Substrate-induced lipase gene expression and aflatoxin production in Aspergillus parasiticus and Aspergillus flavus

AIMS: To establish a relationship between lipase gene expression and aflatoxin production by cloning the lipA gene and studying its expression pattern in several aflatoxigenic and nontoxigenic isolates of Aspergillus flavus and A. parasiticus. METHODS AND RESULTS: We have cloned a gene, lipA, that encodes a lipase involved in the breakdown of lipids from aflatoxin-producing A. flavus, A. parasiticus and two nonaflatoxigenic A. flavus isolates, wool-1 and wool-2. The lipA gene was transcribed under diverse media conditions, however, no mature mRNA was detected unless the growth medium was supplemented with 0.5% soya bean or peanut oil or the fungus was grown in lipid-rich medium such as coconut medium. The expression of the lipase gene (mature mRNA) under substrate-induced conditions correlated well with aflatoxin production in aflatoxigenic species A. flavus (SRRC 1007) and A. parasiticus (SRRC 143). CONCLUSIONS: Substrate-induced lipase gene expression might be indirectly related to aflatoxin formation by providing the basic building block 'acetate' for aflatoxin synthesis. No direct relationship between lipid metabolism and aflatoxin production can be ascertained, however, lipase gene expression correlates well with aflatoxin formation. SIGNIFICANCE AND IMPACT OF THE STUDY: Lipid substrate induces and promotes aflatoxin formation. It gives insight into genetic and biochemical aspects of aflatoxin formation.     

Inhibition of aflatoxin production of Aspergillus parasiticus NRRL 2999 by Bacillus pumilus

Six isolates of Bacillus pumilus were tested for their ability to inhibit aflatoxin production of Aspergillus parasiticus NRRL 2999 in yeast extract sucrose (YES) broth. Aflatoxin production was inhibited in both simultaneous and deferred antagonism assays, suggesting that the inhibitory activity was due to extracellular metabolite(s) produced in cell-free supernatant fluids of cultured broth. The inhibition was not due to organic acids or hydrogen peroxide produced by B. pumilus since the inhibitory activity was not lost after pH adjustment or treatment of supernatant fluids with catalase. A range of media tested for the production of inhibitory metabolite(s) in supernatant fluids showed that all media supported bacterial growth and production of the metabolite(s). The metabolite(s) were produced over a wide range of temperature (25 to 37°C) and pH (4 to 9) of growth of B. pumilus. They were stable over a wide range of pH (4 to 10) and were not inactivated after autoclaving at 121°C for 30 minutes. This revised version was published online in August 2006 with corrections to the Cover Date.     

Effect of nontoxigenic Aspergillus flavus and A. parasiticus on aflatoxin contamination of wounded peanut seeds inoculated with agricultural soil containing natural fungal populations

Peanuts and other seed and grain crops are commonly contaminated with carcinogenic aflatoxins, secondary metabolites produced by Aspergillus flavus and A. parasiticus. Aflatoxin contamination of peanuts in the field can be reduced by 77–98% with biological control through the application of nontoxigenic strains of these species, which competitively exclude native aflatoxin-producing strains from developing peanuts. In this study, viable peanut seeds were artificially wounded and inoculated with field soil containing natural fungal populations that were supplemented with conidia of nontoxigenic A. flavus NRRL 21882 (niaD nitrate-nonutilizing mutant) and A. parasiticus NRRL 21369 (conidial color mutant). Increasing soil densities of applied nontoxigenic strains generally resulted in an increase in the incidence of seed colonization by applied nontoxigenic strains, a decrease in seed colonization by native A. flavus and A. parasiticus, and a decrease in aflatoxin concentration in seeds. Reduction of aflatoxins in peanut seeds depended on both the density and the aflatoxin-producing potential of native populations and on the fungal strain used for biological control. Wild-type strain A. flavus NRRL 21882 and its niaD mutant were equally effective in reducing aflatoxins in peanuts, indicating that nitrate-nonutilizing mutants, which are easily monitored in the field, can be used for evaluating the efficacy of biocontrol strains.     

Association of laccase activity with conidiation in an aflatoxigenic strain of Aspergillus parasiticus

Abstract The relationship between laccase activity and asexual development in Aspergillus parasiticus was established. A. parasiticus produced a laccase activity similar to that reported for Aspergillus nidulans . Laccase activity appeared only in conidiating cultures and was absent from vegetative cultures. Shaking of the cultures inhibited conidiation and suppressed laccase activity. The composition of the media affected the degree of conidiation and the specific activity of laccase. Ammonium sulphate as sole nitrogen source was suppressive, whereas glutamate was highly stimulatory to both conidiation and laccase production. The addition of citrate was also stimulatory to conidia production and, to a lesser degree, laccase activity. There appears to be no quantitative correlation between laccase activity and the number of conidia produced.     

Inhibition of aflatoxin production by trifluoperazine in Aspergillus parasiticus NRRL 2999

Trifluoperazine, an anti-calmodulin agent, inhibited aflatoxin production by Aspergillus parasiticus NRRL 2999, without affecting the growth significantly. Culturing the organism for 3 days in the presence of 0.14mm trifluoperazine resulted in a generalized decrease in the production of all aflatoxins; the production of aflatoxin B1, a potent hepatocarcinogen, was inhibited to 88% under such conditions. Culturing 7-day-old preformed cultures in the presence of higher concentrations of trifluoperazine (>1mm) completely abolished production of all aflatoxins including AFB1. The inhibitory influence of trifluoperazine on aflatoxin production was accompanied by calmodulin-dependent phosphorylation of an 85kDa cytoplasmic calmodulin-binding protein. While the functions of calmodulin in mediating primary events of germination, growth and differentiation in fungi have earlier been reported, the present results indicate a possible role for calmodulin in the production of fungal toxins.     

Aflatoxins in sunflower seeds: Influence of Alternaria alternata on aflatoxin production by Aspergillus parasiticus

The aim of the present work was to determine the influence of Alternaria alternata upon aflatoxin production by Aspergillus parasiticus.A mixture of spores of both strains was inoculated in sunflower seeds at 0,90 a_w, and incubated for 42 days at 28 °C ±1.The cultures were observed and analyzed every 7 days to determine the infection level of the seeds and the production of aflatoxins. Results showed that when the seeds were inoculated only with Aspergillus parasiticus, 100% were infected from the 7th day.When Aspergillus parasiticus and Alternaria alternata were simultaneously inoculated the infection level of the seeds was 100% for Aspergillus parasiticus following 7 days of inoculation and 0% for Alternaria alternata. After the 14th day of inoculation there was no significant difference in the infection percentage of both strains (approximately 80% of each one). As far as toxin production is concerned a remarkable decrease was observed when seeds were inoculated with both strains simultaneously.In accordance to the results, Alternaria alternata would not compete with Aspergillus parasiticus in colonization of seeds but would either degrade the aflatoxins by Aspergillus parasiticus or compete for aflatoxin biosynthesis precursors. Alternaria alternata could also secrete some substance that specifically inhibits aflatoxin synthesis.     

Dillapiol and Apiol as Specific Inhibitors of the Biosynthesis of Aflatoxin G1 in Aspergillus parasiticus

Dillapiol was isolated from the essential oil of dill as a specific inhibitor of aflatoxin G₁ production. It inhibited aflatoxin G₁ production by Aspergillus parasiticus with an IC₅₀ value of 0.15 μM without inhibiting aflatoxin B₁ production or fungal growth. Apiol and myristicin, congeners of dillapiol, showed similar activity with IC₅₀ values of 0.24 and 3.5 μM, respectively.     

Effect of selenite and tellurite on the morphological growth and toxin production of Aspergillus parasiticus var. globosus IMI 120920

Aspergillus parasiticus var. globosus IMI 120920 was able to grow in presence of different concentrations tested (0.052–4.0%) of sodium selenite or concentrations up to 2.0% potassium tellurite. Growth of the fungus was decreased greatly by the increase of metals concentrations. Dark colour colony and black reverse were formed in presence of tellurite while reddish gray to grayish red colony colour and brownish red to orange red reverse were formed in presence of selenite. The fungal biomass was slightly decreased at lower concentrations and highly inhibited at higher concentrations of selenite or tellurite. Selenite slightly stimulated aflatoxin formation at lower concentrations and highly inhibited it at higher concentrations. Aflatoxin production was decreased greatly by increasing tellurite concentrations. Obvious malformations were observed in the morphological features of the fungus in presence of different levels of selenite or tellurite. This revised version was published online in June 2006 with corrections to the Cover Date.     

A highly efficient gene-targeting system for Aspergillus parasiticus

Aims: To establish a system that greatly increases the gene‐targeting frequency in Aspergillus parasiticus. Methods and Results: The ku70 gene, a gene of the nonhomologous end‐joining (NHEJ) pathway, was replaced by the nitrate reductase gene (niaD) in A. parasiticus RHN1 that accumulates O‐methylsterigmatocystin (OMST). The NHEJ‐deficient strain, RHΔku70, produced conidia, sclerotia and OMST normally. It had identical sensitivity as RHN1 to the DNA‐topoisomerase I complex inhibitor, camptothecin, and the DNA‐damaging agent, melphalan. For targeting an aflatoxin biosynthetic pathway gene, adhA, partial restriction enzyme recognition sequences in its flanking regions were manipulated to create homologous ends for integration. Using a linearized DNA fragment that contained Aspergillus oryzae pyrithiamine resistance gene (ptr) marker the adhA‐targeting frequency in RHΔku70 reached 96%. Conclusions: The homologous recombination pathway is primarily responsible for repair of DNA damages in A. parasiticus. The NHEJ‐deficient RHΔku70, easy creation of homologous ends for integration, and the ptr‐based selection form a highly efficient gene‐targeting system. It substantially reduces the time and workload necessary to obtain knockout strains for functional studies. Significance and Impact of the Study: The developed system not only streamlines targeted gene replacement and disruption but also can be used to target specific chromosomal locations like promoters or intergenic regions. It will expedite the progresses in the functional genomic studies of A. parasiticus and Aspergilllus flavus.     

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.     

Effect of peanut tannin extracts on growth of Aspergillus parasiticus and aflatoxin production

Twenty-three peanut (Arachis hypogaea L.) genotypes were evaluated for kernel resistance to Aspergillus parasiticus Spear. colonization and aflatoxin contamination when incubated under high relative humidity. Also, tannin-containing extracts from kernel coats (testae) and cotyledons of these genotypes were prepared and tested for their effect on A. parasiticus growth and aflatoxin production in vitro. The lowest degree of colonization, less than 30% was noted in kernels from the genotypes, Toalson x UF 73-4022 (selections TX-798731 and TX-798736), A72118, SN 55-437, PI337409, and Florunner. Genotypes with low levels of colonization also had the lowest aflatoxin contamination. The coefficient of correlation between infection frequency and aflatoxin contamination was 0.66. Higher levels of tannins were detected in the testae (23.9–97.2 mg g tissue) compared to the cotyledons (0.17–0.82 mg g tissue). Some of the methanol-extracted and water-soluble tannin extracts from testae and cotyledons, when incorporated in yeast extract sucrose liquid medium (100 mg l), significantly inhibited A. parasiticus growth and reduced the levels of aflatoxin produced. There was no overall correlation between the peanut genotypes and the influence of tannin extracts on A. parasiticus growth and aflatoxin production. However, correlations were higher for specific genotypes. For example, the coefficient of correlation between the ability of tannin extracts from testae of genotypes PI337409 and TX-798736 to inhibit aflatoxin production was 0.93 and 0.85 respectively.     

Conidial movement of nontoxigenic Aspergillus flavus and A. parasiticus in peanut fields following application to soil

The use of nontoxigenic strains of Aspergillus flavus and A. parasiticus in biological control effectively reduces aflatoxin in peanuts when conidium-producing inoculum is applied to the soil surface. In this study, the movement of conidia in soil was examined following natural rainfall and controlled precipitation from a sprinkler irrigation system. Conidia of nontoxigenic A. flavus and A. parasiticus remained near the soil surface despite repeated rainfall and varying amounts of applied water from irrigation. In addition, rainfall washed the conidia along the peanut furrows for up to 100 meters downstream from the experimental plot boundary. The dispersal gradient was otherwise very steep upstream along the furrows and in directions perpendicular to the peanut rows. The retention of biocontrol conidia in the upper soil layers is likely important in reducing aflatoxin contamination of peanuts and aerial crops such as corn and cottonseed. This revised version was published online in August 2006 with corrections to the Cover Date.     

Role of mycoferritin from Aspergillus parasiticus (255) in secondary metabolism (aflatoxin production)

Aspergillus parasiticus (255), a non-toxigenic isolate showed the presence of secondary metabolites-aflatoxins (B1, B2, G1, G2) when grown in yeast extract sucrose media but not in basal media, thus demonstrating its toxigenic potential. Native PAGE of the crude protein isolated at different growth periods of A. parasiticus in yeast extract sucrose media containing iron showed prominent expression of mycoferritin from day four onwards. The production of aflatoxins was also maximal on day four, both in the presence and absence of iron. Indicators of oxidative stress metabolites such as reactive oxygen species, thiobarbituric acid reactive species, reduced and oxidized glutathione and antioxidant enzymes like superoxide dismutase and glutathione peroxidase were analyzed both in the presence and absence of iron and the experimental data suggest oxidative stress as a pre-requisite for aflatoxin production. The pro-oxidant role of iron was minimized by induction of mycoferritin and the concomitant alterations in oxidative stress parameters imply an antioxidant role to mycoferritin in secondary metabolism, a finding of significance that has not been reported previously in fungal systems.     

Mycobiota and molecular detection of Aspergillus flavus and A. parasiticus aflatoxin contamination of Strawberry (Fragaria ananassa Duch.) fruits

Strawberry fungi were isolated from fresh fruits and juice on the two types of media (Sabouraud dextrose agar, SDA and potato-dextrose agar, PDA) at 28 °C. Nineteen fungal species belong to 12 genera were isolated from fruits and juice on both isolation media. The most common fungal genera and species were Aspergillus flavus, A. niger, Mucor racemosus, Neurospora crassa, Penicillium chrysogenum, Rhizopus stolonifer and Trichoderma harzianum. Twenty A. flavus and A. parasitics isolates were assayed for their abilities to produce aflatoxins. The concentration of aflatoxins ranged between 25.8–75.2 and 23.6–71.1 ng/ml at 350 and 365 nm, respectively. Among A. flavus and A. parasiticus strains tested, aflatoxin B contributed 30–60% of total isolates. However, G type contributed 85–90%. The R_f values of B₁, B₂, G₁ and G₂ were 0.79, 0.61, 0.44 and 0.32, respectively. High-performance liquid chromatography analysis of extracts revealed the presence of aflatoxins with variable levels.     

Aflatoxins in sunflower seeds: Effect of zinc in aflatoxin production by two strains of Aspergillus parasiticus

Growth and aflatoxin production by Aspergillus parasiticus NRRL 2999 and Aspergillus parasiticus RC 12 were studied both in sunflower seed and a synthetic culture medium (with and without zinc enrichment).On a synthetic culture medium the strains behaved in different ways according to the zinc concentration.In sunflower seed medium the influence of zinc was not so evident. Thus the results show that the influence of zinc is not the same for different strains and substrates.     

Regulation of aflatoxin synthesis by FadA/cAMP/protein kinase A signaling in Aspergillus parasiticus

Analysis of fadA and pkaA mutants in the filamentous fungus Aspergillus nidulans demonstrated that FadA (Galpha) stimulates cyclic AMP (cAMP)-dependent protein kinase A (PKA) activity resulting, at least in part, in inhibition of conidiation and sterigmatocystin (ST) biosynthesis. In contrast, cAMP added to the growth medium stimulates aflatoxin (AF) synthesis in Aspergillus parasiticus. Our goal was to explain these conflicting reports and to provide mechanistic detail on the role of FadA, cAMP, and PKA in regulation of AF synthesis and conidiation in A. parasiticus. cAMP or dibutyryl-cAMP (DcAMP) were added to a solid growth medium and intracellular cyclic nucleotide levels, PKA activity, and nor-1 promoter activity were measured in A. parasiticus D8D3 (nor1::GUS reporter) and TJYP1-22 (fadAGA2R, activated allele). Similar to Tice and Buchanan [34], cAMP or DcAMP stimulated AF synthesis (and conidiation) associated with an AflR-dependent increase in nor-1 promoter activity. However, treatment resulted in a 100-fold increase in intracellular cAMP/DcAMP accompanied by a 40 to 80 fold decrease in total PKA activity. ThefadAG42R allele in TJYP1-22 decreased AF synthesis and conidiation, increased basal PKA activity 10 fold, and decreased total PKA activity 2 fold. In TJYP1-22, intracellular cAMP increased 2 fold without cAMP or DcAMP treatment; treatment did not stimulate conidiation or AF synthesis. Based on these data, we conclude that: (1) FadA/PKA regulate toxin synthesis and conidiation via similar mechanisms in Aspergillus spp.; and (2) intracellular cAMP levels, at least in part, mediate a PKA-dependent regulatory influence on conidiation and AF synthesis.     

Synthesis of sterigmatocystin derivatives and their biotransformation to aflatoxins by a blocked mutant of Aspergillus parasiticus

Seven alkyl and aryl homologues of O-methylsterigmatocystin (OMST) were synthesised and fed in separate experiments to a mutant of Aspergillus parasiticus capable of converting sterigmatocystin (ST) to aflatoxin B₁ (AFB₁). Their conversion to AFB₁ was followed over a time period and it was found that O-propylsterigmatocystin (OPRST) was converted to AFB₁ more rapidly than O-ethylsterigmatocystin (OEST) or OMST or ST itself. The aryl derivative O-benzoylsterigmatocystin (OBzST) was converted at the slowest rate. These results show that alkyl and aryl homologues of OMST may be converted to AFB₁, suggesting that the methylation of ST is not an absolute requirement for its conversion to AFB₁. It seems likely that whatever enzyme(s) are involved in this process exhibit relative specificity. As to whether alkylation of ST is an obligatory step in AFB₁ biosynthesis is neither supported nor disproved as the fungal cells used are presumably capable of methylating ST. The fact that the propyl derivative showed fastest conversion is not necessarily significant as this may be due to faster diffusion of the least polar of the derivatives through the cell membrane. This revised version was published online in August 2006 with corrections to the Cover Date.     

海带渣微生物药肥对土壤中产毒真菌寄生曲霉的抑制作用研究

研究了施用多功能生防芽孢杆菌Hitwh-BA2菌株发酵生产的海带渣微生物药肥对栽培土壤中产毒真菌寄生曲霉的生态防治效果。结果表明：施用海带渣微生物药肥能极显著的降低栽培土壤中寄生曲霉的数量水平,有效降低寄生曲霉占总真菌数的百分含量,其作用效益优于海带渣;施用海带渣微生物药肥能极显著提高栽培土壤中细菌的数量水平,作用效果极显著优于海带渣。研究结果期望为土壤黄曲霉毒素产毒真菌的防控提供参考。     

Aflatoxin B1, zearalenone and deoxynivalenol production by Aspergillus parasiticus and Fusarium graminearum in interactive cultures on irradiated corn kernels

The influence of inoculum size in the production of aflatoxin B1 (AFB1), zearalenone (ZEN) and deoxynivalenol (DON) was determined when Aspergillus parasiticus NRRL 3000 and Fusarium graminearum ITEM 124 were cultured alone and in pairs on irradiated corn kernels at 28 °C and 0.97 water activity (aw). The highest levels of AFB1 produced by A. parasiticus were produced at the lowest levels of the inoculum (103 spores/ml). No significant differences were observed in ZEN and DON production at any inoculum level during the experimental period. When A. parasiticus was co-inoculated with F. graminearum both to the same inocula (106 spores/ml), AFB1 inhibition percentage were 60, 72 and 56% at 10, 20 and 35 days of incubation respectively, while at 106 spores/ml the percentages of inhibition were 34, 84 and 93% at 10, 20 and 35 days. In the mixture cultures A. parasiticus 103 × F. graminearum 106 spores/ml the percentage of inhibition of AFB1 oscillated in 99% during all the incubation. In the interaction A. parasiticus 106 spores/ml × F. graminearum 103 spores/ml the accumulation of AFB1 decreased in 80, 94 and 86% at 10, 20 and 35 days of incubation respectively. In single culture F. graminearum was inoculated with 10³ or 106 spores/ml and the highest levels of ZEN and DON were detected at 35 days of incubation. The levels oscillated in 538–622 μg/kg for ZEN and 870–834 μg/kg for DON respectively. In paired cultures there were no significant differences in the levels regardless of the spore concentrations during the incubation time. This revised version was published online in June 2006 with corrections to the Cover Date.     

Induction of systemic acquired resistance in Zea mays L. by Aspergillus flavus and A. parasiticus derived elicitors

Host defence mechanisms can be elicited by using different elicitors produced from the pathogen/host. In this study, an effort has been made to study the effect of two fungal elicitors derived from Aspergillus flavus and A. parasiticus on induction of various defence-related enzymes in maize (Zea mays L.). Foliar application was done on 20-days-old maize plant with 10% A. flavus fungal culture filtrate (AFFCF) and A. parasiticus fungal culture filtrate (APFCF) as elicitors to trigger systemic acquired resistance (SAR). As a response of SAR, an increase in activities of phenylalanine ammonia lyase (PAL), peroxidase (POX), β-1,3-glucanase, nitrate reductase (NR) and nitrite reductase (NiR), total proteins were found highest on 4th day after treatment (DAT), whereas total carbohydrate and total chlorophyll on 2nd and 6th DAT, respectively, in comparison with the control plants. The SDS PAGE analysis revealed the induction of PR proteins, namely Chitinase (25, 29?kDa) and β-1,3-glucanase (33?kDa), in treated plants in comparison with untreated control plants. The treated plants showed enhanced growth and development as well as increase in yield. About 100% survival rate was found in maize seeds treated with AFFCF and APFCF and grown on respective fungal infested soil than control. The enhanced activities of defence enzymes and elevated protein, carbohydrate, chlorophyll content in treated maize plants suggest the induction of SAR against A. flavus and A. parasiticus by using the same fungal elicitors.