Atoxigenic Strains Research Articles

Biocontrol efficacy of atoxigenic Aspergillus flavus strains against aflatoxin contamination in peanut field in Guangdong province, South China

ABSTRACT Application of atoxigenic strains of Aspergillus flavusto soils is the most successful aflatoxin biological control approach. The objective of this study was to evaluate the efficacies of native non-aflatoxin producing (atoxigenic) strains as a biocontrol agent in peanut field in China. The competitive atoxigenic A. flavus strains (JS4, SI1and SXN) isolated from different crops, in China were used for field evaluation. The strains applied during the growing season (June – October, 2016) in the field at rate of 25 kg inoculum/hectare. The colonization of these biocontrol agents has been investigated and the population of A. flavus communities in soil were determined. The incidences of toxin producing (toxigenic) A. flavus strains and aflatoxin contamination in peanuts were also determined. Treated plots produced significant reductions in the incidence of toxigenic isolates of A. flavus in soil. However, the total fungal densities were not significantly different (p > 0.05) after treatments. Large percentage of aflatoxin reductions, ranging from 82.8% (SXN) up to 87.2% (JS4) were recorded in treated plots. Generally, the results suggest that the strategy can be used to control aflatoxin contamination and continuous evaluation should be done.

Selection of Atoxigenic Aspergillus flavus for Potential Use in Aflatoxin Prevention in Shandong Province, China.

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.

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.

Soil Microbial Communities in Corn Fields Treated with Atoxigenic Aspergillus flavus

Aspergillus flavus refers to a diverse group of saprophytic soil fungi that includes strains producing aflatoxins (toxigenic strains) in the kernels of corn (Zea mays L.) and other crops, causing pre-harvest and post-harvest aflatoxin contamination. Some A. flavus strains are atoxigenic, and the introduction of such strains into the crop environment helps reduce toxigenic aflatoxin contamination. Corn growers in Texas have used the product FourSure™, which contains four atoxigenic strains of A. flavus; however, effects on soil microbial communities associated with these applications are unknown. We compared soil fungal and bacterial communities in corn fields treated with FourSure™ to nearby untreated (control) corn fields in Texas during the summer of 2019. Analysis of soil microbial community structure showed that total fatty acid methyl esters (FAMEs), fungal, and bacterial populations were not significantly different (p = 0.31) between the FourSure™-treated and control fields, yet corn fields located in the northern counties had more (p < 0.05) Gram—bacteria, actinobacteria, and total bacteria than fields in the southernmost county. The Gram—bacteria and actinobacteria were positively correlated (p = 0.04; r = 0.48 and 0.49, respectively) with soil water content. Similar fungal and bacterial abundances between FourSure™-treated and control fields indicated that atoxigenic A. flavus had no negative effects on soil microbial communities.

Bees Occurring in Corn Production Fields Treated with Atoxigenic Aspergillus flavus (Texas, USA)

A saprophytic soil fungus, Aspergillus flavus, produces aflatoxin (toxigenic strains) in the kernels of corn (Zea mays L.) and seeds of many other crops. Many strains of A. flavus do not produce toxigenic aflatoxin, and soil application of these atoxigenic strains is a suppressive control tactic to assist in controlling toxigenic conspecifics. Effects of atoxigenic A. flavus applications on honey bees (Apis mellifera L.) and other bees are unknown, and basic information on bee occurrences in cornfields treated with and without this biological agent is needed to inform integrated pest management in corn. Fields receiving atoxigenic A. flavus applications of FourSureTM were compared to nearby control fields in three counties in corn production regions in eastern Texas. In each cornfield, 20 bee bowl traps were deployed along four equal transects located between corn rows, with contents of the bowls (i.e., bees) retrieved after 24 h. Eleven bee genera from four families were collected from cornfields, with only two honey bees collected and zero honey bees observed in transects. The sweat bee genus Agapostemon (primarily composed of the Texas striped sweat bee A. texanus) was most abundant in cornfields (44% of the total number of bees collected), followed by long-horned bees (Melissodes spp., 24%). The southernmost county (i.e., San Patricio) produced over 80% of the total number of bees collected. Bee numbers occurring in cornfields with applications of atoxigenic A. flavus applications were not significantly different from those of nearby control fields. Although not statistically significant, total numbers of bees tended to be lower in FourSure-treated fields than in control fields. More extensive research on bee abundances in relation to the effect of atoxigenic A. flavus is warranted.

Biological Control of Aflatoxin in Maize Grown in Serbia.

Aspergillus flavus is the main producer of aflatoxin B1, one of the most toxic contaminants of food and feed. With global warming, climate conditions have become favourable for aflatoxin contamination of agricultural products in several European countries, including Serbia. The infection of maize with A. flavus, and aflatoxin synthesis can be controlled and reduced by application of a biocontrol product based on non-toxigenic strains of A. flavus. Biological control relies on competition between atoxigenic and toxigenic strains. This is the most commonly used biological control mechanism of aflatoxin contamination in maize in countries where aflatoxins pose a significant threat. Mytoolbox Af01, a native atoxigenic A. flavus strain, was obtained from maize grown in Serbia and used to produce a biocontrol product that was applied in irrigated and non-irrigated Serbian fields during 2016 and 2017. The application of this biocontrol product reduced aflatoxin levels in maize kernels (51–83%). The biocontrol treatment had a highly significant effect of reducing total aflatoxin contamination by 73%. This study showed that aflatoxin contamination control in Serbian maize can be achieved through biological control methods using atoxigenic A. flavus strains.

Aflatoxins: Producing-Molds, Structure, Health Issues and Incidence in Southeast Asian and Sub-Saharan African Countries.

This review aims to update the main aspects of aflatoxin production, occurrence and incidence in selected countries, and associated aflatoxicosis outbreaks. Means to reduce aflatoxin incidence in crops were also presented, with an emphasis on the environmentally-friendly technology using atoxigenic strains of Aspergillus flavus. Aflatoxins are unavoidable widespread natural contaminants of foods and feeds with serious impacts on health, agricultural and livestock productivity, and food safety. They are secondary metabolites produced by Aspergillus species distributed on three main sections of the genus (section Flavi, section Ochraceorosei, and section Nidulantes). Poor economic status of a country exacerbates the risk and the extent of crop contamination due to faulty storage conditions that are usually suitable for mold growth and mycotoxin production: temperature of 22 to 29 °C and water activity of 0.90 to 0.99. This situation paralleled the prevalence of high liver cancer and the occasional acute aflatoxicosis episodes that have been associated with these regions. Risk assessment studies revealed that Southeast Asian (SEA) and Sub-Saharan African (SSA) countries remain at high risk and that, apart from the regulatory standards revision to be more restrictive, other actions to prevent or decontaminate crops are to be taken for adequate public health protection. Indeed, a review of publications on the incidence of aflatoxins in selected foods and feeds from countries whose crops are classically known for their highest contamination with aflatoxins, reveals that despite the intensive efforts made to reduce such an incidence, there has been no clear tendency, with the possible exception of South Africa, towards sustained improvements. Nonetheless, a global risk assessment of the new situation regarding crop contamination with aflatoxins by international organizations with the required expertise is suggested to appraise where we stand presently.

Spectral-Based Screening Approach Evaluating Two Specific Maize Lines With Divergent Resistance to Invasion by Aflatoxigenic Fungi.

In an effort to control aflatoxin contamination in food and/or feed grains, a segment of research has focused on host resistance to eliminate aflatoxin from susceptible crops, including maize. To this end, screening tools are key to identifying resistant maize genotypes. The traditional field screening techniques, the kernel screening laboratory assay (KSA), and analytical methods (e.g., ELISA) used for evaluating corn lines for resistance to fungal invasion, all ultimately require sample destruction. A technological advancement on the basic BGYF presumptive screening test, fluorescence hyperspectral imaging offers an option for non-destructive and rapid image-based screening. The present study aimed to differentiate fluorescence spectral signatures of representative resistant and susceptible corn hybrids infected by a toxigenic (SRRC-AF13) and an atoxigenic (SRRC-AF36) strain of Aspergillus flavus, at several time points (5, 7, 10, and 14 days), in order to evaluate fluorescence hyperspectral imaging as a viable technique for early, non-invasive aflatoxin screening in resistant and susceptible corn lines. The study utilized the KSA to promote fungal growth and aflatoxin production in corn kernels inoculated under laboratory conditions and to provide actual aflatoxin values to relate with the imaging data. Each time point consisted of 78 kernels divided into four groups (30-susceptible, 30-resistant, 9-susceptible control, and 9-resistant control), per inoculum. On specified days, kernels were removed from the incubator and dried at 60°C to terminate fungal growth. Dry kernels were imaged with a VNIR hyperspectral sensor (image spectral range of 400–1000 nm), under UV excitation centered at 365 nm. Following imaging, kernels were submitted for the chemical AflaTest assay (VICAM). Fluorescence emissions were compared for all samples over 14 days. Analysis of strain differences separating the fluorescence emission peaks of resistant from the susceptible strain indicated that the emission peaks of the resistant strain and the susceptible strains differed significantly (p < 0.01) from each other, and there was a significant difference in fluorescence intensity between the treated and control kernels of both strains. These results indicate a viable role of fluorescence hyperspectral imaging for non-invasive screening of maize lines with divergent resistance to invasion by aflatoxigenic fungi.

The comparative virulence of an atoxigenic strain of Aspergillus flavus (Eurotiales: Trichocomaceae) and the commercial ICIPE 69 Metarhizium anisopliae (Hypocreales: Clavicipitaceae) to the bean leaf beetle Ootheca mutabilis (Coleoptera: Chrysomelidae)

The recent isolation of an atoxigenic strain of Aspergillus flavus (Link) (Eurotiales: Trichocomaceae), which was virulent to all stages of Xylosandrus compactus (Eichhoff) (Coleoptera: Curculionidae) has generated interest to investigate virulence of the entomopathogenic fungus against other crop pests, and how it compares with established commercial biopesticides. In this study, we tested the pathogenicity and virulence of the atoxigenic A. flavus strain to Ootheca mutabilis (Sahlberg) (Coleoptera: Chrysomelidae) in the laboratory compared to the commercial Metarhizium anisopliae (Metschnikoff Sorokin) (Hypocreales: Clavicipitaceae; OD®, ICIPE 69). Serial dilutions of both entomopathogenic fungi (1 × 106–1 × 109 conidia/ml) were evaluated against adult O. mutabilis. Distilled water and cypermethrin 5% EC were included as controls. The treatments were replicated four times and the bioassay was repeated once. Both fungi at 1 × 109 conidia/ml killed 100% of tested O. mutabilis within seven days in the 1st and 2nd trials. The concentration of both fungi that killed 50% of O. mutabilis was the same in both trials (5.3 × 106 conidia/ml). The time taken by A. flavus and ICIPE 69 to kill 50% of O. mutabilis was not significantly different in the 1st trial (3.4 ± 0.1 days and 2.9 ± 0.4 days, respectively). However, ICIPE 69 took a significantly shorter time (2.1 ± 0.4 days) than A. flavus (3.6 ± 0.2 days) to kill 50% of O. mutabilis in the 2nd trial. We concluded that A. flavus is comparably as pathogenic and virulent (in terms of cumulative mortality and LC50) against O. mutabilis as ICIPE 69, and therefore both fungi may be viable biopesticides for commercialization against this pest.

The Atoxigenic Biocontrol Product Aflasafe SN01 Is a Valuable Tool to Mitigate Aflatoxin Contamination of Both Maize and Groundnut Cultivated in Senegal.

Aflatoxin contamination of groundnut and maize infected by Aspergillus section Flavi fungi is common throughout Senegal. The use of biocontrol products containing atoxigenic Aspergillus flavus strains to reduce crop aflatoxin content has been successful in several regions, but no such products are available in Senegal. The biocontrol product Aflasafe SN01 was developed for use in Senegal. The four active ingredients of Aflasafe SN01 are atoxigenic A. flavus genotypes native to Senegal and distinct from active ingredients used in other biocontrol products. Efficacy tests on groundnut and maize in farmers' fields were carried out in Senegal during the course of 5 years. Active ingredients were monitored with vegetative compatibility analyses. Significant (P < 0.05) displacement of aflatoxin producers occurred in all years, districts, and crops. In addition, crops from Aflasafe SN01-treated fields contained significantly (P < 0.05) fewer aflatoxins both at harvest and after storage. Most crops from treated fields contained aflatoxin concentrations permissible in both local and international markets. Results suggest that Aflasafe SN01 is an effective tool for aflatoxin mitigation in groundnut and maize. Large-scale use of Aflasafe SN01 should provide health, trade, and economic benefits for Senegal.[Formula: see text] Copyright © 2020 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Monitoring Aspergillus flavus Genotypes in a Multi-Genotype Aflatoxin Biocontrol Product With Quantitative Pyrosequencing.

Aflatoxins pose significant food security and public health risks, decrease productivity and profitability of animal industries, and hamper trade. To minimize aflatoxin contamination in several crops, a biocontrol technology based on atoxigenic strains of Aspergillus flavus is commercially used in the United States and some African countries. Significant efforts are underway to popularize the use of biocontrol in Africa by various means including incentives. The purpose of this study was to develop quantitative pyrosequencing assays for rapid, simultaneous quantification of proportions of four A. flavus biocontrol genotypes within complex populations of A. flavus associated with maize crops in Nigeria to facilitate payment of farmer incentives for Aflasafe (a biocontrol product) use. Protocols were developed to confirm use of Aflasafe by small scale farmers in Nigeria. Nested PCR amplifications followed by sequence by synthesis pyrosequencing assays were required to quantify frequencies of the active ingredients and, in so doing, confirm successful use of biocontrol by participating farmers. The entire verification process could be completed in 3–4 days proving a savings over other monitoring methods in both time and costs and providing data in a time frame that could work with the commercial agriculture scheme. Quantitative pyrosequencing assays represent a reliable tool for rapid detection, quantification, and monitoring of multiple A. flavus genotypes within complex fungal communities, satisfying the requirements of the regulatory community and crop end-users that wish to determine which purchased crops were treated with the biocontrol product. Techniques developed in the current study can be modified for monitoring other crop-associated fungi.

Spread of Aspergillus flavus and aflatoxin accumulation in postharvested maize treated with biocontrol products

Maize is a major staple crop and calorie source for many people living in Sub-Saharan Africa. In this region, Aspergillus flavus causes ear rot in maize, contributing to food insecurity due to aflatoxin contamination. The biological control principle of competitive exclusion has been applied in both the United States and Africa to reduce aflatoxin levels in maize grain at harvest by introducing atoxigenic strains that out-compete toxigenic strains. The goal of this study was to determine if the efficacy of preharvest biocontrol treatments carry over into the postharvest drying period, the time between harvest and the point when grain moisture is safe for storage. In Sub-Sahara Africa, this period often is extended by weather and the complexities of postharvest drying practices. Maize grain was collected from fields in Texas and North Carolina that were treated with commercial biocontrol products and untreated control fields. To simulate moisture conditions similar to those experienced by farmers during drying in Sub-Sahara Africa, we adjusted the grain to 20% moisture content and incubated it at 28 °C for 6 days. Although the initial number of kernels infected by fungal species was high in most samples, less than 24% of kernels were infected with Aspergillus flavus and aflatoxin levels were low (<4 ppb). Both toxigenic and atoxigenic strains grew and spread through the grain over the incubation period, and aflatoxin levels increased, even in samples from biocontrol-treated fields. Our molecular analysis suggests that applied biocontrol strains from treated fields may have migrated to untreated fields. These results also indicate that the population of toxigenic A. flavus in the harvested grain will increase and produce aflatoxin during the drying period when moisture is high. Therefore, we conclude that preharvest biocontrol applications will not replace the need for better postharvest practices that reduce the drying time between harvest and storage.

Performance of Broilers Fed with Maize Colonized by Either Toxigenic or Atoxigenic Strains of Aspergillus flavus with and without an Aflatoxin-Sequestering Agent.

In warm agricultural areas across the globe, maize, groundnut, and other crops become frequently contaminated with aflatoxins produced primarily by the fungus Aspergillus flavus. Crop contamination with those highly toxic and carcinogenic compounds impacts both human and animal health, as well as the income of farmers and trade. In Nigeria, poultry productivity is hindered by high prevalence of aflatoxins in feeds. A practical solution to decrease crop aflatoxin content is to use aflatoxin biocontrol products based on non-toxin-producing strains of A. flavus. The biocontrol product Aflasafe® was registered in 2014 for use in maize and groundnut grown in Nigeria. Its use allows the production of aflatoxin-safe maize and groundnut. A portion of the maize treated with Aflasafe in Nigeria is being used to manufacture feeds used by the poultry industry, and productivity is improving. One of the conditions to register Aflasafe with the national regulator was to demonstrate both the safety of Aflasafe-treated maize to avian species and the impact of Aflasafe as a public good. Results presented here demonstrate that the use of maize colonized by an atoxigenic strain of Aflasafe resulted in superior (p < 0.05) broiler performance in all evaluated parameters in comparison to broilers fed with toxigenic maize. Use of an aflatoxin-sequestering agent (ASA) was not sufficient to counteract the harmful effects of aflatoxins. Both the safety and public good value of Aflasafe were demonstrated during our study. In Nigeria, the availability of aflatoxin-safe crops as a result of using Aflasafe allows poultry producers to improve their productivity, their income, and the health of consumers of poultry products.

Dual culture of atoxigenic and toxigenic strains of Aspergillus flavus to gain insight into repression of aflatoxin biosynthesis and fungal interaction

Application of atoxigenic strains to compete against toxigenic strains of Aspergillus flavus strains has emerged as one of the practical strategies for reducing aflatoxin contamination in corn, peanut, and tree nuts. The actual mechanism that results in aflatoxin reduction is not fully understood. Real-time RT-PCR and relative quantification of gene expression protocol were applied to elucidate the molecular mechanism. Transcriptional analyses of aflatoxin biosynthetic gene cluster in dual culture of toxigenic and atoxigenic A. flavus strains were carried out. Six targeted genes, aflR, aflJ, omtA, ordA, pksA, and vbs, were downregulated to variable levels depending on paired strains of toxigenic and atoxigenic A. flavus. Consistent with the decreased gene expression levels, the aflatoxin concentrations in dual cultures were reduced significantly in comparison with toxigenic cultures. Fluorescent images showed fungal hyphae in dual culture displayed green fluorescent, and contacts of live hyphae were seen. A coconut agar plate assay was used to show that toxigenic A. flavus colony produced blue fluorescence under long UV exposure, suggesting that aflatoxin is exported outside fungal hyphae. Furthermore, the assay was applied to demonstrate the potential role of thigmo-regulation in fungal interaction.

Effects of a prothioconazole- and tebuconazole-based fungicide on Aspergillus flavus development under laboratory and field conditions

Aspergillus flavus is a fungal pathogen of crops including maize and is frequently associated with the accumulation of aflatoxin, which presents a concern for human and animal health. In maize, good agricultural practices can only partially reduce Aspergillus occurrence, and biological control through atoxigenic strains decreases aflatoxin content, but increasing mold decreases kernel quality. The control of Aspergillus infections by chemicals is not authorized in maize, but recently in Italy, some sterol-biosynthesis inhibitors obtained an emergency authorization for use against Fusarium infections. Therefore, it could be interesting to determine if these inhibitors could also be useful against A. flavus. A mixture of prothioconazole and tebuconazole was tested in vitro on toxigenic and atoxigenic strains and on populations with respect to conidial germination, germinal potential and fungal growth. The fungicide mixture inhibited fungal growth, with a higher effect on toxigenic strains than on atoxigenic strains. All considered parameters were significantly reduced for monoconidial strains at doses lower than those recommended for field application for straw cereals; however, under-dosing should be avoided since it increases aflatoxin production. Field trials under natural infection conditions revealed the efficacy of the fungicide treatment on the reduction of both Aspergillus occurrence and aflatoxin accumulation in maize. In a context in which the sustainable use of pesticides can be guaranteed, our findings support the inclusion of azole-based fungicides in a multiple-strategy approach to control Aspergillus infections in maize.

An atoxigenic L-strain of Aspergillus flavus (Eurotiales: Trichocomaceae) is pathogenic to the coffee twig borer, Xylosandrus compactus (Coleoptera: Curculionidea: Scolytinae).

This study isolated and evaluated virulence of fungal entomopathogens of Xylosandrus compactus - an important pest of Robusta coffee in Sub-Saharan Africa. A survey was conducted in five farming systems in Uganda to isolate entomopathogens associated with X. compactus. Four fungal isolates were screened for virulence against X. compactus in the laboratory at 1 × 107 conidia ml-1 where an atoxigenic L-strain of A. flavus killed 70%-100% of all stages of X. compactus compared with other unidentified isolates which caused 20%-70% mortalities. The time taken by A. flavus to kill 50% of X. compactus eggs, larvae, pupae and adults in the laboratory was 2-3 days; whereas the other unidentified fungal isolates took 4-7 days. The concentrations of A. flavus that killed 50% of different stages of X. compactus were 5 × 105 , 12 × 105 , 17 × 105 and 30 × 105 conidia ml-1 for larvae, eggs, pupae and adults respectively. A formulation of A. flavus in oil caused higher mortalities of X. compactus larvae, pupae and adults in the field (71%-79%) than its formulation in water (33%-47%). The atoxigenic strain of A. flavus could therefore be developed into a safe biopesticide against X. compactus.

Inferring the presence of aflatoxin-producing Aspergillus flavus strains using RNA sequencing and electronic probes as a transcriptomic screening tool.

E-probe Diagnostic for Nucleic acid Analysis (EDNA) is a bioinformatic tool originally developed to detect plant pathogens in metagenomic databases. However, enhancements made to EDNA increased its capacity to conduct hypothesis directed detection of specific gene targets present in transcriptomic databases. To target specific pathogenicity factors used by the pathogen to infect its host or other targets of interest, e-probes need to be developed for transcripts related to that function. In this study, EDNA transcriptomics (EDNAtran) was developed to detect the expression of genes related to aflatoxin production at the transcriptomic level. E-probes were designed from genes up-regulated during A. flavus aflatoxin production. EDNAtran detected gene transcripts related to aflatoxin production in a transcriptomic database from corn, where aflatoxin was produced. The results were significantly different from e-probes being used in the transcriptomic database where aflatoxin was not produced (atoxigenic AF36 strain and toxigenic AF70 in Potato Dextrose Broth).

ТРАНСКРИПЦИОННЫЙ АНАЛИЗ ЭКСПРЕССИИ ГЕНОВ ОСТРОВКА VcB V. CHOLERAE МЕТОДОМ ПОЛНОГЕНОМНОГО СЕКВЕНИРОВАНИЯ

The aim of the study was to analyze the expression of V. cholerae genes that are part of the VcB island by means of full-genomic sequencing of the transcriptome. The VcB island is localized on the second chromosome in all toxigenic vibrios studied and is absent in the atoxigenic apiliated strains. Two strains of V. cholerae O1 ctxA+ tcpA+ and one strain V. cholerae O1 ctxA– tcpA– were studied. The pool of total RNA vibrios was isolated by a technique based on differential precipitation in the presence of lithium ions. In the total pool of sequenced RNA, RNA encoded in the order of 3 500 by known cholera vibrio genes was identified. In a pool of total RNA from two ctx + tcpA + strains RNA transcripts were found for the five genes included in the VcB island, excluding the VCA0282 gene, previously identified as the ISVch5-transposase. In the the pool of total RNA from the ctx– tcpA– strain no transcripts of the two genes previously described as VCA0282-transposase and VCA0283 were detected. A possible explanation may be the existence of copies of these genes in other parts of the genome of the ctxA– tcpA– V. cholerae.

Cultural and Genetic Approaches to Manage Aflatoxin Contamination: Recent Insights Provide Opportunities for Improved Control.

Aspergillus flavus is a morphologically complex species that can produce the group of polyketide derived carcinogenic and mutagenic secondary metabolites, aflatoxins, as well as other secondary metabolites such as cyclopiazonic acid and aflatrem. Aflatoxin causes aflatoxicosis when aflatoxins are ingested through contaminated food and feed. In addition, aflatoxin contamination is a major problem, from both an economic and health aspect, in developing countries, especially Asia and Africa, where cereals and peanuts are important food crops. Earlier measures for control of A. flavus infection and consequent aflatoxin contamination centered on creating unfavorable environments for the pathogen and destroying contaminated products. While development of atoxigenic (nonaflatoxin producing) strains of A. flavus as viable commercial biocontrol agents has marked a unique advance for control of aflatoxin contamination, particularly in Africa, new insights into the biology and sexuality of A. flavus are now providing opportunities to design improved atoxigenic strains for sustainable biological control of aflatoxin. Further, progress in the use of molecular technologies such as incorporation of antifungal genes in the host and host-induced gene silencing, is providing knowledge that could be harnessed to develop germplasm that is resistant to infection by A. flavus and aflatoxin contamination. This review summarizes the substantial progress that has been made to understand the biology of A. flavus and mitigate aflatoxin contamination with emphasis on maize. Concepts developed to date can provide a basis for future research efforts on the sustainable management of aflatoxin contamination.

Development of a droplet digital PCR assay for population analysis of aflatoxigenic and atoxigenic Aspergillus flavus mixtures in soil.

Aflatoxin B1 is a potent hepatotoxin and carcinogen that poses a serious safety hazard to both humans and animals. Aspergillus flavus is the most common aflatoxin-producing species on corn, cotton, peanuts, and tree nuts. Application of atoxigenic strains to compete against aflatoxigenic strains of A. flavus has emerged as one of the most practical strategies for ameliorating aflatoxin contamination in food. Genes directly involved in aflatoxin biosynthesis are clustered on an 82-kb region of the genome. Three atoxigenic strains (CA12, M34, and AF123) were each paired with each of four aflatoxigenic strains (CA28, CA42, CA90, and M52), inoculated into soil and incubated at 28°C for 2weeks and 1month. TaqMan probes, omtA-FAM, and norA-HEX were designed for developing a droplet digital PCR (ddPCR) assay to analyze the soil population of mixtures of A. flavus strains. DNA was extracted from each soil sample and used for ddPCR assays. The data indicated that competition between atoxigenic and aflatoxigenic was strain dependent. Variation in competitive ability among different strains of A. flavus influenced the population reduction of the aflatoxigenic strain by the atoxigenic strain. Higher ratios of atoxigenic to aflatoxigenic strains increased soil population of atoxigenic strains. This is the first study to demonstrate the utility of ddPCR to quantify mixtures of both atoxigenic and aflatoxigenic A. flavus strains in soil and allows for rapid and accurate determination of population sizes of atoxigenic and aflatoxigenic strains. This method eliminates the need for isolation and identification of individual fungal isolates from experimental soil samples.