Variation in the morphology and effector profiles of Exserohilum turcicum isolates associated with the Northern Corn Leaf Blight of maize in Nigeria.

BACKGROUND: Maize production in lowland agro-ecologies in West and Central Africa is constrained by the fungus Exserohilum turcicum, causal agent of Northern Corn Leaf Blight (NCLB). Breeding for resistance to NCLB is considered the most effective management strategy. The strategy would be even more effective if there is adequate knowledge of the characteristics of E. turcicum in a target region. Maize leaves showing NCLB symptoms were collected during field surveys in three major maize growing areas in Nigeria: Ikenne, Ile-Ife, and Zaria during 2018/2019 and 2019/2020 growing seasons to characterize E. turcicum populations interacting with maize using morphological and molecular criteria. RESULTS: A total of 217 E. turcicum isolates were recovered. Most of the isolates (47%) were recovered from the Ikenne samples while the least were obtained from Zaria. All isolates were morphologically characterized. A subset of 124 isolates was analyzed for virulence effector profiles using three primers: SIX13-like, SIX5-like, and Ecp6. Inter- and intra-location variations among isolates was found in sporulation, growth patterns, and presence of the effectors. Candidate effector genes that condition pathogenicity and virulence in E. turcicum were found but not all isolates expressed the three effectors. CONCLUSION: Morphological and genetic variation among E. turcicum isolates was found within and across locations. The variability observed suggests that breeding for resistance to NCLB in Nigeria requires selection for quantitative resistance to sustain the breeding efforts.

Abstracts of Presentations to the Working Session on Improving Predictive Modeling of Mycotoxin Risk for Africa Held at the 3rd ASM2022 on 7 September 2022, in Stellenbosch, South Africa.

In 2008, the African Postharvest Losses Information Systems project (APHLIS, accessed on 6 September 2022) developed an algorithm for estimating the scale of cereal postharvest losses (PHLs). The relevant scientific literature and contextual information was used to build profiles of the PHLs occurring along the value chains of nine cereal crops by country and province for 37 sub-Saharan African countries. The APHLIS provides estimates of PHL figures where direct measurements are not available. A pilot project was subsequently initiated to explore the possibility of supplementing these loss estimates with information on the aflatoxin risk. Using satellite data on drought and rainfall, a time series of agro-climatic aflatoxin risk warning maps for maize was developed covering the countries and provinces of sub-Saharan Africa. The agro-climatic risk warning maps for specific countries were shared with mycotoxin experts from those countries for review and comparison with their aflatoxin incidence datasets. The present Work Session was a unique opportunity for African food safety mycotoxins experts, as well as other international experts, to meet and deepen the discussion about prospects for using their experience and their data to validate and improve agro-climatic risk modeling approaches.

Detoxification of Aflatoxins in Fermented Cereal Gruel (Ogi) by Probiotic Lactic Acid Bacteria and Yeasts with Differences in Amino Acid Profiles.

Toxigenic members of Aspergillus flavus contaminate cereal grains, resulting in contamination by aflatoxin, a food safety hazard that causes hepatocellular carcinoma. This study identified probiotic strains as aflatoxin detoxifiers and investigated the changes to the grain amino acid concentrations during fermentation with probiotics in the presence of either A. flavus La 3228 (an aflatoxigenic strain) or A. flavus La 3279 (an atoxigenic strain). Generally, higher concentrations (p < 0.05) of amino acids were detected in the presence of toxigenic A. flavus La 3228 compared to the atoxigenic A. flavus La 3279. Compared to the control, 13/17 amino acids had elevated (p < 0.05) concentrations in the presence of the toxigenic A. flavus compared to the control, whereas in systems with the atoxigenic A. flavus 13/17 amino acids had similar (p > 0.05) concentrations to the control. There were interspecies and intraspecies differences in specific amino acid elevations or reductions among selected LAB and yeasts, respectively. Aflatoxins B1 and B2 were detoxified by Limosilactobacillus fermentum W310 (86% and 75%, respectively), Lactiplantibacillus plantarum M26 (62% and 63%, respectively), Candida tropicalis MY115 (60% and 77%, respectively), and Candida tropicalis YY25, (60% and 31%, respectively). Probiotics were useful detoxifiers; however, the extent of decontamination was species- and strain-dependent. Higher deviations in amino acid concentrations in the presence of toxigenic La 3228 compared to atoxigenic La 3279 suggests that the detoxifiers did not act by decreasing the metabolic activity of the toxigenic strain.

Aflatoxin Contamination of Maize, Groundnut, and Sorghum Grown in Burkina Faso, Mali, and Niger and Aflatoxin Exposure Assessment.

Aflatoxin contamination of staple crops by Aspergillus flavus and closely related fungi is common across the Sahel region of Africa. Aflatoxins in maize, groundnut, and sorghum collected at harvest or from farmers' stores within two weeks of harvest from Burkina Faso, Mali, and Niger were quantified. Thereafter, aflatoxin exposure values were assessed using per capita consumption rates of those crops. Mean aflatoxin concentrations in maize were high, 128, 517, and 659 µg/kg in Mali, Burkina Faso, and Niger, respectively. The estimated probable daily intake (PDI) of aflatoxins from maize ranged from 6 to 69, 29 to 432, and 310 to 2100 ng/kg bw/day in Mali, Burkina Faso, and Niger, respectively. Similarly, mean aflatoxin concentrations in sorghum were high, 76 and 259 µg/kg in Mali and Niger, respectively, with an estimated PDI of 2-133 and 706-2221. For groundnut, mean aflatoxin concentrations were 115, 277, and 628 µg/kg in Mali, Burkina Faso, and Niger, respectively. Aflatoxin exposure values were high with an estimated 9, 28, and 126 liver cancer cases/100,000 persons/year in Mali, Burkina Faso, and Niger, respectively. Several samples were extremely unsafe, exceeding manyfold regulatory levels of diverse countries (up to 2000 times more). Urgent attention is needed across the Sahel for integrated aflatoxin management for public health protection, food and nutrition security, and access to trade opportunities.

Identification of Early and Extra-Early Maturing Tropical Maize Inbred Lines with Multiple Disease Resistance for Enhanced Maize Production and Productivity in Sub-Saharan Africa.

Maize, a staple for millions across sub-Saharan Africa (SSA), faces major biotic constraints affecting production and safety of the crop. These include northern corn leaf blight (NCLB), southern corn leaf blight (SCLB), Curvularia leaf spot (CLS), and aflatoxin contamination by Exserohilum turcicum, Bipolaris maydis, Curvularia lunata, and Aspergillus flavus, respectively. Farmers in SSA would benefit tremendously if high-yielding maize hybrids with multiple disease resistance (MDR) were developed and commercialized. In all, 49 early-maturing (EM; 90 to 95 days to physiological maturity) and 55 extra-early-maturing (EEM, 80 to 85 days to physiological maturity) inbred lines developed by the International Institute of Tropical Agriculture were identified as resistant to NCLB in field evaluations in multiple agroecologies of Nigeria in 2017 and 2018. From each maturity group, the 30 most resistant inbreds were selected for evaluation for resistance to SCLB and CLS using a detached-leaf assay. Additionally, the inbreds were screened for resistance to kernel rot and aflatoxin contamination using a kernel screening assay. In all, 7 EM and 6 EEM maize inbreds were found to be highly resistant to the three foliar pathogens while 10 inbreds were resistant to the foliar pathogens and supported significantly less (P = 0.01) aflatoxin accumulation than other inbreds. Inbreds having MDR should be tested extensively in hybrid combinations and commercialized. Large-scale use of maize hybrids with MDR would (i) increase maize production and productivity and (ii) reduce losses caused by aflatoxin contamination. Overall, planting of EM and EEM maize hybrids with MDR would contribute to food security, reduced aflatoxin exposure, and increased incomes of maize farmers in SSA.[Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Can it be all more simple? Manufacturing aflatoxin biocontrol products using dry spores of atoxigenic isolates of Aspergillus flavus as active ingredients.

Aflatoxin contamination of staple crops, commonly occurring in warm areas, negatively impacts human and animal health, and hampers trade and economic development. The fungus Aspergillus flavus is the major aflatoxin producer. However, not all A. flavus genotypes produce aflatoxins. Effective aflatoxin control is achieved using biocontrol products containing spores of atoxigenic A. flavus. In Africa, various biocontrol products under the tradename Aflasafe are available. Private and public sector licensees manufacture Aflasafe using spores freshly produced in laboratories adjacent to their factories. BAMTAARE, the licensee in Senegal, had difficulties to obtain laboratory equipment during its first year of production. To overcome this, a process was developed in Ibadan, Nigeria, for producing high-quality dry spores. Viability and stability of the dry spores were tested and conformed to set standards. In 2019, BAMTAARE manufactured Aflasafe SN01 using dry spores produced in Ibadan and sent via courier and 19 000 ha of groundnut and maize in Senegal and The Gambia were treated. Biocontrol manufactured with dry spores was as effective as biocontrol manufactured with freshly produced spores. Treated crops contained safe and significantly (P < 0.05) less aflatoxin than untreated crops. The dry spore innovation will make biocontrol manufacturing cost-efficient in several African countries.

Does Use of Atoxigenic Biocontrol Products to Mitigate Aflatoxin in Maize Increase Fumonisin Content in Grains?

In the tropics and subtropics, maize (Zea mays) and other crops are frequently contaminated with aflatoxins by Aspergillus flavus. Treatment of crops with atoxigenic isolates of A. flavus formulated into biocontrol products can significantly reduce aflatoxin contamination. Treated crops contain up to 100% fewer aflatoxins compared with untreated crops. However, there is the notion that protecting crops from aflatoxin contamination may result in increased accumulation of other toxins, particularly fumonisins produced by a few Fusarium species. The objective of this study was to determine if treatment of maize with aflatoxin biocontrol products increased fumonisin concentration and fumonisin-producing fungi in grains. Over 200 maize samples from fields treated with atoxigenic biocontrol products in Nigeria and Ghana were examined for fumonisin content and contrasted with maize from untreated fields. Apart from low aflatoxin levels, most treated maize also harbored fumonisin levels considered safe by the European Union (<1 part per million; ppm). Most untreated maize also harbored equally low fumonisin levels but contained higher aflatoxin levels. In addition, during one year, we detected considerably lower Fusarium spp. densities in treated maize than in untreated maize. Our results do not support the hypothesis that treating crops with atoxigenic isolates of A. flavus used in biocontrol formulations results in higher grain fumonisin levels.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

"Ground-Truthing" Efficacy of Biological Control for Aflatoxin Mitigation in Farmers' Fields in Nigeria: From Field Trials to Commercial Usage, a 10-Year Study.

In sub-Saharan Africa (SSA), diverse fungi belonging to Aspergillus section Flavi frequently contaminate staple crops with aflatoxins. Aflatoxins negatively impact health, income, trade, food security, and development sectors. Aspergillus flavus is the most common causal agent of contamination. However, certain A. flavus genotypes do not produce aflatoxins (i.e., are atoxigenic). An aflatoxin biocontrol technology employing atoxigenic genotypes to limit crop contamination was developed in the United States. The technology was adapted and improved for use in maize and groundnut in SSA under the trademark Aflasafe. Nigeria was the first African nation for which an aflatoxin biocontrol product was developed. The current study includes tests to assess biocontrol performance across Nigeria over the past decade. The presented data on efficacy spans years in which a relatively small number of maize and groundnut fields (8-51 per year) were treated through use on circa 36,000 ha in commercially-produced maize in 2018. During the testing phase (2009-2012), fields treated during one year were not treated in the other years while during commercial usage (2013-2019), many fields were treated in multiple years. This is the first report of a large-scale, long-term efficacy study of any biocontrol product developed to date for a field crop. Most (>95%) of 213,406 tons of maize grains harvested from treated fields contained <20 ppb total aflatoxins, and a significant proportion (>90%) contained <4 ppb total aflatoxins. Grains from treated plots had preponderantly >80% less aflatoxin content than untreated crops. The frequency of the biocontrol active ingredient atoxigenic genotypes in grains from treated fields was significantly higher than in grains from control fields. A higher proportion of grains from treated fields met various aflatoxin standards compared to grains from untreated fields. Results indicate that efficacy of the biocontrol product in limiting aflatoxin contamination is stable regardless of environment and cropping system. In summary, the biocontrol technology allows farmers across Nigeria to produce safer crops for consumption and increases potential for access to premium markets that require aflatoxin-compliant crops.

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.

Metabolites Identified during Varied Doses of Aspergillus Species in Zea mays Grains, and Their Correlation with Aflatoxin Levels.

Aflatoxin contamination is associated with the development of aflatoxigenic fungi such as Aspergillus flavus and A. parasiticus on food grains. This study was aimed at investigating metabolites produced during fungal development on maize and their correlation with aflatoxin levels. Maize cobs were harvested at R3 (milk), R4 (dough), and R5 (dent) stages of maturity. Individual kernels were inoculated in petri dishes with four doses of fungal spores. Fungal colonisation, metabolite profile, and aflatoxin levels were examined. Grain colonisation decreased with kernel maturity: milk-, dough-, and dent-stage kernels by approximately 100%, 60%, and 30% respectively. Aflatoxin levels increased with dose at dough and dent stages. Polar metabolites including alanine, proline, serine, valine, inositol, iso-leucine, sucrose, fructose, trehalose, turanose, mannitol, glycerol, arabitol, inositol, myo-inositol, and some intermediates of the tricarboxylic acid cycle (TCA—also known as citric acid or Krebs cycle) were important for dose classification. Important non-polar metabolites included arachidic, palmitic, stearic, 3,4-xylylic, and margaric acids. Aflatoxin levels correlated with levels of several polar metabolites. The strongest positive and negative correlations were with arabitol (R = 0.48) and turanose and (R = −0.53), respectively. Several metabolites were interconnected with the TCA; interconnections of the metabolites with the TCA cycle varied depending upon the grain maturity.

In vitro experimental environments lacking or containing soil disparately affect competition experiments of Aspergillus flavus and co-occurring fungi in maize grains.

In vitro experimental environments are used to study interactions between microorganisms, and to predict dynamics in natural ecosystems. This study highlights that experimental in vitro environments should be selected to match closely the natural environment of interest during in vitro studies to strengthen extrapolations about aflatoxin production by Aspergillus and competing organisms. Fungal competition and aflatoxin accumulation were studied in soil, cotton wool or tube (water-only) environments, for Aspergillus flavus competition with Penicillium purpurogenum, Fusarium oxysporum or Sarocladium zeae within maize grains. Inoculated grains were incubated in each environment at two temperature regimes (25 and 30°C). Competition experiments showed interaction between the main effects of aflatoxin accumulation and the environment at 25°C, but not so at 30°C. However, competition experiments showed fungal populations were always interacting with their environments. Fungal survival differed after the 72-h incubation in different experimental environments. Whereas all fungi incubated within the soil environment survived, in the cotton wool environment none of the competitors of A. flavus survived at 30°C. With aflatoxin accumulation, F. oxysporum was the only fungus able to interdict aflatoxin production at both temperatures. This occurred only in the soil environment and fumonisins accumulated instead. Smallholder farmers in developing countries face serious mycotoxin contamination of their grains, and soil is a natural reservoir for the associated fungal propagules, and a drying and storage surface for grains on these farms. Studying fungal dynamics in the soil environment and other environments in vitro can provide insights into aflatoxin accumulation post-harvest.