Atoxigenic Strains Research Articles

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.

Farmer perceptions of aflatoxin management strategies in lower Eastern Kenya

Aflatoxins pose a serious problem to maize grains, rendering them unsuitable for human and livestock consumption. This study assessed farmers’ attitude toward, and knowledge and perceptions of, the nature, causes and use of biological technologies in aflatoxin control in Embu, Machakos, Makueni and Kitui Counties of Kenya. A total of 480 households were randomly selected from two districts per county using a GPS system. The tools used in the survey included pre-tested semi-structured questionnaires, focused group discussions, key informant interviews and secondary data exploration. The information collected included crop production constraints, existing aflatoxin management strategies, farmers’ experiences with use of atoxigenic strains of Aspergillus and other household farm production activities. The study found that farmers were largely aware of the aflatoxin problem and its negative effects, factors that contribute to its occurrence, and available options to address it. The main pre-harvest aflatoxin control technologies used were crop rotation, irrigation, use of resistant crop varieties and pest control. The main post-harvest aflatoxin control technologies used were proper storage, drying, sorting, and use of pesticides to manage pests. Modern aflatoxin control technologies such as ammoniation and use of hydrogels were not in use mainly due to unavailability, high cost and safety concerns (especially regarding grains meant for human consumption). Effective management of aflatoxin contamination requires that farmers are aware of the problem and use a combination of strategies targeting the crop before and after harvesting, and understanding farmers’ perceptions and knowledge is a vital step to finding a local solution for aflatoxin management. The study recommends the strengthening of existing public extension service system to enable it deliver up-to-date information, through a variety of channels, on aflatoxin and its management to farmers in a more effective and timely manner. Key words: Aflatoxin, Aflasafe, farmers, perceptions, Kenya.

Method for monitoring deletions in the aflatoxin biosynthesis gene cluster of Aspergillus flavus with multiplex PCR.

The report presents a rapid, inexpensive and simple method for monitoring indels with influence on aflatoxin biosynthesis within Aspergillus flavus populations. PCR primers were developed for 32 markers spaced approximately every 5kb from 20kb proximal to the aflatoxin biosynthesis gene cluster to the telomere repeat. This region includes gene clusters required for biosynthesis of aflatoxins and cyclopiazonic acid; the resulting data were named cluster amplification patterns (CAPs). CAP markers are amplified in four multiplex PCRs, greatly reducing the cost and time to monitor indels within this region across populations. The method also provides a practical tool for characterizing intraspecific variability in A.flavus not captured with other methods. Aflatoxins, potent naturally-occurring carcinogens, cause significant agricultural problems. The most effective method for preventing contamination of crops with aflatoxins is through use of atoxigenic strains of Aspergillus flavus to alter the population structure of this species and reduce incidences of aflatoxin producers. Cluster amplification pattern (CAP) is a rapid multiplex PCR method for identifying and monitoring indels associated with atoxigenicity in A.flavus. Compared to previous techniques, the reported method allows for increased resolution, reduced cost, and greater speed in monitoring the stability of atoxigenic strains, incidences of indel mediated atoxigenicity and the structure of A.flavus populations.

A critical evaluation of cultural methods for the identification of atoxigenic Aspergillus flavus isolates for aflatoxin mitigation in pistachio orchards of Iran

Aflatoxin contamination of tree nuts is a growing concern for pistachio producing countries. Development of competitive exclusion strategies through application of atoxigenic Aspergillus flavus iso- lates is a highly effective route of natural aflatoxin mitigation. Aflatoxin assays conducted on a high num- ber of native A. flavus isolates are a first step to identify potential biological control isolates. Many cultural methods for the rapid and visual identification of atoxigenic A. flavus isolates have been described. The current study identified atoxigenic A. flavus isolates from Iranian pistachio orchards using and contrasting cultural, analytical and molecular methods. Ammonium vapour (AV) and fluorescence detection (FD), two rapid aflatoxin assays, were directly compared using various media preparations to screen 524 A. flavus isolates ob- tained from Iranian pistachio orchards. Percentages of false negatives were high using FD assays for all media preparationsrangingfrom13to15%.Thisincontrastto AV assays. Here incidences of false negatives ranged from 0 % (using coconut agar medium) to 7.2 % (using potato dextrose agar). Aflatoxin-producing ability of all isolates was further confirmed using thin layer- and high-performance liquid chromatography. Sixty three atoxigenic A. flavus isolates were identified as atoxigenic in all assays. For these isolates, five loci across the aflatoxin biosynthesis cluster path- way were compared to identify genetic defects explaining atoxigenicity. Genetic deletions in at least one of five loci in the aflatoxin biosynthesis path- way were found for 97 % of isolates. Frequencies of atoxigenic strains ranged from 7.1 to 37.5 % with the lowest incidence detected in the Kerman prov- ince. Proper identification of atoxigenic isolates is considered a first step in the development of biolog- ical control strategies. Ability of identified isolates to competitively exclude aflatoxin-producing fungi has to be further investigated.

Biodiversity of Aspergillus section Flavi in Europe in relation to the management of aflatoxin risk.

Aflatoxins and the producing fungi Aspergillus section Flavi are widely known as the most serious and dangerous mycotoxin issue in agricultural products. In Europe, before the outbreak of aflatoxins on maize (2003–2004) due to new climatic conditions, their contamination was confined to imported foods. Little information is available on molecular biodiversity and population structure of Aspergillus section Flavi in Europe. Preliminary reports evidenced the massive presence of Aspergillus flavus L -morphotype as the predominant species in maize field, no evidence of the highly toxigenic S-morphotype and of other aflatoxigenic species are reported. The risk of a shift in traditional occurrence areas for aflatoxins is expected in the world and in particular in South East of Europe due to the increasing average temperatures. Biological control of aflatoxin risk in the field by atoxigenic strains of A. flavus starts to be widely used in Africa and USA. Studies are necessary on the variation of aflatoxin production in populations of A. flavus to characterize stable atoxigenic A. flavus strains. The aim of present article is to give an overview on biodiversity and genetic variation of Aspergillus section Flavi in Europe in relation to the management of aflatoxins risk in the field.

Evaluation of the Atoxigenic Aspergillus flavus Strain AF36 in Pistachio Orchards.

The atoxigenic strain Aspergillus flavus AF36, which has been extensively used as a biocontrol agent in commercial corn and cotton fields to reduce aflatoxin contamination, was applied in research pistachio orchards from 2002 to 2005 and in commercial pistachio orchards from 2008 to 2011. AF36 was applied as hyphae-colonized steam-sterilized wheat seed (the same product and same application rate as used in cotton fields). In all orchards, applying the wheat-AF36 product substantially increased the proportion of vegetative compatibility group (VCG) YV36, the VCG to which AF36 belongs, within A. flavus soil communities. Application of the AF36 product in additional years further increased YV36 in the soil until it composed 93% of the A. flavus isolates in treated commercial orchards. Nonetheless, application of the AF36 product did not result in increased incidence of kernel decay of the nuts. For nuts harvested from commercial orchards, reductions in percentages of samples contaminated with aflatoxin from treated orchards (relative to that for untreated orchards) ranged from 20 to 45%, depending on the year. Because of the high value of pistachio nuts and the costs associated with rejection of shipments due to aflatoxin contamination, these reductions are significant and valuable to the pistachio industry.

Molecular characterization of atoxigenic Aspergillus flavus isolates collected in China

Aspergillus flavus strains were isolated from peanut fields of Liaoning, Shandong, Hubei and Guangdong Provinces in China, and identified through phenotypic and molecular approaches. Of the 323 A. flavus strains isolated, 76 strains did not produce aflatoxins detectable by UPLC. The incidence of atoxigenic A. flavus strains decreased with increase in temperature and increased with increase in latitude in different geographical locations. Amplification of all the aflatoxin genes in the aflatoxin gene cluster in the atoxigenic isolates showed that there were 25 deletion patterns (A-Y), with 22 deletion patterns identified for the first time. Most of the atoxigenic A. flavus isolates with gene deletions (97%) had deletions in at least one of the four genes (aflT, nor-1, aflR, and hypB), indicating that these four genes could be targeted for rapid identification of atoxigenic strains. The atoxigenic isolates with gene deletions, especially the isolates with large deletions, are potential candidates for aflatoxin control.

Use of benzo analogs to enhance antimycotic activity of kresoxim methyl for control of aflatoxigenic fungal pathogens

The aim of this study was to examine two benzo analogs, octylgallate (OG) and veratraldehyde (VT), as antifungal agents against strains of Aspergillus parasiticus and A.flavus (toxigenic or atoxigenic). Both toxigenic and atoxigenic strains used were capable of producing kojic acid, another cellular secondary product. A. fumigatus was used as a genetic model for this study. When applied independently, OG exhibits considerably higher antifungal activity compared to VT. The minimum inhibitory concentrations (MICs) of OG were 0.3–0.5 mM, while that of VT were 3.0–5.0 mM in agar plate-bioassays. OG or VT in concert with the fungicide kresoxim methyl (Kre-Me; strobilurin) greatly enhanced sensitivity of Aspergillus strains to Kre-Me. The combination with OG also overcame the tolerance of A. fumigatus mitogen-activated protein kinase (MAPK) mutants to Kre-Me. The degree of compound interaction resulting from chemosensitization of the fungi by OG was determined using checkerboard bioassays, where synergistic activity greatly lowered MICs or minimum fungicidal concentrations. However, the control chemosensitizer benzohydroxamic acid, an alternative oxidase inhibitor conventionally applied in concert with strobilurin, did not achieve synergism. The level of antifungal or chemosensitizing activity was also “compound—strain” specific, indicating differential susceptibility of tested strains to OG or VT, and/or heat stress. Besides targeting the antioxidant system, OG also negatively affected the cell wall-integrity pathway, as determined by the inhibition of Saccharomyces cerevisiae cell wall-integrity MAPK pathway mutants. We concluded that certain benzo analogs effectively inhibit fungal growth. They possess chemosensitizing capability to increase efficacy of Kre-Me and thus, could reduce effective dosages of strobilurins and alleviate negative side effects associated with current antifungal practices. OG also exhibits moderate antiaflatoxigenic activity.

Field efficacy of a mixture of atoxigenic Aspergillus flavus Link:Fr vegetative compatibility groups in preventing aflatoxin contamination in maize (Zea mays L.)

Competitive exclusion of aflatoxin producers by endemic atoxigenic strains of Aspergillus flavus is a proven tool for aflatoxin management being adapted for use in Africa. Field efficacy of an experimental formulation consisting of four native atoxigenic strains (La3303, La3304, La3279 and Ka16127) was evaluated on maize in 2007 and 2008 in four agroecological zones in Nigeria. The four atoxigenic strains were individually formulated on sterile sorghum grain and subsequently mixed in equal proportions. The blended product was applied on soil (40kg/ha), 2–3weeks before flowering. Grains from treated and untreated fields were analyzed for aflatoxins at harvest and after storage. Proportions of the A. flavus population composed of each of the four applied strains in soil before treatment and in harvested grains were determined using vegetative compatibility analyses. Application of the strain mixture resulted in reduced aflatoxin content and significantly (P<0.05) increased the combined frequencies of the vegetative compatibility groups (VCGs) of the applied strains recovered from the soil and grain. Aflatoxin reductions of 67–95% were associated with a 74–80% combined incidence of the VCGs of the four atoxigenic strains on the treated crops. The applied atoxigenic strains remained with the crop into storage and reduced postharvest increases in contamination. The results suggest that the evaluated multi-strain formulated product has potential to contribute to reduced aflatoxin contamination in Nigeria. This is the first report of a field evaluation of an endemic strain mixture effective at reducing aflatoxin contamination during crop development.

The results of the monitoring of vibrio cholerae in the Taganrog Bay of the Azov sea in 2011-2012

The complication of cholera epidemic situation in Ukraine (2011) caused the reinforcement measures on monitoring Vibrio cholerae in seawater in Taganrog Bay in the framework of measures for prevention of the spread of infection over the territory of the Rostov region and the country. Phenotypic and genotypic features of 3 selected atoxigenic strains of Vibrio cholerae eltor O1 and 1 toxigenic strain of V. cholerae eltor Inaba № 301, containing the hybrid prophage were characterized. With great probability there was established the absence of association of this strain with the outbreak of infection within the territory of the neighboring country, but there was revealed his relationship with to imported strains (1999, 2005). Atoxigenic strains were similar to strains previously detected in water bodies of the Rostov region. The implementation of the set of measures for the strengthening of epidemiological control permitted to prevent the importation of infection into the territory of the Russian Federation in 2011 - 2012.

Comparison of growth, nutritional utilisation patterns, and niche overlap indices of toxigenic and atoxigenic Aspergillus flavus strains

The effects of temperatures (20–30 °C) and water activity (0.90–0.99 aw) on the lag phase duration, mycelial growth, and nutritional utilisation patterns of two toxigenic (AFL1+ & AFL2+) and three atoxigenic (AFL1−, AFL2−, & AFL3−) Aspergillus flavus strains were evaluated in vitro. Both temperature and aw and their interactions had a significant influence on the growth and nutritional utilisation patterns (p < 0.05). There were no significant differences between toxigenic and atoxigenic strains in terms of lag phase prior to growth and mycelial growth rates. Based on carbon source (CS) utilisation patterns, toxigenic and atoxigenic strains' niche size was greater at higher temperatures and in wetter conditions. Additionally, based on niche overlap indices (NOIs), regardless of temperature, when water was freely available, atoxigenic and toxigenic strains co-existed. However, under moisture stress, the nutritional competitiveness was variable. Temporal carbon utilisation sequences (TCUS) of toxigenic and atoxigenic strains were compared. At 0.99 aw most CS sources were utilised by the strains and the time to detection (TTD) of each strain was shortest on monosaccharides at the same level of aw. Conversely, under moisture stress the least number of CS was utilised. The current study has demonstrated that carbon utilisation patterns are equally important as are other determinants of competitiveness and that growth rate alone is not a key attribute which determines competitiveness.

Effect of environmental factors on in vitro and in situ interactions between atoxigenic and toxigenic A. flavus strains and control of aflatoxin contamination of maize

The potential of three atoxigenic strains from different geographical origins in Africa were examined for in vitro and in situ competitiveness against two toxigenic strains of Aspergillus flavus and subsequent inhibition of aflatoxin B1 (AFB1) production under different environmental conditions. Temperature, water activity (a w) and substrate influenced the types of interaction between the three AFL− and two AFL+ strains. The competitiveness and AFB1 reduction ability of the three atoxigenic strains when interacting with the two toxigenic strains were evaluated by inoculation of 100, 25:75, 50:50, 75:25 and 100% ratios of mixed spore suspensions in vitro on malt extract and milled maize agars over 28 days and in situ on stored maize grain for 14 days, respectively at 0.99, 0.96 and 0.90 a w. For all the treatments, the effect of a w and inoculum ratio and their interaction was highly significant. Toxin inhibition was >80% in vitro at both 0.99 and 0.96 a w. In situ AFB1 reduction was influenced by the toxigenic strain assayed, a w and the inoculum ratio. Where control was achieved, it was more variable at 0.96 a w, while with more stringent water stress conditions (0.90 a w) the percentage inhibition was up to 77.2%. The study shows the importance of including environmental factors in screening and identifying effective atoxigenic strains for control of AFs (aflatoxins).

Characterization of nonochratoxigenic strains of Aspergillus carbonarius from grapes

Aspergillus carbonarius is the main responsible source of ochratoxin A (OTA) in food commodities such as wine, grapes or dried vine fruits from main viticultural regions worldwide. Besides, OTA production is a very consistent property of this species and for this reason atoxigenic isolates of A. carbonarius are very rarely found in natural environments. In the present study, for the first time, three nonochratoxigenic wild strains of A. carbonarius have been discovered, unambiguously identified, characterized in deep and compared to ochratoxigenic strains of the same species. In addition, polyketide synthase (pks) genes suggested to be involved in OTA biosynthesis were also screened in these strains. The identification of the strains was confirmed by ITS-5.8S rRNA, β-tubulin and calmodulin gene sequencing. The three atoxigenic strains did not produce OTA in a conducive culture medium at any of the temperatures and times of incubation tested. Five ketosynthase domains from pks genes previously described in A. carbonarius were detected both in ochratoxigenic and in nonochratoxigenic strains. Atoxigenic strains of A. carbonarius could be useful as biotechnological agents to be used in food industry and as biological agents for control of OTA production in vineyards and other crops.

Detecting maize inoculated with toxigenic and atoxigenic fungal strains with fluorescence hyperspectral imagery

Naturally occurring Aspergillus flavus strains can be either toxigenic or atoxigenic, indicating their ability to produce aflatoxin or not. The objective was to assess, with the use of a hyperspectral sensor, the difference in fluorescence emission between maize kernels inoculated with toxigenic and atoxigenic inoculums of A. flavus. Maize ears were inoculated with AF13, a toxigenic strain of A. flavus, and AF38, an atoxigenic strain of A. flavus, at dough stage of development and harvested 8 weeks after inoculation. After harvest, single kernels were divided into three groups prior to imaging: control, adjacent, and glowing. Both sides of the kernel, germ and endosperm, were imaged separately using a fluorescence hyperspectral imaging system. After imaging each single kernel was processed with affinity column fluorimetry to determine aflatoxin level. Results from discriminant analysis of the imaging data found that the classification accuracies of the three visually designated groups were not promising. The separation of maize kernels based on different fungal inoculums yielded better results. The best results were achieved with the germ side of the maize kernels. The kernels were later grouped into ‘contaminated’ and ‘healthy’ with 20 ppb and 100 ppb thresholds. The contaminated kernels all had longer peak wavelength than did the healthy ones. Results from the discriminant analysis classification indicated overall higher classification accuracy for the 100 ppb threshold on the germ side (94.4%). The germ side was also more useful at discriminating healthy from contaminated kernels for the 20 ppb threshold.

Influence of plant host species on intraspecific competition during infection by Aspergillus flavus

Compositions of Aspergillus flavus populations determine the extent to which crops become contaminated with aflatoxins. In the current study, influences of diverse crop hosts on competition among A. flavus isolates were quantified with pyrosequencing. Maize, cotton, soyabean and sorghum supported different levels of sporulation, but intraspecific differences in sporulation were not detected on any host. However, hosts differentially influenced competition during infection, allowing greater sporulation by some isolates and increased host tissue invasion by others. Furthermore, competitive interactions during host invasion did not predict isolate success during sporulation. Isolates were similarly competitive on maize and sorghum, the two most closely related hosts. Host‐specific influences on intraspecific competition may dictate compositions of A. flavus populations and, as a result, the severity of aflatoxin contamination. Host factors should be considered when designing and implementing aflatoxin management strategies including biocontrol with atoxigenic strains.

Structure of an Aspergillus flavus population from maize kernels in northern Italy

In order to gain insight into the causal agents of aflatoxin contamination of maize in Italy, populations of Aspergillus flavus on maize produced in the most affected area were characterized. Forty-six percent of A. flavus, isolated from maize kernels collected in 5 districts of northern Italy between 2003 and 2010, were unable to produce detectable levels of aflatoxins. The genetic diversity of the population was assessed by analysis of vegetative compatibility groups (VCGs) and presence or absence of several aflatoxin biosynthesis genes. Forty-eight VCGs were identified through complementation between nitrate non-utilizing mutants. Twenty-five VCGs contained only atoxigenic isolates, and the remaining 23 only aflatoxin producers. Members of the largest atoxigenic VCG (IT6) were found in 4 of the 5 districts sampled. Six deletion patterns of genes in the aflatoxin biosynthesis gene cluster were detected. No deletions in the cluster were detected for twelve atoxigenic isolates and 10 had the entire cluster deleted. One isolate had a deletion pattern only seen once before in Nigeria. The basis for initial selection of endemic atoxigenic strains of A. flavus for biological control of aflatoxin contamination of maize in Italy is provided.

ATOXIGENIC ASPERGILLUS FLAVUS ISOLATES AS CANDIDATE BIOCONTROL AGENTS IN MAIZE

In Italy, nearly 90% of the maize is produced in five regions in the north of the country (Emilia Romagna, Friuli Venezia Giulia, Lombardy, Piedmont and Veneto). Although contaminations by fumonisins are very fre- quent in these areas, high levels of aflatoxins (AFs) were detected for the first time in 2003, a situation that turned worse in 2012. Aflatoxins are secondary metabolites pro- duced by several members of the genus Aspergillus sec- tion Flavi. Among these, aflatoxin B1 (AFB1) is the most dangerous and has been classified by the International Agency for Research on Cancer as class 1 toxin, due to its confirmed carcinogenic and teratogenic activity in hu- mans. The main responsible of maize AFB1 contamina- tion is Aspergillus flavus, a filamentous fungus with a vegetative incompatibility system that limits hyphal fu- sion and gene flow between individuals belonging to dif- ferent vegetative compatibility groups (VCGs). Isolates are assigned to VCGs with functional vegetative compat- ibility analyses (VCAs) typically using nitrate nonutiliz- ing auxotrophs (nit- mutants). The ability to synthesize AFs varies very much within A. flavus population, with fungal isolates that produce anywhere from over 1000 ppm AF to no toxin at all (atoxigenic isolates). The genes involved in AFs biosynthesis (confirmed 25) are contained within a 65 to 70 kb cluster, and several dis- tinct lesions within this cluster have been described that may each be responsible for atoxigenicity in various iso- lates. The main objectives of this study were: (i) VCAs of A. flavus isolates from northern Italy, (ii) selection of atoxigenic isolates potentially useful as biocontrol agents to reduce AFB1 contamination in maize. A high number (45%) of the isolates tested on autoclaved maize were unable to produce detectable levels of aflatoxins. Nit- mutants were obtained from all isolates within 10 days on chlorate-amended medium. More than 1500 mutant sectors were obtained and phenotyped revealing 72% to be niaD, 21% nirA and 7% cnx. Forty-six VCGs were identified; 24 contained only atoxigenic isolates, and the remaining 22 only aflatoxin producers. Members of the largest atoxigenic VCG were found in four of the five ar- eas sampled and in different years of sampling. Molecu- lar analysis of the genes in the aflatoxin biosynthesis gene cluster showed six deletion patterns. In particular, no deletions were detected in 10 atoxigenic isolates, whereas eight isolated had the entire cluster deleted. The capability of 18 atoxigenic isolates to reduce AFB1 pro- duction by a toxigenic strain was evaluated on maize. Each atoxigenic strain was co-inoculated with a toxi- genic, in the same proportion (1:1) or in a reduced amount (1 atoxigenic : 4 toxigenic). The extent of reduc- tion of AFB1 ranged from 60% to 89%, and the best five isolates, belonging to five different VCGs, showed a percentage of reduction similar to or greater than that achieved by a A. flavus strain registered in the USA as a biocontrol agent. Although through the proportion 1:1 (atoxigenic versus toxigenic) the highest percentage of reduction was achieved, the efficiency, intended as the ability of the inoculum unit of an atoxigenic strain to re- duce AFB1 produced by a toxigenic one, was greater in the 1:4 proportion. This study analysed for the first time the VCG structure of a A. flavus population isolated in the major maize-producing districts of Italy. Several atoxigenic fungal strains were identified as potential bio- control agents for mitigating aflatoxin contamination, thus laying the basis for the development of a reportedly very effective biocontrol strategy in Italy.

Aspergillus flavusdiversity on crops and in the environment can be exploited to reduce aflatoxin exposure and improve health

Humans and animals are exposed to aflatoxins, toxic carcinogenic fungal metabolites, through consumption of contaminated food and feed. Aspergillus flavus, the primary causal agent of crop aflatoxin contamination, is composed of phenotypically and genotypically diverse vegetative compatibility groups (VCGs). Molecular data suggest that VCGs largely behave as clones with certain VCGs exhibiting niche preference. VCGs vary in aflatoxin-producing ability, ranging from highly aflatoxigenic to atoxigenic. The prevalence of individual VCGs is dictated by competition during growth and reproduction under variable biotic and abiotic conditions. Agronomic practices influence structures and average aflatoxin-producing potentials of A. flavus populations and, as a result, incidences and severities of crop contamination. Application of atoxigenic strains has successfully reduced crop aflatoxin contamination across large areas in the United States. This strategy uses components of the endemic diversity to alter structures of A. flavus populations and improve safety of food, feed, and the overall environment.

Incidence and consumer awareness of toxigenic Aspergillus section Flavi and aflatoxin B1 in peanut cake from Nigeria

Peanut cake samples were collected from major markets in five states of Nigeria and evaluated for incidence of toxigenic Aspergillus section Flavi populations, and aflatoxin B1 (AFB1) levels by liquid chromatography tandem mass spectrometry (LC–MS/MS). The awareness of consumers to the presence of aflatoxin in the snack and potential health risks of its regular ingestion was evaluated by questionnaire analysis. Aspergillus section Flavi populations were recovered from 83% of the peanut cake samples. Aspergillus flavus L-strain was the most predominant (>56%) across the states while Aspergillus tamarii had the least mean incidence (2.7%). The incidence of atoxigenic strains was significantly (p < 0.05) higher than that of toxigenic strains in samples from Lagos and Kaduna, while the toxigenic strains had a significantly (p < 0.05) higher incidence than the atoxigenic strains in Niger. All analyzed cake samples contained AFB1 in concentrations exceeding the NAFDAC recommended level for AFB1 in food and reaching up to 2824 μg/kg. There was a weak positive correlation (r = 0.32, p = 0.03) for the relationship between the incidence of toxigenic strains in the samples and AFB1 concentration. The consumer awareness data showed that 64% of the respondents consumed peanut cake; majority of who are youth of economic and reproductive age. Eighty-five percent of the consumers lacked awareness of aflatoxin contamination in the snack and possible health risks associated with its ingestion.

Proteomic Analysis Reveals an Aflatoxin-Triggered Immune Response in Cotyledons of Arachis hypogaea Infected with Aspergillus flavus

An immune response is triggered in host cells when host receptors recognize conserved molecular motifs, pathogen-associated molecular patterns (PAMPs), such as β-glucans, and chitin at the cell surface of a pathogen. Effector-triggered immunity occurs when pathogens deliver effectors into the host cell to suppress the first immune signaling. Using a differential proteomic approach, we identified an array of proteins responding to aflatoxins in cotyledons of peanut (Arachis hypogaea) infected with aflatoxin-producing (toxigenic) but not nonaflatoxin-producing (atoxigenic) strains of Aspergillus flavus. These proteins are involved in immune signaling and PAMP perception, DNA and RNA stabilization, induction of defense, innate immunity, hypersensitive response, biosynthesis of phytoalexins, cell wall responses, peptidoglycan assembly, penetration resistance, condensed tannin synthesis, detoxification, and metabolic regulation. Gene expression analysis confirmed the differential abundance of proteins in peanut cotyledons supplemented with aflatoxins, with or without infection with the atoxigenic strain. Similarly, peanut germination and A. flavus growth were altered in response to aflatoxin B1. These findings show an additional immunity initiated by aflatoxins. With the PAMP- and effector-triggered immune responses, this immunity constitutes the third immune response of the immune system in peanut cotyledon cells. The system is also a three-grade coevolution of plant-pathogen interaction.