghum supported different levels of sporulation, but intraspecific differences in sporulation were not detected on any host. However, hosts differentially influenced competition during infection, allowing greater sporulation by some isolates and increased host tissue invasion by others. Furthermore, competitive interactions during host invasion did not predict isolate success during sporulation. Isolates were similarly competitive on maize and sorghum, the two most closely related hosts. Host‐specific influences on intraspecific competition may dictate compositions of A. flavus populations and, as a result, the severity of aflatoxin contamination. Host factors should be considered when designing and implementing aflatoxin management strategies including biocontrol with atoxigenic strains.     

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.