Genetic diversity in Aspergillus parasiticus population from the peanut agroecosystem in Argentina

AIMS: The aims of this work were to identify potential sources of Aspergillus parasiticus inoculum and to evaluate the sclerotial and toxigenic profiles of this species from the peanut agroecosystem in Argentina. Likewise, the genetic diversity of A. parasiticus population was analysed using vegetative compatibility group (VCG) analysis. METHODS AND RESULTS: The A. parasiticus strains were isolated from soil, debris and peanut seeds in Córdoba Province, Argentina. A. parasiticus was recovered from the three sources analysed. Only 11 of 185 A. parasiticus isolates (5.9%) did not produce aflatoxins, while 57% produced sclerotia. Twenty-four VCG were identified from 63 isolates. The VCG diversity index for A. parasiticus, expressed as the number of groups divided by the total number of isolates, was 0.31. In general, there were significant differences among VCG in aflatoxin production. CONCLUSIONS: The presence of aflatoxigenic strains of A. parasiticus in the three substrates suggests that they may be an important source of aflatoxin in Argentina's peanut agroecosystem. The A. parasiticus population shows a low genetic diversity. SIGNIFICANCE AND IMPACT OF THE STUDY: The present study showed data on inoculum distribution, aflatoxin and sclerotia production and genetic diversity in an A. parasiticus population isolated from the peanut agroecosystem in Argentina.     

A multiplex RT-PCR approach to detect aflatoxigenic strains of Aspergillus flavus

AIMS: To develop a multiplex reverse transciption-polymerase chain reaction (RT-PCR) protocol to discriminate aflatoxin-producing from aflatoxin-nonproducing strains of Aspergillus flavus. METHODS AND RESULTS: The protocol was first optimized on a set of strains obtained from laboratory collections and then validated on A. flavus strains isolated from corn grains collected in the fields of the Po Valley (Italy). Five genes of the aflatoxin gene cluster of A. flavus, two regulatory (aflR and aflS) and three structural (aflD, aflO and aflQ), were targeted with specific primers to highlight their expression in mycelia cultivated under inducing conditions for aflatoxins production. 48-h-old cultures expressed the complete set of the genes analysed here whereas 24-h-old ones did not. Genomic PCR (quadruplex PCR) was also performed in parallel using chromosomal DNA extracted from the same set of strains to correlate the integrity of the genes with their expression. CONCLUSIONS: We show that a good correlation exists between gene expression of the aflatoxin genes, here analysed by multipex RT-PCR, and aflatoxin production, except for one strain that apparently transcribed all the relevant genes but did not produce aflatoxin in the medium. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first example of the application of a combination of multiplex PCR and RT-PCR approaches to screen a population of A. flavus for the presence of aflatoxigenic and nonaflatoxigenic strains. The proposed protocol will be helpful in evaluating the risk posed by A. flavus in natural environments and might also be a useful tool to monitor its presence during the processing steps of food and feed commodities.     

Osmotic and matric potential effects on growth, sugar alcohol and sugar accumulation by Aspergillus section Flavi strains from Argentina

AIMS: The effect of osmotic and matric potential stress on growth and sugar alcohols (polyols: glycerol, erythritol, arabitol and mannitol) and sugars (trehalose and glucose) accumulation in toxigenic and nontoxigenic colonies of Aspergillus flavus and A. parasiticus was evaluated. METHODS AND RESULTS: Growth of Aspergillus section Flavi with significant reductions at 20 and 30 degrees C was more sensitive to changes in matric potential, between 60 and 100% in the range of -7 to -14 MPa. No significant differences were found between toxigenic and nontoxigenic strains for both species. Total polyol accumulation in unamended maize meal agar medium (-0.75 MPa water potential) was higher at 30 than 20 degrees C. The major change in concentrations of endogenous sugars and total polyols was in matrically amended medium (with PEG 8000) at -7 and -10 MPa. Accumulation of glucose, arabitol, mannitol and erythritol content of A. flavus and A. parasiticus mycelial colonies was greater in normal unstressed maize meal agar medium (-0.75 Mpa) at 20 degrees C. This was modified by solute and matric stress. CONCLUSIONS: The data showed relative sensitivity to osmotic and matric potential, and temperature, and the impact on growth rates, polyol and sugar accumulation in mycelia of A. flavus and A. parasiticus. SIGNIFICANCE AND IMPACT OF THE STUDY: The matric potential effects on growth may be of particular importance for growth and survival in environments with low-matric potential stress. The tolerance of spoilage fungi such as Aspergillus section Flavi to such modifications could increase the potential for spoilage and mycotoxin production in such substrates. This knowledge is important for understanding the relative ecological fitness of these aflatoxigenic species and in the development of prevention strategies for their control.     

Cloning of the Aspergillus parasiticus apa-2 gene associated with the regulation of aflatoxin biosynthesis.

An Aspergillus parasiticus gene, designated apa-2, was identified as a regulatory gene associated with aflatoxin biosynthesis. The apa-2 gene was cloned on the basis of overproduction of pathway intermediates following transformation of fungal strains with cosmid DNA containing the aflatoxin biosynthetic genes nor-1 and ver-1. Transformation of an O-methylsterigmatocystin-accumulating strain, A. parasiticus SRRC 2043, with a 5.5-kb HindIII-XbaI DNA fragment containing apa-2 resulted in overproduction of all aflatoxin pathway intermediates analyzed. Specific enzyme activities associated with the conversion of norsolorinic acid and sterigmatocystin were increased approximately twofold. The apa-2 gene was found to complement an A. flavus afl-2 mutant strain for aflatoxin production, suggesting that apa-2 is functionally homologous to afl-2. Comparison of the A. parasiticus apa-2 gene DNA sequence with that of the A. flavus afl-2 gene (G. A. Payne, G. J. Nystorm, D. Bhatnagar, T. E. Cleveland, and C. P. Woloshuk, Appl. Environ. Microbiol. 59:156-162, 1993) showed that they shared > 95% DNA homology. Physical mapping of cosmid subclones placed apa-2 approximately 8 kb from ver-1.     

Effect of pyridazinone herbicides on growth and aflatoxin release by Aspergillus flavus and Aspergillus parasiticus

The influence of pyridazinone herbicides on aflatoxin production by Aspergillus flavus and A. parasiticus was studied in liquid media. Mycelia production was not affected by 20, 40, or 60 micrograms of herbicide per ml; however, aflatoxin production by A. parasiticus was higher in media with herbicide, whereas A. flavus produced lower aflatoxin levels.     

Effect of pyridazinone herbicides on growth and aflatoxin release by Aspergillus flavus and Aspergillus parasiticus.

The influence of pyridazinone herbicides on aflatoxin production by Aspergillus flavus and A. parasiticus was studied in liquid media. Mycelia production was not affected by 20, 40, or 60 micrograms of herbicide per ml; however, aflatoxin production by A. parasiticus was higher in media with herbicide, whereas A. flavus produced lower aflatoxin levels.     

Characterization of aflatoxin-producing fungi outside of Aspergillus section Flavi

Most aspergilli that produce aflatoxin are members of Aspergillus section Flavi, however isolates of several Aspergillus species not closely related to section Flavi also have been found to produce aflatoxin. Two of the species, Aspergillus ochraceoroseus and an undescribed Aspergillus species SRRC 1468, are morphologically similar to members of Aspergillus section Circumdati. The other species have Emericella teleomorphs (Em. astellata and an undescribed Emericella species SRRC 2520) and are morphologically distinctive in having ascospores with large flanges. All these aflatoxin-producing isolates were from tropical zones near oceans, and none of them grew on artificial media at 37 C. Aflatoxins and sterigmatocystin production were quantified by high-pressure liquid chromatography (HPLC) and confirmed by HPLC-mass spectrometry (LC-MS) detection. Phylogenetic analyses were conducted on these four species using A. parasiticus and Em. nidulans, (which produce aflatoxin and the aflatoxin precursor sterigmatocystin, respectively) for comparison. Two aflatoxin/sterigmatocystin biosynthesis genes and the beta tubulin gene were used in the analyses. Results showed that of the new aflatoxin-producers, Aspergillus SRRC 1468 forms a strongly supported clade with A. ochraceoroseus as does Emericella SRRC 2520 with Em. astellata SRRC 503 and 512.     

Aflatoxin production by entomopathogenic isolates of Aspergillus parasiticus and Aspergillus flavus

Fourteen isolates of Aspergillus parasiticus and 2 isolates of Aspergillus flavus isolated from the mealybug Saccharicoccus sacchari were analyzed for production of aflatoxins B1, B2, G1, and G2 in liquid culture over a 20-day period. Twelve Aspergillus isolates including 11 A. parasiticus and 1 A. flavus produced aflatoxins which were extracted from both the mycelium and culture filtrate. Aflatoxin production was detected at day 3 and was detected continually for up to day 20. Aflatoxin B1 production was greatest between 7 and 10 days and significantly higher quantities were produced by A. flavus compared to A. parasiticus. Aflatoxin production was not a stable trait in 1 A. parasiticus isolate passaged 50 times on agar. In addition to loss of aflatoxin production, an associated loss in sporulation ability was also observed in this passaged isolate, although it did maintain pathogenicity against S. sacchari. An aflatoxin B1 concentration of 0.16 micrograms/mealybug (14.2 micrograms/g wet wt) was detected within the tissues of infected mealybugs 7 days after inoculation. In conclusion, the ability of Aspergillus isolates to produce aflatoxins was not essential to the entomopathogenic activity of this fungus against its host S. sacchari.     

Detection of toxigenic isolates of Aspergillus flavus and related species on coconut cream agar

A new readily-prepared medium, coconut cream agar, was developed for the detection of aflatoxin production by isolates of Aspergillus flavus and related species. Coconut cream agar, which comprised coconut cream (50%) and agar (1.5%), detected isolates of A. flavus more effectively than the synthetic media tested and was as effective as media containing desiccated coconut. Fluorescence colouring of colonies grown on coconut cream agar could be used to differentiate A. flavus from A. parasiticus and A. nomius. In addition, conidial colour of A. flavus and A. nomius was quite distinct from that of A. parasiticus.     

Molecular characterization of Aspergillus section Flavi isolates collected from peanut fields in Argentina using AFLPs

AIMS: The objectives of this study were: (i) to evaluate genetic relatedness among Aspergillus section Flavi strains isolated from soil and peanut seeds in Argentina; (ii) to determine if AFLP molecular markers could be useful to identify isolates up to species level, and to correlate these markers with the isolates' toxigenic potentials and/or vegetative compatibility group (VCG) affiliations. METHODS AND RESULTS: Amplified fragment length polymorphism (AFLPs) analysis was applied to compare 82 isolates of Aspergillus section Flavi. Cluster analysis showed a clear separation of A. flavus and A. parasiticus, and comparison of fingerprints revealed several specific markers for each group of isolates. AFLP analysis indicates that no genotypical differences can be established between aflatoxigenic and nonaflatoxigenic producers in both species analysed. In addition, candidate AFLP markers associated with a particular VCG were not found. CONCLUSIONS: There was a concordance between morphological identification and separation up to species level using molecular markers. The findings of specific bands for A. flavus and A. parasiticus may be useful for the design of specific PCR primers in order to differentiate these species and detect them in food. SIGNIFICANCE AND IMPACT OF THE STUDY: The present study provides new data on molecular characterization of Aspergillus section Flavi in Argentina.     

Cloning and characterization of a cDNA from Aspergillus parasiticus encoding an O-methyltransferase involved in aflatoxin biosynthesis.

Aflatoxins are polyketide-derived secondary metabolites produced by the fungi Aspergillus flavus and Aspergillus parasiticus. Among the catalytic steps in the aflatoxin biosynthetic pathway, the conversion of sterigmatocystin to O-methylsterigmatocystin and the conversion of dihydrosterigmatocystin to dihydro-O-methylsterigmatocystin are catalyzed by an S-adenosylmethionine-dependent O-methyltransferase. A cDNA library was constructed by using RNA isolated from a 24-h-old culture of wild-type A. parasiticus SRRC 143 and was screened by using polyclonal antiserum raised against a purified 40-kDa O-methyltransferase protein. A clone that harbored a full-length cDNA insert (1,460 bp) containing the 1,254-bp coding region of the gene omt-1 was identified by the antiserum and isolated. The complete cDNA sequence was determined, and the corresponding 418-amino-acid sequence of the native enzyme with a molecular weight of 46,000 was deduced. This 46-kDa native enzyme has a leader sequence of 41 amino acids, and the mature form of the enzyme apparently consists of 377 amino acids and has a molecular weight of 42,000. Direct sequencing of the purified mature enzyme from A. parasiticus SRRC 163 showed that 19 of 22 amino acid residues were identical to the amino acid residues in an internal region of the deduced amino acid sequence of the mature protein. The 1,460-bp omt-1 cDNA was cloned into an Escherichia coli expression system; a Western blot (immunoblot) analysis of crude extracts from this expression system revealed a 51-kDa fusion protein (fused with a 5-kDa beta-galactosidase N-terminal fragment).(ABSTRACT TRUNCATED AT 250 WORDS)     

Molasses supplementation promotes conidiation but suppresses aflatoxin production by small sclerotial Aspergillus flavus

AIMS: To find a supplemental ingredient that can be added to routinely used growth media to increase conidial production and decrease aflatoxin biosynthesis in small sclerotial (S strain) isolates of Aspergillus flavus. METHODS AND RESULTS: Molasses was added to three commonly used culture media: coconut agar (CAM), potato dextrose agar (PDA), and vegetable juice agar (V8) and production of conidia, sclerotia, and aflatoxins by A. flavus isolate CA43 was determined. The effect of nitrogen sources in molasses medium (MM) on production of conidia, sclerotia and aflatoxins was examined. Water activity and medium pH were also measured. Conidia harvested from agar plates were counted using a haemocytometer. Sclerotia were weighed after drying at 45 degrees C for 5 days. Aflatoxins B(1) and B(2) were quantified by high-performance liquid chromatography. Addition of molasses to the media did not change water activity or the pH significantly. Supplementing CAM and PDA with molasses increased conidial production and decreased aflatoxins. Two-fold increased yield of conidia was found on MM, which, like V8, did not support aflatoxin production. Adding ammonium to MM significantly increased the production of sclerotia and aflatoxins, but slightly decreased conidial production. Adding urea to MM significantly increased the production of conidia, sclerotia and aflatoxins. CONCLUSIONS: Molasses stimulated conidial production and inhibited aflatoxin production. Its effect on sclerotial production was medium-dependent. Water activity and medium pH were not related to changes in conidial, sclerotial or aflatoxin production. Medium containing molasses alone or molasses plus V8 juice were ideal for conidial production by S strain A. flavus. SIGNIFICANCE AND IMPACT OF THE STUDY: Insight into molecular events associated with the utilization of molasses may help to elucidate the mechanism(s) that decreases aflatoxin biosynthesis. Targeting genetic parameters in S strain A. flavus isolates may reduce aflatoxin contamination of crops by reducing the survival and toxigenicity of these strains.     

传统发酵豆瓣中产毒黄曲霉高效拮抗菌的筛选

从自然发酵的豆瓣中筛选出对产毒黄曲霉菌的生长及其毒素合成均有抑制作用的细菌, 在蚕豆天然培养基(BAM)上利用菌落对峙实验初筛和滤纸片复筛得到1株有较高抑制产毒黄曲霉活性的菌株L4。对L4进行形态学、生理生化特征及16S rRNA序列同源性分析, 鉴定此菌株为枯草芽孢杆菌(Bacillus subtilis)。在抑制黄曲霉生长和黄曲霉毒素B1 (AFB1)合成的研究中表明, 在L4与黄曲霉菌共同培养15 d后, 黄曲霉菌丝产量和黄曲霉毒素B1 产量均比黄曲霉单独培养时显著降低(P < 0.01), AFB1合成受到明显抑制, 抑制率达93.7%。当黄曲霉孢子液与L4发酵上清液1: 1 (V/V)混合后接种在玉米粒上时, 黄曲霉在玉米上的生长和孢子萌发均得到完全抑制。     

Fungicidal and anti-aflatoxigenic effects of the essential oil of Cymbopogon citratus (DC.) Stapf. (lemongrass) against Aspergillus flavus Link. isolated from stored rice

AIMS: To develop a natural fungicide against aflatoxigenic fungi, to protect stored rice, using the essential oil of lemongrass. METHODS AND RESULTS: Aspergillus flavus Link. was isolated from stored rice and identified as an aflatoxigenic strain. Lemongrass oil was tested against A. flavus and the test oil was fungistatic and fungicidal against the test pathogen at 0.6 and 1.0 mg ml(-1), respectively. Aflatoxin production was completely inhibited at 0.1 mg ml(-1). The results obtained from the thin layer chromatographic bioassay and gas chromatography indicated citral a and b as the fungicidal constituents in lemongrass oil. During the fumigant toxicity assay of lemongrass oil, the sporulation and the mycelial growth of the test pathogen were inhibited at the concentrations of 2.80 and 3.46 mg ml(-1), respectively. CONCLUSION: Lemongrass oil could be used to manage aflatoxin formation and fungal growth of A. flavus in stored rice. SIGNIFICANCE AND IMPACT OF THE STUDY: Currently, fungicides are not used to control fungal pests or mycotoxin production on stored rice. Rice treated with the essential oil of lemongrass could be used to manage fungal pests as well as the insect pests in stored rice. The essential oil is chemically safe and acceptable to consumers, as synthetic chemical fungicides can cause adverse health effects to consumers.     

Cloning of a sugar utilization gene cluster in Aspergillus parasiticus

At one end of the 70 kb aflatoxin biosynthetic pathway gene cluster in Aspergillus parasiticus and Aspergillus flavus reported earlier, we have cloned a group of four genes that constitute a well-defined gene cluster related to sugar utilization in A. parasiticus: (1) sugR, (2) hxtA, (3) glcA and (4) nadA. No similar well-defined sugar gene cluster has been reported so far in any other related Aspergillus species such as A. flavus, A. nidulans, A. sojae, A. niger, A. oryzae and A. fumigatus. The expression of the hxtA gene, encoding a hexose transporter protein, was found to be concurrent with the aflatoxin pathway cluster genes, in aflatoxin-conducive medium. This is significant since a close linkage between the two gene clusters could potentially explain the induction of aflatoxin biosynthesis by simple sugars such as glucose or sucrose.     

Divergent regulation of aflatoxin production at acidic pH by two <Emphasis Type="Italic">Aspergillus</Emphasis> strains

Production of aflatoxins (AF) by Aspergillus flavus and A. parasiticus is known to occur only at acidic pH. Although typical A. flavus isolates produced more AF as the external pH became increasingly acidic, an atypical strain from West Africa produced less. The lower AF production was not well correlated with decreases in expression of the aflatoxin pathway regulatory gene, aflR, or of two other biosynthesis genes.     

Species identification of Aspergillus section Flavi isolates from Portuguese almonds using phenotypic, including MALDI-TOF ICMS, and molecular approaches

AIMS: Section Flavi is one of the most significant sections in the genus Aspergillus. Taxonomy of this section currently depends on multivariate approaches, entailing phenotypic and molecular traits. This work aimed to identify isolates from section Flavi by combining various classic phenotypic and genotypic methods as well as the novel approach based on spectral analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF ICMS) and to evaluate the discriminatory power of the various approaches in species identification. METHODS AND RESULTS: Aspergillus section Flavi isolates obtained from Portuguese almonds were characterized in terms of macro- and micromorphology, mycotoxin pattern, calmodulin gene sequence and MALDI-TOF protein fingerprint spectra. For each approach, dendrograms were created and results were compared. All data sets divided the isolates into three groups, corresponding to taxa closely related to Aspergillus flavus, Aspergillus parasiticus and Aspergillus tamarii. In the A. flavus clade, molecular and spectral analyses were not able to resolve between aflatoxigenic and nonaflatoxigenic isolates. In the A. parasiticus cluster, two well-resolved clades corresponded to unidentified taxa, corresponding to those isolates with mycotoxin profile different from that expected for A. parasiticus.