Search Results (11)

Aspergillus flavus can produce aflatoxins, posing a threat of contamination to peanuts. To mitigate this issue, the use of biocontrol isolates, which do not produce aflatoxins (AF⁻), has been considered to reduce aflatoxin levels. In this study, we evaluated five different AF⁻ isolates belonging to different vegetative compatibility groups, all of which exhibited varying degrees of deletion in aflatoxin biosynthesis gene clusters. One isolate that exhibited poor competitive ability against toxigenic A. flavus was eliminated, and the remaining four isolates were formulated as biocontrol agents and applied to a peanut field in Tai’an, Shandong, as a combination. Three months after application, the soil aflatoxin content was reduced from 0.62 ± 0.01 to 0.19 ± 0.03 μg/kg (inhibition rate: 69.35%). Among filamentous fungi in the soil, the proportion of AF⁻ isolates increased from 0% to 4.33%. Using SSR-specific primers, the microbial agents were recovered. We discovered that among the four AF⁻ isolates, CA04 had a lower colonization rate compared to the other three (only 12.00% of the total AF⁻ population), suggesting that the absence of sclerotia may result in poor reversibility and weaker dispersal ability. We utilized Illumina sequencing to investigate the changes in soil fungal ecology. The results showed a reduction in the population density of harmful fungi, such as Fusarium spp. (66.18%) and Plectosphaerella spp. (79.90%), but an increase in the density of Nothopassalora personata. This is the first study on the dispersal distance and soil fungal community structure following the application of AF⁻ agents in peanut fields in China. Full article

Aflatoxins (AFs) are considered to play important functions in species of Aspergillus section Flavi including an antioxidative role, as a deterrent against fungivorous insects, and in antibiosis. Atoxigenic Flavi are known to degrade AF-B1 (B1). To better understand the purpose of AF degradation, we investigated the degradation of B1 and AF-G1 (G1) in an antioxidative role in Flavi. Atoxigenic and toxigenic Flavi were treated with artificial B1 and G1 with or without the antioxidant selenium (Se), which is expected to affect levels of AF. After incubations, AF levels were measured by HPLC. To estimate which population would likely be favoured between toxigenic and atoxigenic Flavi under Se, we investigated the fitness, by spore count, of the Flavi as a result of exposure to 0, 0.40, and 0.86 µg/g Se in 3%-sucrose cornmeal agar (3gCMA). Results showed that levels B1 in medium without Se were reduced in all isolates, while G1 did not significantly change. When the medium was treated with Se, toxigenic Flavi significantly digested less B1, while levels of G1 significantly increased. Se did not affect the digestion of B1 in atoxigenic Flavi, and also did not alter levels of G1. Furthermore, atoxigenic strains were significantly fitter than toxigenic strains at Se 0.86 µg/g 3gCMA. Findings show that while atoxigenic Flavi degraded B1, toxigenic Flavi modulated its levels through an antioxidative mechanism to levels less than they produced. Furthermore, B1 was preferred in the antioxidative role compared to G1 in the toxigenic isolates. The higher fitness of atoxigenic over toxigenic counterparts at a plant non-lethal dose of 0.86 µg/g would be a useful attribute for integration in the broader biocontrol prospects of toxigenic Flavi. Full article

In order to study the relationship between the distribution and aflatoxin production capacity of Aspergillus species and soil types, 35 soil samples were collected from the main peanut planting areas in Xiangyang, which has 19.7 thousand square kilometers and is located in a special area with different soil types. The soil types of peanut planting areas in Xiangyang are mainly sandy loam and clay loam, and most of the soil is acidic, providing unique nature conditions for this study. The results showed that the Aspergillus sp. population in clay loam (9050 cfu/g) was significantly larger than that in sandy loam (3080 cfu/g). The percentage of atoxigenic Aspergillus strains isolated from sandy loam samples was higher than that from clay loam samples, reaching 58.5%. Meanwhile the proportion of high toxin-producing strains from clay loam (39.7%) was much higher than that from sandy loam (7.3%). Under suitable culture conditions, the average aflatoxin production capacity of Aspergillus isolates from clay loam samples (236.97 μg/L) was higher than that of strains from sandy loam samples (80.01 μg/L). The results inferred that under the same regional climate conditions, the density and aflatoxin production capacity of Aspergillus sp. in clay loam soil were significantly higher than that in sandy loam soil. Therefore, peanuts from these planting areas are at a relatively higher risk of contamination by Aspergillus sp. and aflatoxins. Full article

Crop yield and plant products quality are directly or indirectly affected by climate alterations. Adverse climatic conditions often promote the occurrence of different abiotic stresses, which can reduce or enhance the susceptibility to pests or pathogens. Aflatoxin producing fungi, in particular, whose diffusion and deleterious consequences on cereals commodities have been demonstrated to highly depend on the temperature and humidity conditions that threaten increasingly larger areas. Biological methods using intraspecific competitors to prevent fungal development and/or toxin production at the pre-harvest level are particularly promising, even if their efficacy could be affected by the ecological interaction within the resident microbial population. A previously characterized Aspergillus flavus atoxigenic strain was applied in two maize fields to validate its effectiveness as a biocontrol agent against aflatoxin contamination. At one month post-application, at the harvest stage, its persistence within the A. flavus population colonizing the maize kernels in the treated area was assessed, and its efficacy was compared in vitro with a representation of the isolated atoxigenic population. Results proved that our fungal competitor contained the aflatoxin level on maize grains as successfully as a traditional chemical strategy, even if representing less than 30% of the atoxigenic strains re-isolated, and achieved the best performance (in terms of bio-competitive potential) concerning endogenous atoxigenic isolates. Full article

Aspergillus flavus is a common filamentous fungus widely present in the soil, air, and in crops. This facultative pathogen of both animals and plants produces aflatoxins, a group of mycotoxins with strong teratogenic and carcinogenic properties. Peanuts are highly susceptible to aflatoxin contamination and consumption of contaminated peanuts poses serious threats to the health of humans and domestic animals. Currently, the competitive displacement of aflatoxin-producers from agricultural environments by atoxigenic A. flavus is the most effective method of preventing crop aflatoxin contamination. In the current study, 47 isolates of A. flavus collected from peanut samples originating in Shandong Province were characterized with molecular methods and for aflatoxin-producing ability in laboratory studies. Isolates PA04 and PA10 were found to be atoxigenic members of the L strains morphotype. When co-inoculated with A. flavus NRRL3357 at ratios of 1:10, 1:1, and 10:1 (PA04/PA10: NRRL3357), both atoxigenic strains were able to reduce aflatoxin B₁ (AFB₁) levels, on both culture media and peanut kernels, by up to 90%. The extent to which atoxigenic strains reduced contamination was correlated with the inoculation ratio. Abilities to compete of PA04 and PA10 were also independently verified against local aflatoxin-producer PA37. The results suggest that the two identified atoxigenic strains are good candidates for active ingredients of biocontrol products for the prevention of aflatoxin contamination of peanuts in Shandong Province. Full article

Aflatoxin contamination of important food and feed crops occurs frequently in warm tropical and subtropical regions. The contamination is caused mainly by Aspergillus flavus and A. parasiticus. Aflatoxin contamination negatively affects health and trade sectors and causes economic losses to agricultural industries. Many pre- and post-harvest technologies can limit aflatoxin contamination but may not always reduce aflatoxin concentrations below tolerance thresholds. However, the use of atoxigenic (non-toxin producing) isolates of A. flavus to competitively displace aflatoxin producers is a practical strategy that effectively limits aflatoxin contamination in crops from field to plate. Biocontrol products formulated with atoxigenic isolates as active ingredients have been registered for use in the US, several African nations, and one such product is in final stages of registration in Italy. Many other nations are seeking to develop biocontrol products to protect their crops. In this review article we present an overview of the biocontrol technology, explain the basis to select atoxigenic isolates as active ingredients, describe how formulations are developed and tested, and describe how a biocontrol product is used commercially. Future perspectives on formulations of aflatoxin biocontrol products, along with other important topics related to the aflatoxin biocontrol technology are also discussed. Full article

Aspergillus flavus colonisation of maize can produce mycotoxins that are detrimental to both human and animal health. Screening of maize lines, resistant to A. flavus infection, together with a biocontrol strategy, could help minimize subsequent aflatoxin contamination. We developed a qPCR assay to measure A. flavus biomass and showed that two African maize lines, GAF4 and KDV1, had different fungal loads for the aflatoxigenic isolate (KSM014), fourteen days after infection. The qPCR assay revealed no significant variation in A. flavus biomass between diseased and non-diseased maize tissues for GAF4, while KDV1 had a significantly higher A. flavus biomass (p < 0.05) in infected shoots and roots compared to the control. The biocontrol strategy using an atoxigenic isolate (KSM012) against the toxigenic isolate (KSM014), showed aflatoxin production inhibition at the co-infection ratio, 50:50 for both maize lines (KDV1 > 99.7% and GAF ≥ 69.4%), as confirmed by bioanalytical techniques. As far as we are aware, this is the first report in Kenya where the biomass of A. flavus from maize tissue was detected and quantified using a qPCR assay. Our results suggest that maize lines, which have adequate resistance to A. flavus, together with the appropriate biocontrol strategy, could limit outbreaks of aflatoxicoses. Full article

The dichlorvos–ammonia (DV–AM) method is a sensitive method for distinguishing aflatoxigenic fungi by detecting red (positive) colonies. In this study, the DV–AM method was applied for the isolation of aflatoxigenic and atoxigenic fungi from soil samples from a maize field in Mexico. In the first screening, we obtained two isolates from two soil subsamples of 20 independent samples and, in the second screening, we obtained two isolates from one subsample of these. Morphological and phylogenic analyses of the two isolates (MEX-A19-13, MEX-A19-2^nd-5) indicated that they were Aspergillus flavus located in the A. flavus clade. Chemical analyses demonstrated that one isolate could produce B-type aflatoxins, while the other produced no aflatoxins. These results demonstrate that the DV–AM method is useful for the isolation of both aflatoxigenic and atoxigenic Aspergilli. Full article

The emergence of super-toxigenic strains by recombination is a risk from an intensive use of intraspecific aflatoxin (AF) biocontrol agents (BCAs). Periodical alternation with interspecific-BCAs will be safer since they preclude recombination. We are developing an AF-biocontrol system using rice-associated Bacilli reported previously (RABs). More than 50% of RABs inhibited the growth of multiple A. flavus strains, with RAB4R being the most inhibitory and RAB1 among the least. The fungistatic activity of RAB4R is associated with the lysis of A. flavus hyphal tips. In field trails with the top five fungistatic RABs, RAB4R consistently inhibited AF contamination of maize by Tox4, a highly toxigenic A. flavus strain from Louisiana corn fields. RAB1 did not suppress A. flavus growth, but strongly inhibited AF production. Total and HPLC-fractionated lipopeptides (LPs) isolated from culture filtrates of RAB1 and RAB4R also inhibited AF accumulation. LPs were stable in vitro with little loss of activity even after autoclaving, indicating their potential field efficacy as a tank-mix application. A. flavus colonization and AF were suppressed in RAB1- or RAB4R-coated maize seeds. Since RAB4R provided both fungistatic and strong anti-mycotoxigenic activities in the laboratory and field, it can be a potent alternative to atoxigenic A. flavus strains. On the other hand, RAB1 may serve as an environmentally safe helper BCA with atoxigenic A. flavus strains, due its lack of strong fungistatic and hemolytic activities. Full article

Several atoxigenic Aspergillus flavus isolates, including some being used as biocontrol agents, and one toxigenic isolate were surveyed for the ability to produce extracellular xylanolytic and pectinolytic hydrolases. All of the tested isolates displayed good production of endoxylanases when grown on a medium utilizing larch xylan as a sole carbon substrate. Four of the tested isolates produced reasonably high levels of esterase activity, while the atoxigenic biocontrol agent NRRL 21882 isolate esterase level was significantly lower than the others. Atoxigenic A. flavus isolates 19, 22, K49, AF36 (the latter two are biocontrol agents) and toxigenic AF13 produced copious levels of pectinolytic activity when grown on a pectin medium. The pectinolytic activity levels of the atoxigenic A. flavus 17 and NRRL 21882 isolates were significantly lower than the other tested isolates. In addition, A. flavus isolates that displayed high levels of pectinolytic activity in the plate assay produced high levels of endopolygalacturonase (pectinase) P2c, as ascertained by isoelectric focusing electrophoresis. Isolate NRRL 21882 displayed low levels of both pectinase P2c and pectin methyl esterase. A. flavus appears capable of producing these hydrolytic enzymes irrespective of aflatoxin production. This ability of atoxigenic isolates to produce xylanolytic and pectinolytic hydrolases mimics that of toxigenic isolates and, therefore, contributes to the ability of atoxigenic isolates to occupy the same niche as A. flavus toxigenic isolates. Full article

Drought stress in the field has been shown to exacerbate aflatoxin contamination of maize and peanut. Drought and heat stress also produce reactive oxygen species (ROS) in plant tissues. Given the potential correlation between ROS and exacerbated aflatoxin production under drought and heat stress, the objectives of this study were to examine the effects of hydrogen peroxide (H₂O₂)-induced oxidative stress on the growth of different toxigenic (+) and atoxigenic (−) isolates of Aspergillus flavus and to test whether aflatoxin production affects the H₂O₂ concentrations that the isolates could survive. Ten isolates were tested: NRRL3357 (+), A9 (+), AF13 (+), Tox4 (+), A1 (−), K49 (−), K54A (−), AF36 (−), and Aflaguard (−); and one A. parasiticus isolate, NRRL2999 (+). These isolates were cultured under a H₂O₂ gradient ranging from 0 to 50 mM in two different media, aflatoxin-conducive yeast extract-sucrose (YES) and non-conducive yeast extract-peptone (YEP). Fungal growth was inhibited at a high H₂O₂ concentration, but specific isolates grew well at different H₂O₂ concentrations. Generally the toxigenic isolates tolerated higher concentrations than did atoxigenic isolates. Increasing H₂O₂ concentrations in the media resulted in elevated aflatoxin production in toxigenic isolates. In YEP media, the higher concentration of peptone (15%) partially inactivated the H₂O₂ in the media. In the 1% peptone media, YEP did not affect the H₂O₂ concentrations that the isolates could survive in comparison with YES media, without aflatoxin production. It is interesting to note that the commercial biocontrol isolates, AF36 (−), and Aflaguard (−), survived at higher levels of stress than other atoxigenic isolates, suggesting that this testing method could potentially be of use in the selection of biocontrol isolates. Further studies will be needed to investigate the mechanisms behind the variability among isolates with regard to their degree of oxidative stress tolerance and the role of aflatoxin production. Full article