Spatial Relationships of Soil Texture and Crop Rotation to Aspergillus flavus Community Structure in South Texas

ABSTRACT Aspergillus flavus, the causal agent of aflatoxin contamination of cottonseed, is a natural inhabitant of soils. A. flavus can be divided into the S and L strains, of which the S-strain isolates, on average, produce greater quantities of aflatoxins than the L-strain isolates. Aflatoxin contamination can be severe in several crops in South Texas. The structure of A. flavus communities residing in soils of South Texas was determined from 326 soil samples collected from 152 fields located from the Rio Grande Valley in the south to Fort Bend County in the north from 2001 through 2003. Analysis of variance indicated significant differences in the incidence of A. flavus isolates belonging to the S strain (percent S) among regions. The Coastal Bend (30.7%) and Upper Coast (25.5%) regions had significantly higher percent S incidence than the Rio Grande Valley (4.8%). No significant differences in percent S among years were detected. The CFU per gram of soil were not significantly different among regions. Strain S incidence was positively correlated with clay content and negatively correlated with sand content. Fields cropped to cotton the previous year had a higher S-strain incidence, whereas fields cropped to corn had greater total quantities of A. flavus propagules. Maps of S-strain patterns show that the S strain constitutes >30% of the overall A. flavus community in the area extending from the central Coastal Bend region to the central Upper Coast region. The west Rio Grande Valley had the lowest S-strain incidence (<10%). Geographic variation in S-strain incidence may influence the distribution of aflatoxin contamination in South Texas.     

Aflatoxin contamination of commercial cottonseed in South Texas

ABSTRACT Aflatoxins are toxic fungal metabolites produced by several members of Aspergillus section Flavi. U.S. federal regulations limit the use of aflatoxin-contaminated cottonseed. Cottonseed with aflatoxin content of 20 ng/g or higher may not enter the profitable dairy market. Between 4,472 and 9,949 truckloads of cottonseed from 31 to 35 gins in South Texas were analyzed for aflatoxin content each year from 1997 to 2001 upon receipt at the Valley Co-op Oil Mill in Harlingen, TX. The highest levels of contamination occurred in 1999, with an average aflatoxin content of 112 ng/g and 66% of the cottonseed truckloads exceeding 20 ng/g. Years 1997 and 2000 had the lowest aflatoxin levels, averaging 24 ng/g, with the lowest incidence (16%) of the truckloads exceeding 20 ng/g in 1997. In general, aflatoxin contamination increased as the ginning season progressed. Rainfall after boll opening correlated highly with aflatoxin content, with rainfall in July explaining over 50% of the observed variability in aflatoxin content. South Texas was divided into four regions: Rio Grande Valley, Coastal Bend, Upper Coast, and Winter Garden. Geostatistical analyses revealed recurrent patterns of high and low contamination. The greatest contamination occurred from the central Coastal Bend region through the southern Upper Coast region. The Rio Grande Valley region experienced the least contamination during the study period.     

Formation of Sclerotia and Aflatoxins in Developing Cotton Bolls Infected by the S Strain of Aspergillus flavus and Potential for Biocontrol with an Atoxigenic Strain

ABSTRACT Aspergillus flavus can be divided into the S and L strains on the basis of sclerotial morphology. On average, S strain isolates produce greater quantities of aflatoxins than do L strain isolates. Sclerotia of the S strain were observed in commercial seed cotton from western Arizona. Greenhouse tests were performed to better define sclerotial formation in developing bolls. Eight S strain isolates were inoculated into developing bolls via simulated pink bollworm exit holes. All eight isolates formed sclerotia on locule surfaces, and seven of eight isolates produced sclerotia within developing seed. Boll age at inoculation influences formation of sclerotia. More sclerotia formed within bolls that were less than 31 days old at inoculation than in bolls older than 30 days at inoculation. Frequent formation of sclerotia during boll infection may both favor S strain success within cotton fields and increase toxicity of A. flavus-infected cottonseed. Atoxigenic A. flavus L strain isolate AF36 reduced formation of both sclerotia and aflatoxin when coinoculated with S strain isolates. AF36 formed no sclerotia in developing bolls and was more effective at preventing S strain isolates than L strain isolates from contaminating developing cottonseed with aflatoxins. The use of atoxigenic L strain isolates to prevent contamination through competitive exclusion may be particularly effective where S strain isolates are common. In addition to aflatoxin reduction, competitive exclusion of S strain isolates by L strain isolates may result in reduced overwintering by S strain isolates and lower toxicity resulting from sclerotial metabolites.     

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.     

Use of pyrosequencing to quantify incidence of a specific Aspergillus flavus strain within complex fungal communities associated with commercial cotton crops

Atoxigenic strains of Aspergillus flavus have been used as aflatoxin management tools on over 50,000 hectares of commercial crops since 2000. To assess treatment efficacy, atoxigenic strain incidence is routinely monitored by vegetative compatibility analyses (VCA) that require culturing, generation of auxotrophs, and complementation with tester mutants. Two pyrosequencing assays (PA) that require no culturing were developed for monitoring incidences of atoxigenic strains on ginned cottonseed. The assays, which quantify frequencies of characteristic single nucleotide polymorphisms (SNPs) in the aflR and pksA genes, were validated against standard VCA on cottonseed collected from commercial gins in South Texas, Arizona, and Southern California where the atoxigenic strain AF36 is used to manage aflatoxin contamination. Cottonseed washings were subjected to both VCA and PA. PA was performed directly on DNA isolated from particulates pelleted from the wash water by centrifugation. Addition of CaCl(2) and diatomaceous earth prior to pelleting increased the amount of DNA isolated. Accuracy and reproducibility of the PA were contrasted with those for the VCA that has been used for over a decade. Correlation coefficients between VCA and PA indicated good correspondence between the results from the two assays (r = 0.91 for aflR assay and r = 0.80 for pksA assay). PAs were highly variable for samples with low incidences of A. flavus due to variability in the initial polymerase chain reaction step. This held for both DNA isolated from cottonseed washes and for mixtures of purified DNA. For samples yielding low quantities of A. flavus DNA, averaging of results from 4 to 5 replicates was required to achieve acceptable correlations with VCA. Pyrosequencing has the potential to become a powerful tool for monitoring atoxigenic strains within complex A. flavus communities without limitations imposed by traditional culturing methods.     

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.     

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.     

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

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

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.     

生物防治黄曲霉毒素污染研究进展

黄曲霉毒素（aflatoxin,AFT）是由黄曲霉（Aspergillus flavus）和寄生曲霉（A. parasiticus）产生的一类强致癌真菌毒素。生物防治是防治黄曲霉毒素污染的经济和有效方法之一,近年来,生物防治黄曲霉毒素污染的研究取得较大进展,并在生产上得到广泛应用。本文就近年来生物防治黄曲霉毒素方面的研究进行综述。     

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.     

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.     

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.     

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.     

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.     

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.     

Deadly strains of Kenyan <Emphasis Type="Italic">Aspergillus</Emphasis> are distinct from other aflatoxin producers

Aflatoxin contamination of crops is a world-wide problem. Lethal aflatoxicosis of humans has been associated with maize produced in Kenya for over three decades. The S strain morphotype of Aspergillus flavus was identified as the primary cause of aflatoxin contamination events occurring between 2004 and 2006 in Kenya. Because the S strain was first described in the U.S., it was suggested that the agent causing lethal levels of aflatoxins was introduced to Kenya with maize. DNA sequence comparisons among 68 S strain isolates from Kenya, the Americas, Asia, and Australia suggest the Kenyan isolates are distinct from those causing aflatoxin contaminations in the U.S. Analyses of 4.06 kb representing three loci from distinct chromosomes indicate that most S strain isolates from the U.S. resolved into a clade distinct from one containing the 30 Kenyan isolates. S strain isolates from Kenya were more closely related to the recently described species A. minisclerotigenes than to A. flavus. Furthermore, failure of the Kenyan isolates to produce G aflatoxins was attributed to a previously undescribed deletion in the cypA gene, suggesting that different deletion events led to loss of G aflatoxin production in S strain isolates from the U.S. and Kenya. Thus, although the Kenyan isolates have S strain morphology and produce large quantities of only B aflatoxins like A. flavus S strain isolates, these isolates are phylogenetically divergent from those described from other regions. The molecular characteristics of the Kenyan S strain isolates described herein are valuable tools to identify and track these highly aflatoxigenic fungi.     

Neotropical Monogenoidea. 40. Protorhinoxenus prochilodi gen. n., sp. n. (Monogenoidea: Ancyrocephalinae), parasite of prochilodus lineatus (Characiformes: Prochilodontidae) from south Brazil

The monotypic Protorhinoxenus gen. n. is proposed to accommodate a species with the following characteristics: 1) tubular sclerotised vagina, 2) vaginal pore dextrolateral, 3) ventral and dorsal anchors with undifferentiated elongate shaft and base (representing approximately 2/3 of the length of anchor), and 4) superficial and deep roots of ventral and dorsal anchors lacking. Protorhinoxenus prochilodi sp. n. is described from the gills of Prochilodus lineatus (Valenciennes) of the Represa Capivari-Cachoeira, Municipality of Campina Grande do Sul, metropolitan area of Curitiba, Paraná. Specimens of other probable new species of Protorhinoxenus are reported from Prochilodus lineatus of the Rio Paranapanema, Municipality of Salto Grande, S?o Paulo; Hoplias spp. of the Rio Dois de Fevereiro, Municipality of Antonina, Paraná, and the Rio Piraquara, metropolitan area of Curitiba, Parana; Leporinus elongatus Valenciennes of the Rio Tibagi, Municipality of Jataizinho, Parana; and Schizodon fasciatum Agassiz of the Rio Solim?es, island of Marchantaria, near Manaus, Amazonas. Protorhinoxenus appears to be a sister group of Rhinoxenus Kritsky, Boeger et Thatcher, 1988 based on the following apparent synapomorphies: 1) ventral and dorsal anchors lacking superficial and deep roots, 2) ventral and dorsal anchors with elongate shaft, and 3) male copulatory organ with counterclockwise rings.