Non-aflatoxigenic Strains Of Aspergillus Flavus Research Articles

Detection of Aflatoxin B1-producing Aspergillus flavus strains from pistachio orchards soil in Iran by multiplex polymerase chain reaction method.

The current study aimed to report a multiplex polymerase chain reaction (PCR) assay as a monitoring technique to differentiate aflatoxigenic from non-aflatoxigenic strains of Aspergillus flavus isolated from pistachio orchards soil. In total, 25 A. flavus strains were isolated from soil samples of pistachio orchards. To test the strains for Aflatoxin B1 (AFB1)-producing ability, thin-layer chromatography (TLC) was used and the amounts of AFB1 were measured by high-performance liquid chromatography (HPLC). Multiplex PCR was used as a genome-based method to detect genes responsible for AFB1 production by A. flavus and the results were analyzed in terms of speed and specificity of detection. A set of four primers was designed specifically for the omtA, omtB, ver-1, and aflR genes which are commonly present in aflatoxin biosynthetic pathways. The AFB1 production by the A. flavus strains ranged from 0 to 321 ρg/μl. Four-band patterns of the primer sets were observed only in AFB1-producing A. flavus strains. Moreover, 18 out of the 25 strains showed all four bands belonging to omtA, omtB, ver-1, and aflR, whereas 7 strains did not display omtA, or aflR-related bands, in non-toxigenic and low toxin-producing A. flavus. The multiplex PCR is a supplementary strategy to current conventional mycotoxin analytical techniques, such as TLC and HPLC. It could be used as an efficient method to differentiate aflatoxigenic from non-aflatoxigenic strains of A. flavus. This achievement is crucial to minimize fungal contamination of food, feed, and agricultural commodities, thereby reducing the risk of subsequent aflatoxin consumption.

Effect of non-aflatoxigenic strains of Aspergillus flavus on aflatoxin contamination of pre-harvest peanuts in fields in China

Aflatoxin contamination of peanuts is one of the most concerns in peanut production in China. Applying non-aflatoxigenic Aspergillus flavus strains, based on competitive exclusion, has been proved to be a promising strategy to reduce aflatoxin contamination in pre-harvest peanuts. Two non-aflatoxigenic A. flavus strains collected in China, which have been proved effectively reducing aflatoxin in the laboratory, were mixed with high aflatoxin producer to the soil in peanut growing season. The two non-aflatoxigenic strains significantly (P ​< ​0.05) reduced aflatoxin contamination in peanut kernels under both normal and drought stresses in two fields. Compared to control, the total aflatoxin (sum of aflatoxin B1 and B2) was reduced 26.7–99.12% in field 1, and 84.96–99.33% in field 2. The aflatoxin was reduced 84.96–99.33% under drought stress in two fields. The present study indicated the non-aflatoxigenic A. flavus strains could be potential biocontrol agents for reducing aflatoxin contamination under field condition.

Co-Inoculation of Aflatoxigenic and Non-Aflatoxigenic Strains of Aspergillus flavus to Assess the Efficacy of Non-Aflatoxigenic Strains in Growth Inhibition and Aflatoxin B1 Reduction

The pre-harvest biocontrol approach currently used includes laboratory inoculations using non-aflatoxigenic strains of Aspergillus flavus. This strategy effectively suppresses the indigenous aflatoxigenic strains and reduces aflatoxin accumulation in sweetcorn. The current in vitro study’s main objective is to determine the diametric growth rates of both Aflatoxin (AF)+ and AF− strains and improve the understanding of competitive relationships among these strains in sweetcorn (Zea mays). Sweetcorn kernels inoculated with AF+ strains only, AF− strains only, and co-inoculated with AF+ + AF− strains were investigated for aflatoxin concentrations. The diametric growth results revealed that growth rates of AF− strains at 25 and 30 °C were much greater than AF+ strains, which was in line with previous studies. The in vitro findings showed that the AKR5− and AKL34− biocontrol strains effectively inhibited the colony propagation and subsequent AFB1 contamination (up to 79%) of AF+ strains. On the other hand, the AKR1− and AKL35− were least effective in reducing AFB1 contents only by 58% and 60%, respectively. There was a significant difference (p &lt; 0.05) in the reduction of AFB1 contents achieved by AF− strains of A. flavus. The findings of the present study indicated the reduction in AFB1 with population expressions of AF+ strains by the AF− strains and supports the notion of competitive exclusion through vigorous development and propagation of the non-aflatoxigenic fungi.

Mechanism of inhibition of aflatoxin synthesis by non-aflatoxigenic strains of Aspergillus flavus

Aflatoxins are toxic secondary metabolites primarily produced by Aspergillus flavus and A. paraciticus. Exposure to these mycotoxins through contaminated food and feed may cause oxidative stress and liver toxicity in animals. One of the promising strategies to mitigate aflatoxin accumulation is the biological management during pre-harvest using non-aflatoxigenic A. flavus. The mechanism offered by these strains in mitigating aflatoxin is still unclear. Thus, the aim of the present study is to delineate the mechanism of intraspecific inhibition of aflatoxin production. Among the 18 non-aflatoxigenic strains evaluated, six strains were able to reduce more than 50% of the aflatoxins produced by the native aflatoxigenic strains. The non-aflatoxigenic strains used in this study failed to degrade the aflatoxins. Eventhough, the non-aflatoxigenic strains were not able to inhibit the synthesis of aflatoxins completely. Four non-aflatoxigenic isolates could competitively excluded the aflatoxigenic strain. Furthermore, when non-aflatoxigenic and an aflatoxigenic isolate were separated by 0.4 and 3 μm filters, aflatoxin synthesis was not significantly reduced. However, when the pore size was 8 μm, there was a significant decrease in aflatoxin production. This results suggest the role of physical contact between the hyphae, thigmoregulation, in the inhibition of aflatoxin production. Additionally, to better understand the transcriptional level control of this phenomenon, we analyzed the gene expression profile of aflatoxin biosynthesis genes in the aflatoxigenic strain. The aflatoxin biosynthesis genes were down regulated in the aflatoxigenic strain in contact with non-aflatoxigenic strain group when compared to the control. This is the first evidence of the combined action of competitive exclusion and thigmodownregulation which led to the intraspecific inhibition of aflatoxin production.

Field Displacement of Aflatoxigenic Aspergillus flavus Strains Through Repeated Biological Control Applications.

A popular pre-harvest strategy to mitigate aflatoxin contamination of corn involves field application of non-aflatoxigenic strains of Aspergillus flavus. The basis of this biological control may involve multiple factors, but competitive displacement of aflatoxigenic strains by the biocontrol strains is a likely mechanism. Three biocontrol strains (NRRL 21882, 18543, and 30797) were applied annually, over a 4-year period, to the same 3.2-ha commercial corn field in the Mississippi Delta, where we monitored their post-release establishment, spread, and persistence. Within 2 months of the first biocontrol application, the percentage of soil-inhabiting aflatoxigenic A. flavus strains in some plots was reduced from 48 to 9% of the population. The frequency of aflatoxigenic A. flavus strains was also significantly reduced in the rest of field. After 4 years, neighboring plots that had never received a biocontrol treatment, and distanced from our treatment plots by at least 20 meters, had less than 20% aflatoxigenic isolates. This significant halo effect might be attributed to movement of soil through tillage operations, but the aflatoxigenicity shift could be detected in the untreated plots within 2 months of the initial applications, at a time when there was no tillage. The A. flavus populations that colonized the grain were also monitored and found to be less than 15% toxigenic in the fourth year for all treatments. Over all treatments and years, less than 2 ppb of aflatoxin was detected, which could be a consequence of the field-wide shift of the inherent A. flavus population to predominately non-aflatoxigenic strains. This study supports the efficacy of using non-aflatoxigenic A. flavus strains as pre-harvest biocontrol, and shows that most of its effectiveness occurs with the first application.

Biocontrol Strains Differentially Shift the Genetic Structure of Indigenous Soil Populations of Aspergillus flavus.

Biocontrol using non-aflatoxigenic strains of Aspergillus flavus has the greatest potential to mitigate aflatoxin contamination in agricultural produce. However, factors that influence the efficacy of biocontrol agents in reducing aflatoxin accumulation under field conditions are not well-understood. Shifts in the genetic structure of indigenous soil populations of A. flavus following application of biocontrol products Afla-Guard and AF36 were investigated to determine how these changes can influence the efficacy of biocontrol strains in reducing aflatoxin contamination. Soil samples were collected from maize fields in Alabama, Georgia, and North Carolina in 2012 and 2013 to determine changes in the population genetic structure of A. flavus in the soil following application of the biocontrol strains. A. flavus L was the most dominant species of Aspergillus section Flavi with a frequency ranging from 61 to 100%, followed by Aspergillus parasiticus that had a frequency of <35%. The frequency of A. flavus L increased, while that of A. parasiticus decreased after application of biocontrol strains. A total of 112 multilocus haplotypes (MLHs) were inferred from 1,282 isolates of A. flavus L using multilocus sequence typing of the trpC, mfs, and AF17 loci. A. flavus individuals belonging to the Afla-Guard MLH in the IB lineage were the most dominant before and after application of biocontrol strains, while individuals of the AF36 MLH in the IC lineage were either recovered in very low frequencies or not recovered at harvest. There were no significant (P > 0.05) differences in the frequency of individuals with MAT1-1 and MAT1-2 for clone-corrected MLH data, an indication of a recombining population resulting from sexual reproduction. Population mean mutation rates were not different across temporal and spatial scales indicating that mutation alone is not a driving force in observed multilocus sequence diversity. Clustering based on principal component analysis identified two distinct evolutionary lineages (IB and IC) across all three states. Additionally, patristic distance analysis revealed phylogenetic incongruency among single locus phylogenies which suggests ongoing genetic exchange and recombination. Levels of aflatoxin accumulation were very low except in North Carolina in 2012, where aflatoxin levels were significantly (P < 0.05) lower in grain from treated compared to untreated plots. Phylogenetic analysis showed that Afla-Guard was more effective than AF36 in shifting the indigenous soil populations of A. flavus toward the non-toxigenic or low aflatoxin producing IB lineage. These results suggest that Afla-Guard, which matches the genetic and ecological structure of indigenous soil populations of A. flavus in Alabama, Georgia, and North Carolina, is likely to be more effective in reducing aflatoxin accumulation and will also persist longer in the soil than AF36 in the southeastern United States.

Aspergillus flavus NRRL 35739, a Poor Biocontrol Agent, May Have Increased Relative Expression of Stress Response Genes.

Biocontrol of the mycotoxin aflatoxin utilizes non-aflatoxigenic strains of Aspergillus flavus, which have variable success rates as biocontrol agents. One non-aflatoxigenic strain, NRRL 35739, is a notably poor biocontrol agent. Its growth in artificial cultures and on peanut kernels was found to be slower than that of two aflatoxigenic strains, and NRRL 35739 exhibited less sporulation when grown on peanuts. The non-aflatoxigenic strain did not greatly prevent aflatoxin accumulation. Comparison of the transcriptomes of aflatoxigenic and non-aflatoxigenic A. flavus strains AF36, AF70, NRRL 3357, NRRL 35739, and WRRL 1519 indicated that strain NRRL 35739 had increased relative expression of six heat shock and stress response proteins, with the genes having relative read counts in NRRL 35739 that were 25 to 410 times more than in the other four strains. These preliminary findings tracked with current thought that aflatoxin biocontrol efficacy is related to the ability of a non-aflatoxigenic strain to out-compete aflatoxigenic ones. The slower growth of NRRL 35739 might be due to lower stress tolerance or overexpression of stress response(s). Further study of NRRL 35739 is needed to refine our understanding of the genetic basis of competitiveness among A. flavus strains.

Characterization of non-aflatoxigenic strains of Aspergillus flavus as potential biocontrol agent for the management of aflatoxin contamination in groundnut.

In the present study, nine non-aflatoxigenic Aspergillus flavus strains were evaluated for their potential to reduce A.flavus infection and aflatoxin contamination in groundnut. Genetic characterization of these strains revealed six different deletion patterns (A-F) for thirteen examined genes from the aflatoxin biosynthesis pathway. Strain AFGS5 recorded maximum number of gene deletion (F) which included 12 out of 13 tested genes. Our findings indicated that aflR was the most frequently absent gene among the observed deletion patterns. A dendrogram inferred from combining random amplified polymorphic DNA and microsatellite data showed three of the non-aflatoxigenic strains segregating from other sampled isolates (aflatoxigenic and non-aflatoxigenic) tested. Greenhouse experiments, involving non-aflatoxigenic strains as biocontrol agents resulted in two strains, AFGS5 and AFGS12, which significantly reduced the population of aflatoxigenic fungi and the level of total aflatoxins in the rhizosphere/geocarposphere of soil samples as well as in groundnut seeds. Based on our findings, the use of these native non-aflatoxigenic strains; AFGS5 or AFGS12 in particular, as biopesticides could offer efficacious mitigation of aflatoxin contamination.

Characterization of Iranian nonaflatoxigenic strains of Aspergillus flavus based on microsatellite-primed PCR.

Out of fifty-two Iranian nonaflatoxigenic strains of Aspergillus flavus,collected from various substrates (soil and kernel) and sources (peanut, corn and pistachio), fifteen representatives were selected according to their different geographical origins (six provinces: Guilan and Golestan, Ardebil, Fars, Kerman and Semnan) and vegetative compatibility groups (VCGs, IR1 to IR15) for microsatellite-primed PCR analysis. Two inter-simple sequence repeat (ISSR) primers AFMPP and AFM13 were used to determine polymorphism and the relationship among strain isolates. A. flavus isolates were identified by their morphologies and their identities were confirmed by PCR amplification using the specific primer pair ITS1 and ITS4. The results revealed variations in the percentages of polymorphisms. In the ISSR analysis, primers AFMPP and AFM13 generated a total of 18 and 23 amplicons among the fungal strains, out of which 12 (66.7%) and 22 (95.7%) were polymorphic, respectively. Cluster analysis of the ISSR data was carried out using 1 D DNA gel image analysis. The two dendrograms obtained through these markers showed six different clusterings of testing nonaflatoxigenic A. flavus L strains, but we noticed that some clusters were different in some cases. The microsatellite-primed PCR data revealed that the Iranian nonaflatoxigenic isolates of A. flavus were not clustered according to their origins and sources. This study is the first to characterize Iranian nonaflatoxigenic isolates of A. flavususing ISSR markers.

Biological control of aflatoxin is effective and economical in Mississippi field trials

Aflatoxin contamination of corn reduces grain quality and can negatively impact the economic well-being of Mississippi's corn farmers. Mitigation of aflatoxin contamination in corn through pre-harvest field application of non-aflatoxigenic strains of Aspergillus flavus holds promise as an effective management strategy. In this study, field trials were conducted at fourteen sites over three years, in experimental plots and within commercial fields, to evaluate the efficacy of two biocontrol products at 11 or 22 kg ha−1 application rates and at various timings, including two vegetative-stages (V5 and V10), tasseling (VT), a reproductive application made at blister (R2) and two split-applications. The greatest reduction in aflatoxin generally occurred with applications at the earliest developmental stages. Furthermore, six treatments proved more cost-effective across multiple fields and years. These observations support the use of atoxigenic strains of A. flavus to promote the safety and marketability of corn grown in areas susceptible to aflatoxin contamination.

Discrimination of moldy peanuts with reference to aflatoxin using FTIR-ATR system

Abstract This study demonstrated the potential use of Fourier transform infrared spectroscopy, coupled with attenuated total reflectance unit (FTIR-ATR) for determination of aflatoxigenic and non-aflatoxigenic strains of Aspergillus flavus and Aspergillus parasiticus invasion in peanuts. Threshold mold density on peanut paste samples was 2.7 Log CFU/g peanut corresponding to legislative limiting aflatoxin (AF) level of 20 ppb. Classification was performed to separate the “Acceptable” stream (AF ≤ 20 ppb) from so called “Moldy” (20 1200 ppb). All of the samples (n = 164) were classified correctly when discriminant analysis technique was employed. Second threshold value was set at 300 ppb aflatoxin to further sort out the samples in the “Moldy” class into mildly (which can be used for feed) or highly toxic (which has to be discarded). Correct separation was observed at 98.5% with only one misclassified sample. Growth profiles of both strains of A. flavus and A. parasiticus were interpreted with respect to spectral changes. Even when spectral alterations for aflatoxin presence were not clearly identifiable, similar secondary metabolites of both aflatoxigenic species led to clusters in distance plots and showed the potential usage of the developed method to separate safer peanuts (e.g. AF ≤ 20 ppb) in a lot when implemented. Partial least squares (PLS) regression models were developed to predict AF level with maximum correlation coefficient of determination ( R C 2 = 99.98% for both AF–producing Aspergillus spp.). The fingerprint region (1800–800 cm−1) was used for regression analysis and corresponding bands were interpreted.

Co-inoculation of aflatoxigenic and non-aflatoxigenic strains of Aspergillus flavus to study fungal invasion, colonization, and competition in maize kernels.

A currently utilized pre-harvest biocontrol method involves field inoculations with non-aflatoxigenic Aspergillus flavus strains, a tactic shown to strategically suppress native aflatoxin-producing strains and effectively decrease aflatoxin contamination in corn. The present in situ study focuses on tracking the invasion and colonization of an aflatoxigenic A. flavus strain (AF70), labeled with green fluorescent protein (GFP), in the presence of a non-aflatoxigenic A. flavus biocontrol strain (AF36), to better understand the competitive interaction between these two strains in seed tissue of corn (Zea mays). Corn kernels that had been co-inoculated with GFP-labeled AF70 and wild-type AF36 were cross-sectioned and observed under UV and blue light to determine the outcome of competition between these strains. After imaging, all kernels were analyzed for aflatoxin levels. There appeared to be a population difference between the co-inoculated AF70-GFP+AF36 and the individual AF70-GFP tests, both visually and with pixel count analysis. The GFP allowed us to observe that AF70-GFP inside the kernels was suppressed up to 82% when co-inoculated with AF36 indicating that AF36 inhibited progression of AF70-GFP. This was in agreement with images taken of whole kernels where AF36 exhibited a more robust external growth compared to AF70-GFP. The suppressed growth of AF70-GFP was reflected in a corresponding (upto 73%) suppression in aflatoxin levels. Our results indicate that the decrease in aflatoxin production correlated with population depression of the aflatoxigenic fungus by the biocontrol strain supporting the theory of competitive exclusion through robust propagation and fast colonization by the non-aflatoxigenic fungus.

Aflatoxin Reduction in Corn Through Field Application of Competitive Fungi

Soil in corn plots was inoculated with nonaflatoxigenic strains of Aspergillus flavus and A. parasiticus during crop years 1994 to 1997 to determine the effect of application of the nontoxigenic strains on preharvest aflatoxin contamination of corn. Corn plots in a separate part of the field were not inoculated and served as controls. Inoculation resulted in significant increases in the total A. flavus/parasiticus soil population in treated plots, and that population was dominated by the applied strain of A. parasiticus (NRRL 21369). In the years when weather conditions favored aflatoxin contamination (1996 and 1997), corn was predominately colonized by A. flavus as opposed to A. parasiticus. In 1996, colonization by wild-type A. flavus was significantly reduced in treated plots compared with control plots, but total A. flavus/parasiticus colonization was not different between the two groups. A change to a more aggressive strain of A. flavus (NRRL 21882) as part of the biocontrol inoculum in 1997 resulted in a significantly (P < 0.001) higher colonization of corn by the applied strain. Weather conditions did not favor aflatoxin contamination in 1994 and 1995. In 1996, the aflatoxin concentration in corn from treated plots averaged 24.0 ppb, a reduction of 87% compared with the aflatoxin in control plots that averaged 188.4 ppb. In 1997, aflatoxin was reduced by 66% in treated corn (29.8 ppb) compared with control corn (87.5 ppb). Together, the data indicated that although the applied strain of A. parasiticus dominated in the soil, the nonaflatoxigenic strains of A. flavus were more responsible for the observed reductions in aflatoxin contamination. Inclusion of a nonaflatoxigenic strain of A. parasiticus in a biological control formulation for aflatoxin contamination may not be as important for airborne crops, such as corn, as for soilborne crops, such as peanuts.

Production of cyclopiazonic acid by aflatoxigenic and non-aflatoxigenic strains of Aspergillus flavus.

96 strains of Aspergillus flavus isolated from samples of stored grain and smoke-dried meat products were examined for ability to produce cyclopiazonic acid and aflatoxins, grown on mycological broth medium and malt extract agar. Five strains produced cyclopiazonic acid in the range of 0.5-30 mg/kg and 9 produced aflatoxin B1 (0.1-14.8 mg/kg) but none of them produced both cyclopiazonic acid and aflatoxins.

Glutathione Levels and γ-Glutamyl Transpeptidase Activities in Aflatoxigenic and Non-Aflatoxigenic Strains of Aspergillus Flavus

AbstractGlutathione (GSH) levels and activity of the enzyme γ-glutamyl transpeptidase (γ-GT) was assessed in aflatoxigenic and non-aflatoxigenic strains of Aspergillus flavus. The aflatoxigenic strains (NRRL 3240 and NRRL 2999) generally contained enhanced levels of glutathione while it was low in non-aflatoxigenic strains (NRRL 3537 and NRRL 5565) although considerable strain to strain difference existed. In order to examine factors controlling glutathione levels in this organism, all the strains were grown in sucrose low salt (SLS) medium and glucose ammonium nitrate (GAN) medium supporting high and low aflatoxin production respectively. The glutathione level varied for each strain with respect to the medium, suggesting a possible role of media related factors in controlling the formation of glutathione. Aflatoxigenic and non-aflatoxigenic strains were also compared for the activity of γ-GT. Significantly higher activity of γ-GT was associated with aflatoxigenic strains compared to non-aflatoxigenic str...

Response of Turkey Poults to Aerosolized Spores of Aspergillus fumigatus and Aflatoxigenic and Nonaflatoxigenic Strains of Aspergillus flavus

Groups of 3-week-old turkey poults were exposed to aerosols containing spores of either Aspergillus fumigatus or aflatoxigenic or nonaflatoxigenic strains of Aspergillus flavus. Approximately 5 X 10(5) spores of A. fumigatus/gm of lung tissue killed about 50% of the turkey poults with A. funigatus, whereas doses approximating this level with either of the A. flavus strains did not cause any mortalities for up to 8 weeks postexposure. Fungi were isolated from lung tissue of 60% of the birds surviving for 8 weeks after exposure to spores of A fumigatus, but A. flavus was isolated from lung tissue from only 20% of the birds 8 weeks after exposure to either A. flavus strain. Almost all birds exposed to A. fumigatus developed precipitating antibodies, whereas none of the birds exposed to A. flavus developed precipitating antibodies. The lesions in the lung tissue from birds exposed to A. fumigatus were larger, were more numerous, and involved a greater percentage of tissue than was observed in lung tissue from birds exposed to A. flavus. There were no apparent differences in pathogenicity between the A. flavus strains. Aflatoxin was not detected in tissues from birds exposed to the aflatoxigenic strain of A. flavus, and there were no signs of aflatoxigenic when sections of liver from these birds were examined histopathologically.

Production of Sclerotia by Aflatoxigenic and Nonaflatoxigenic Strains of Aspergillus Flavus and A. Parasiticus

Production of Sclerotia by Aflatoxigenic and Nonaflatoxigenic Strains of <i>Aspergillus Flavus</i> and <i>A. Parasiticus</i>

Production of Sclerotia by Aflatoxigenic and Nonaflatoxigenic Strains of Aspergillus flavus and A. parasiticus

Previous reports suggest that sclerotial strains of Aspergillus flavus and A. parasiticus may be high aflatoxin producers. We studied 14 strains that differed in both aflatoxin- and sclerotium-prod...

Cyclopiazonic acid production by aflatoxigenic and non-aflatoxigenic strains of Aspergillus flavus.

Twenty-eight of 54 isolates of Aspergillus flavus grown on autoclaved agricultural commodities such as wheat, rice and corn were found to produce the mycotoxin cyclopiazonic acid. Eighteen of the A. flavus isolates produced aflatoxin, and fourteen isolates produced both cyclopiazonic acid and aflatoxin. A preliminary screening of some aflatoxin-contaminated corn samples revealed for the first time the natural occurrence of cyclopiazonic acid in agricultural commodities.