Non-aflatoxigenic Strains Research Articles

Biocontrol of Aflatoxin-Producing Aspergillus flavus ATCC 22546 by a Non-Aflatoxigenic Aspergillus flavus ATCC 9643

The biological control of Aspergillus flavus and A. parasiticus by non-aflatoxigenic strains has been introduced in cotton- and peanut-cultivating fields and proven successful at reducing aflatoxin (AFB) contamination of crops and agricultural soils. In this study, a non-aflatoxigenic strain, A. flavus ATCC 9643 (ATCC 9643), was evaluated for its ability to competitively inhibit the growth of an aflatoxigenic strain, A. flavus ATCC 22546 (ATCC 22546), and mitigate AFB production in ATCC 22546 during competitive growth. To comparatively analyze the suppressive effect of ATCC 9643 on ATCC 22546, a non-aflatoxigenic strain (A. flavus ATCC 96045, known as AF36) was used as a positive control in some experiments. The two non-aflatoxigenic strains did not produce AFB1 or AFB2 owing to the absence of several AFB biosynthesis-related genes, especially aflK and aflL, which encode versicolorin B synthase and desaturase, respectively. To create a competitive growth environment, ATCC 9643 and ATCC 22546 were co-inoculated into a solid agar medium, and they grew at similar rates when added at a 1:1 ratio. Increasing the inoculum rate of ATCC 9643 (1:1, 1:3, 1:5) dramatically inhibited ATCC 22546 growth, and AFB production was effectively decreased by about 84%, 95%, and 97% by treatment with ATCC 9643. On rice, ATCC 22546 attenuated ATCC 9643 growth only when the rice was submerged in distilled water, whereas agar addition enhanced it. Taken together, ATCC 9643 is a promising candidate biological agent for suppressing aflatoxigenic A. flavus strain growth and alleviating AFB contamination. Further studies on AFB reduction in crop fields, including cotton-cultivation and maize-cultivation fields, are warranted.

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.

Characterization and Determination of Aflatoxigenic and Non-aflatoxigenic Aspergillus flavus Isolated from Bakery Food Products from Frontier around Davangere District

Mycotoxins are secondary metabolites produced by filamentous fungi in food and feed due to several conditions that affect fungal growth and mycotoxin production in different ways. Mycotoxin contamination in bakery food products is seriously dangerous to humans and animals. This study aimed to morphologically characterize and determine the aflatoxigenic and non-aflatoxigenic Aspergillus flavus isolates. Thirty three isolates of A.flavus were obtained from bakery food products collected from bakeries in rural areas like Bathi, Kodaganur, Bada, sasalu, Mayakonda, Avargere, Anaberu, Alur, Attigere, Echagatta around Davangere city. They were cultured on Potato Dextrose Agar medium (PDA), Rose Bengal Agar medium (RBA), Czapec(dox) agar medium (CZA) and Sabouraud’s dextrose agar medium (SDA). By observing the colony color and texture macromorphological characteristics were determined and micromorphological characteristics were determined by observing the spore color, size, structure, conidiophore structure, conidia, vesical shape under microscope. The production of aflatoxin was determined by UV fluorescence of isolates on coconut cream agar (CCA) medium plates. In aflatoxin detection 20 (60.60%) isolates of A.flavus shows positive results and 13 (39.39%) isolates of A.flavus shows negative results. The highest incidence of A.flavus was recorded in samples collected from Attigere followed by Bathi, Alur, Echagatta, Kodaganur, Bada, Anaberu, Sasalu, Mayakonda, Avargere. The incidence of A.flavus that produces aflatoxin highlights the necessity of taking action to eliminate their presence in food. The non-aflatoxigenic strains are being used in a biological control strategy to outcompete the aflatoxigenic ones.

Multiple Year Influences of the Aflatoxin Biocontrol Product AF-X1 on the A. flavus Communities Associated with Maize Production in Italy.

AF-X1 is a commercial aflatoxin biocontrol product containing the non-aflatoxigenic (AF-) strain of Aspergillus flavus MUCL54911 (VCG IT006), endemic to Italy, as an active ingredient. The present study aimed to evaluate the long-term persistence of VCG IT006 in the treated fields, and the multi-year influence of the biocontrol application on the A. flavus population. Soil samples were collected in 2020 and 2021 from 28 fields located in four provinces in north Italy. A vegetative compatibility analysis was conducted to monitor the occurrence of VCG IT006 on the total of the 399 isolates of A. flavus that were collected. IT006 was present in all the fields, mainly in the fields treated for 1 yr or 2 consecutive yrs (58% and 63%, respectively). The densities of the toxigenic isolates, detected using the aflR gene, were 45% vs. 22% in the untreated and treated fields, respectively. After displacement via the AF- deployment, a variability from 7% to 32% was noticed in the toxigenic isolates. The current findings support the long-term durability of the biocontrol application benefits without deleterious effects on each fungal population. Nevertheless, based on the current results, as well as on previous studies, the yearly applications of AF-X1 to Italian commercial maize fields should continue.

Variation with In Vitro Analysis of Volatile Profiles among Aspergillus flavus Strains from Louisiana

Volatile organic compounds (VOCs) produced by A. flavus strains were first captured and identified to discern between non-aflatoxigenic and toxigenic phenotypes, and more recently to help with detecting fungal infection, but not with the goal of using VOCs produced by non-aflatoxigenic strains to inhibit growth and/or production of one or more mycotoxins (e.g., aflatoxin and cyclopiazonic acid) by toxigenic aspergilli. In this study, four Aspergillus strains from Louisiana (one non-aflatoxigenic and three toxigenic) were grown on various substrates and had their headspaces captured and analyzed by solid-phase microextraction/gas chromatography/mass spectroscopy (SPME/GC/MS), to find biocontrol and biomarker compounds. Here, we present a collection of nearly 100 fungus-related VOCs, many of which were substrate dependent. Thirty-one were produced across multiple replicates and the rest were observed in a single replicate. At least three VOCs unique to non-aflatoxigenic strain LA1 can be tested for biocontrol properties (e.g., euparone, 4-nonyne), and at least four VOCs unique to toxigenic strains LA2-LA4 can be explored as biomarkers (e.g., 2-heptanone, glycocyamidine) to detect their presence while infecting crops in the field or in storage.

Application of Non-Aflatoxigenic Aspergillus flavus for the Biological Control of Aflatoxin Contamination in China.

Biological control through the application of competitive non-aflatoxigenic Aspergillus flavus (A. flavus) to the soil during peanut growth is a practical method for controlling aflatoxin contamination. However, appropriate materials need to be found to reduce the cost of biocontrol products. In this study, a two-year experiment was conducted under field conditions in China, using a native non-aflatoxigenic strain to explore its effect. After three months of storage under high humidity, aflatoxin levels remained low in peanuts from fields treated with the biocontrol agent. Three types of substrates were tested with the biocontrol agent: rice grains, peanut meal (peanut meal fertilizer) and peanut coating. Compared to untreated fields, these formulations resulted in reductions of 78.23%, 67.54% and 38.48%, respectively. Furthermore, the ratios of non-aflatoxigenic A. flavus recovered in the soils at harvest in the treated fields were between 41.11% and 96.67% higher than that in untreated fields (25.00%), indicating that the rice inoculum was the most effective, followed by the peanut meal fertilizer and peanut coating. In 2019, the mean aflatoxin content of freshly harvested peanuts in untreated fields was 19.35 µg/kg higher than that in the fields treated with 7.5 kg/ha rice inoculum, which was 1.37 µg/kg. Moreover, no aflatoxin was detected in the two other plots treated with 10 and 15 kg/ha rice inoculum. This study showed that the native Chinese non-aflatoxigenic strain of A. flavus (18PAsp-zy1) had the potential to reduce aflatoxin contamination in peanuts. In addition, peanut meal can be used as an alternative substrate to replace traditional grains, reducing the cost of biocontrol products.

Antagonism of nonaflatoxigenic Aspergillus flavus isolated from peanuts against aflatoxigenic A.flavus growth and aflatoxin B1 production in vitro.

Aspergillus section Flavi constitutes several species of opportunistic fungi, notable among them are A.flavus and A.parasiticus, capable of surviving harsh conditions and colonizing a wide range of agricultural products pre- and postharvest. Physical and chemical control methods are widely applied in order to mitigate the invasion of A.flavus in crops. However, physical control is not suitable for large scale and chemical control often leads to environmental pollution, whereas biological control offers a safer, environmentally friendly, and economical alternative. The present study aimed to investigate the antagonism of several non-aflatoxigenic A.flavus strains against the aflatoxigenic ones in vitro (semisynthetic peanut growth medium; MPA) in terms of colony growth rate and AFB1 inhibition. Different peanut concentrations were used to obtain the optimum peanut concentration in the formulated growth medium. A dual culture assay was performed to assess the antagonism of nonaflatoxigenic strains against the aflatoxigenic ones. Results revealed that 9% MPA exhibited the highest growth and AFB1 inhibition by nonaflatoxigenic strains. It was also found that different nonaflatoxigenic strains exhibited different antagonism against the aflatoxigenic ones which ranged from 11.09 ± 0.65% to 14.06 ± 0.14% for growth inhibition, and 53.97 ± 2.46% to 72.64 ± 4.54% for AFB1 inhibition. This variability could be due to the difference in antagonistic metabolites produced by different nonaflatoxigenic strains assessed in the present study. Metabolomics study to ascertain the specific metabolites that conferred the growth and aflatoxin inhibition is ongoing.

Aspergillus flavus La3279, a component strain of the Aflasafe™ biocontrol product, contains a partial aflatoxin biosynthesis gene cluster followed by a genomic region highly variable among A. flavus isolates

Aspergillus flavus communities in agricultural fields consist of isolates with varying abilities to produce aflatoxins, which are highly toxic and carcinogenic to humans and animals. Biological control using multiple non-aflatoxigenic strains as a formulation to outcompete aflatoxigenic A. flavus has become a mainstream strategy. Aflasafe™ is a biocontrol product composed of four strains, Ka16127, La3279, La3304 and Og0222. It was first developed in Nigeria and is now widely used on maize and groundnut. In this study, phylogenetic analyses based on genome-wide single nucleotide polymorphisms showed that Ka16127 and La3304 were more closely related to each other than both were to La3279, and the three were distantly related to Og0222. Detailed molecular characterization of La3279 indicated that its genome, contradictory to the published report, lacked approximately half of the aflatoxin gene cluster as well as the entire cyclopiazonic acid gene cluster. La3279 was a member of the previously known “pattern E” group, which includes A. flavus and Aspergillus oryzae isolates that have the aforementioned deletion followed by a 3.8-kb “E block” sequence insertion. In comparison to the E block, corresponding regions in typical aflatoxigenic S-morphotype/genotype isolates as well as Ka16127 and La3304 were found to lack 1.1 kb of the 5′ portion whereas L-morphotype/genotype isolates contained a complete nonhomologous region characterized by 2.5 copies of A. flavus telomeric repeat sequence at one end. Regions corresponding to the E block were highly variable and were useful for classifying A. flavus isolates into groups that mostly contained both mating types. The presence of both mating-type genes in genetically closely related A. flavus suggests a previously active sexual cycle. It could facilitate the development of a refined biocontrol strategy such as deploying biocontrol strains with the same mating-type that is predominant in a field A. flavus population.

Spatial and Temporal Population Dynamics of Aspergillus flavus in Commercial Pistachio Orchards in Arizona

Aspergillus flavus infects a wide range of crops, including pistachio, and subsequent aflatoxin contamination results in significant economic losses. Application of biocontrol products based on nonaflatoxigenic (atoxigenic) strains of A. flavus is one of the most effective tactics for controlling aflatoxins in crops. Both risk of aflatoxin contamination and effectiveness of biocontrol are influenced by the extent to which A. flavus spores move into pistachio tree canopies during periods of nut development. Thus, the purpose of this study was to evaluate spatial and temporal population dynamics of A. flavus, including the applied biocontrol strain AF36, in canopies of pistachio orchards in Arizona. Propagule densities of A. flavus were quantified on leaf samples collected from lower, middle, and upper canopies from spring through harvest in 2018 and 2019. A. flavus propagule densities peaked during periods of high temperature and rainfall in 2018 (up to 600 CFU/g) and 2019 (up to 23 CFU/g), which coincided with nut development and maturation. The applied biocontrol strain AF36 was detected at all canopy heights but overall propagule densities were greater in the upper and middle canopy (mean = 70 CFU/g) compared with the lower canopy (mean = 47 CFU/g). Results suggest that June to August is the period during which A. flavus inoculum increases in Arizona pistachio orchards and, to most effectively displace aflatoxin-producing fungi in tree canopies, biocontrol applications should precede this period. In addition, this study demonstrates that soil-applied biocontrol strains can successfully disperse throughout the canopies of commercial tree nut orchards.

Analysis of the competitiveness between a non-aflatoxigenic and an aflatoxigenic Aspergillus flavus strain on maize kernels by droplet digital PCR

Non-aflatoxigenic Aspergillus flavus strains are used as a biocontrol system on maize fields to decrease the aflatoxin biosynthesis of aflatoxigenic A. flavus strains. A. flavus strain AF36 was the first commercially available biocontrol strain and is authorized for use on maize fields by the US Environmental Protection Agency, e.g., in Texas and Arizona. A droplet digital PCR (ddPCR) assay was developed to analyze the mechanisms of competition and interaction of aflatoxigenic and non-aflatoxigenic A. flavus strains. This assay enables the parallel identification and quantification of the biocontrol strain A. flavus AF36 and the aflatoxigenic A. flavus strain MRI19. To test the assay, spores of both strains were mixed in varying ratios and were incubated on maize-based agar or maize kernels for up to 20 days. Genomic equivalent ratios (genome copy numbers) of both strains were determined by ddPCR at certain times after incubation and were compared to the spore ratios used for inoculation. The aflatoxin biosynthesis was also measured. In general, A. flavus MRI19 had higher competitiveness in the tested habitats compared to the non-aflatoxigenic strain, as indicated by higher final genomic equivalent ratios of this strain compared to the spore ratios used for inoculation. Nevertheless, A. flavus AF36 effectively controlled aflatoxin biosynthesis of A. flavus MRI19, as a clear aflatoxin inhibition was already seen by the inoculation of 10% spores of the biocontrol strain mixed with 90% spores of the aflatoxigenic strain compared to samples inoculated with only spores of the aflatoxigenic A. flavus MRI19.

Bio-competitive exclusion: efficacy of non-aflatoxigenic Aspergillus section Flavi-L morphotypes in control of aflatoxigenic Aspergillus flavus in groundnuts (Arachis hypogaea L.)

BackgroundThe biological control mechanism of Aspergillus flavus (aflatoxigenic) strains in groundnuts with atoxigenic strains from the same species through competitive exclusion employed the use of endemic and well-adapted strains within the agro-ecological zones of Zimbabwe. The selected elite non-aflatoxigenic isolates of A. flavus native to Zimbabwe were evaluated for their capability to reduce aflatoxin contamination in groundnuts under laboratory conditions.ResultsAverage reduction percentages in aflatoxin B concentration for the 2019 and 2020 set of experiments ranged from 91.6 ± 3.4 to 95.8 ± 3.1% and 90.29 ± 3.6% to 95.29 ± 4.1%, respectively. Levels of aflatoxin in the co-inoculation research experiments administered were significantly reduced in all the experimental units carried out. Treatment efficiencies of the tested isolates in this study at 4:1 and 2:1 ranged from 1.20 to 2.52 and from 1.02 to 1.21, respectively. The efficacy of the tested non-aflatoxigenic strains against the aflatoxigenic strain native to Zimbabwe (ZMW 0127) indicates that the non-aflatoxigenic isolates of A. flavus. have sound practical applications against vast communities of aflatoxin-producing fungi across all the agro-ecological zones in Zimbabwe.ConclusionThe recognized non-aflatoxigenic isolates will be of an incentive as dynamic active ingredients in biocontrol formulations for the decrease in aflatoxins in groundnuts grown in Zimbabwe.

Chromatographic Analysis of Aflatoxigenic Aspergillus flavus Isolated from Malaysian Sweet Corn

High-performance liquid chromatography (HPLC) provides a quick and efficient tool for accurately characterizing aflatoxigenic and non-aflatoxigenic isolates of Aspergillus flavus. This method also provides a quantitative analysis of AFs in Aspergillus flavus. The method’s recovery was assessed by spiking a mixture of AF at different concentrations to the testing medium. The validity of the method was confirmed using aflatoxigenic and non-aflatoxigenic strains of A. flavus. The HPLC system, coupled with a fluorescence detector and post-column photochemical reactor, showed high sensitivity in detecting spiked AFs or AFs produced by A. flavus isolates. Recovery from medium spiked with 10, 20, 60, and 80 ppb of AFs was found to be 73–86% using this approach. For AFB1 and AFB2, the limit of detection was 0.072 and 0.062 ppb, while the limit of quantification was 0.220 and 0.189 ppb, respectively. The AFB1 concentrations ranged from 0.09 to 50.68 ppb, while the AFB2 concentrations ranged between 0.33 and 9.23 ppb. The findings showed that six isolates produced more AFB1 and AFB2 than the acceptable limit of 5 ppb. The incidence of aflatoxigenic isolates of A. flavus in sweet corn and higher concentrations of AFB1 and AFB2 emphasize the need for field trials to explore their real potential for AF production in corn.

Biocontrol of Aflatoxins Using Non-Aflatoxigenic Aspergillus flavus: A Literature Review.

Aflatoxins (AFs) are mycotoxins, predominantly produced by Aspergillus flavus, A. parasiticus, A. nomius, and A. pseudotamarii. AFs are carcinogenic compounds causing liver cancer in humans and animals. Physical and biological factors significantly affect AF production during the pre-and post-harvest time. Several methodologies have been developed to control AF contamination, yet; they are usually expensive and unfriendly to the environment. Consequently, interest in using biocontrol agents has increased, as they are convenient, advanced, and friendly to the environment. Using non-aflatoxigenic strains of A. flavus (AF−) as biocontrol agents is the most promising method to control AFs’ contamination in cereal crops. AF− strains cannot produce AFs due to the absence of polyketide synthase genes or genetic mutation. AF− strains competitively exclude the AF+ strains in the field, giving an extra advantage to the stored grains. Several microbiological, molecular, and field-based approaches have been used to select a suitable biocontrol agent. The effectiveness of biocontrol agents in controlling AF contamination could reach up to 99.3%. Optimal inoculum rate and a perfect time of application are critical factors influencing the efficacy of biocontrol agents.

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.

Morphological Characterization and Determination of Aflatoxigenic and Non-Aflatoxigenic Aspergillus flavus Isolated from Sweet Corn Kernels and Soil in Malaysia

This study aimed to morphologically characterize and determine the aflatoxigenic and non-aflatoxigenic Aspergillus flavus isolates. Forty isolates of A. flavus were obtained from sweet corn kernels and soil samples collected from Kampong Raja, Rose Valley, Kea, and Klebang farms in Malaysia. They were cultured on potato dextrose agar (PDA), dichloran rose-bengal chloramphenicol (DRBC), Aspergillusflavus and Aspergillus parasiticus agar (AFPA), and coconut cream agar (CCA). Macromorphological characteristics were determined by observing the colony color and texture, while the micromorphological characteristics were determined by examining the spore color, size, structure, conidiophore structure, and vesicle shape. The production of aflatoxin was determined by direct visualization of the UV fluorescence of A. flavus colonies on CCA. Aflatoxin was qualitatively detected in 18 (45%) isolates of A. flavus using UV fluorescence screening while the remaining 22 (55%) isolates did not exhibit any aflatoxin production. The highest incidence of A. flavus (30%) and aflatoxin production (15%) was recorded in samples from Kampong Raja. On the other hand, isolates from Rose Valley (17%) and Kea (12%) were non-aflatoxigenic. Klebang recorded a 25% incidence of A. flavus in which 15% were aflatoxigenic while 10% were non-aflatoxigenic. The occurrence of aflatoxin-producing A. flavus emphasizes the need for the measure to eradicate their presence in food crops. A biological control treatment utilizing the non-aflatoxigenic strains to compete with the aflatoxigenic ones is underway. Validation of aflatoxin production through high performance liquid chromatography is also ongoing.

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.

Rapid isolation of non-aflatoxigenicAspergillus flavus strains

In the present study, a method for screening non-aflatoxigenicAspergillus flavus in soil samples collected from major peanut-growing regions of China was developed. The single colonies were picked and cultured onAspergillus flavus andparasiticus agar (AFPA). If the reverse side of the colony on AFPA was orange-coloured, it was consideredA. flavus orAspergillus parasiticus. After the genomic DNA of each strain was extracted, 28S rRNA and calmodulin were amplified and sequenced to determine the species. The key gene,aflR, was amplified and digested via polymerase chain reaction-restriction fragment length polymorphism. The aflatoxigenicA. flavus and the non-aflatoxigenicA. flavus andA. parasiticus were distinguished by enzyme digestion ofaflR. 156 strains ofA. flavus were screened, which consisted of 135 aflatoxigenic and 21 non-aflatoxigenic strains. The aflatoxin producing ability of each strain was confirmed using solid-state fermentation experiments. Using the method developed in the present study, we confirmed that the non-aflatoxigenicA. flavus strains isolated lost their capacity to produce aflatoxins. Considering there could be some alterations in other functional genes, some non-aflatoxigenic strains could be identified inaccurately as aflatoxigenic strains, although that did not occur in the present study. The growth of non-aflatoxigenicA. flavus was observed, and the most rapidly growing non-aflatoxigenic strain was selected for plate confrontation assays and toxic mixed culture experiments. The inhibition rate of non-aflatoxigenicA. flavus against aflatoxigenicA. flavus was 55.4 and 72.6% in potato dextrose agar (PDA) plate and natural soybean medium, respectively. The screened non-aflatoxigenicA. flavus strains provide a microbial resource for biological control of aflatoxin contamination.

Molecular profile of non-aflatoxigenic phenotype in native strains of Aspergillus flavus.

Aflatoxins are the most common mycotoxin contaminant reported in food and feed. Aflatoxin B1, the most toxic among different aflatoxins, is known to cause hepatocellular carcinoma in animals. Aspergillus flavus and A. parasiticus are the main producers of aflatoxins and are widely distributed in tropical countries. Even though several robust strategies have been in use to control aflatoxin contamination, the control at the pre-harvest level is primitive and incompetent. Therefore, the aim of the study was to isolate and identify the non-aflatoxigenic A. flavus and to delineate the molecular mechanism for the loss of aflatoxin production by the non-aflatoxigenic isolates. Eighteen non-aflatoxigenic strains were isolated from various biological sources using cultural and analytical methods. Among the 18 isolates, 8 isolates produced sclerotia and 17 isolates had type I deletion in norB-cypA region. The isolates were confirmed as A. flavus using gene-specific PCR and sequencing of the ITS region. Later, aflatoxin gene-specific PCR revealed that the defect in one or more genes has led to non-aflatoxigenic phenotype. The strain R9 had maximum defect, and genes avnA and verB had the highest frequency of defect among the non-aflatoxigenic strains. Further, qRT-PCR confirmed that the non-aflatoxigenic strains had high frequency of defect or downregulation in the late pathway genes compared to early pathway genes. Thus, these non-aflatoxigenic strains can be the potential candidates for an effective and proficient strategy for the control of pre-harvest aflatoxin contamination.

Development of an ELISA-based method for testing aflatoxigenicity and aflatoxigenic variability among Aspergillus species in culture

Aflatoxins contaminate foodstuff posing a severe threat to human health because chronic exposure is linked to liver cancer while acute exposure may cause death. Therefore, it is of interest to reduce the contamination of crops by aflatoxins in the field and post-harvest. Among the current technologies being developed is the deployment of non-aflatoxigenic strains of Aspergillus species to competitively exclude aflatoxigenic conspecifics from crops in the field thereby curtailing aflatoxin production by the former. The success in this endeavor makes the non-aflatoxigenic fungi good candidates for biological control programs. However, the current techniques for segregating non-aflatoxigenic from aflatoxigenic fungi suffer two main drawbacks: they are based on morphological and chemical tests with a combination of visual color changes detected in a culture plate which suffer some degree of inaccuracy. Secondly, the existing methods are incapable of accurately quantifying aflatoxin production by fungi in culture. We developed a culture system for inducing aflatoxin production by Aspergillus using maize kernels as growth substrate followed by quantification using ELISA. The method was compared to the Dichlorvos-Ammonia (DV-AM) method for determining aflatoxigenicity. Our findings encapsulate a method more robust than the currently used DV-AM approach because, for the first time, we are able to assess aflatoxigenicity and aflatoxigenic variability among Aspergillus species earlier classified as non-aflatoxigenic by the DV-AM method. Furthermore, the new method presents an opportunity to attribute the toxin production by actively growing fungal cultures. We believe this method when further developed presents a chance to study and predict fungal behavior prior to field trials for biological control programs.