Impact of storage conditions on the shelf life of aflatoxin biocontrol products containing atoxigenic isolates of Aspergillus flavus as active ingredient applied in various countries in Africa.

Aflatoxin contamination significantly threatens food safety and security, particularly in tropical and sub-tropical regions where staple crops such as maize, groundnut, and sorghum become frequently affected. This contamination is primarily caused by the fungus Aspergillus flavus. The contamination causes adverse health effects, reduced income, and trade restrictions. In response to this challenge, various technologies have been developed to mitigate the impacts of aflatoxin. Among these, biocontrol products containing atoxigenic isolates of A. flavus as the active ingredient can effectively reduce aflatoxin levels both at pre- and post-harvest. A notable example of such products is Aflasafe, which contains four atoxigenic isolates native to specific target regions. These products have undergone rigorous testing, have received regulatory approval, and are commercially available in multiple African countries. However, their manufacturing processes have evolved, and comprehensive shelf life studies for current formulations are lacking. Evaluations of the spore production ability of atoxigenic A. flavus isolates in Aflasafe products over 4 years, under various storage conditions, revealed a significant linear decrease in sporulation with storage months (P < 0.001; R 2 = 0.203), with no significant differences observed between treatments. However, this marginal decline (P = 0.398) is unlikely to be sufficient to prevent the effectiveness in limiting aflatoxin. In addition, storing the products for 2 weeks at 54 °C did not affect (P > 0.05) the ability of the coated fungi to produce spores compared to when the products were stored at 24 °C. The findings contribute valuable insights for manufacturers and users of atoxigenic-based aflatoxin biocontrol products, informing best practices for product storage and utilization to ensure prolonged effectivenes in aflatoxin mitigation efforts.

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.

Aflatoxin contamination of maize and groundnut in Burundi: Distribution of contamination, identification of causal agents and potential biocontrol genotypes of Aspergillus flavus.

Aflatoxin contamination of the staples maize and groundnut is a concern for health and economic impacts across sub-Saharan Africa. The current study (i) determined aflatoxin levels in maize and groundnut collected at harvest in Burundi, (ii) characterized populations of Aspergillus section Flavi associated with the two crops, and (iii) assessed aflatoxin-producing potentials among the recovered fungi. A total of 120 groundnut and 380 maize samples were collected at harvest from eight and 16 provinces, respectively. Most of the groundnut (93%) and maize (87%) contained aflatoxin below the European Union threshold, 4 µg/kg. Morphological characterization of the recovered Aspergillus section Flavi fungi revealed that the L-morphotype of A. flavus was the predominant species. Aflatoxin production potentials of the L-morphotype isolates were evaluated in maize fermentations. Some isolates produced over 137,000 µg/kg aflatoxin B1. Thus, despite the relatively low aflatoxin levels at harvest, the association of both crops with highly toxigenic fungi poses significant risk of post-harvest aflatoxin contamination and suggests measures to mitigate aflatoxin contamination in Burundi should be developed. Over 55% of the L-morphotype A. flavus did not produce aflatoxins. These atoxigenic L-morphotype fungi were characterized using molecular markers. Several atoxigenic genotypes were detected across the country and could be used as biocontrol agents. The results from the current study hold promise for developing aflatoxin management strategies centered on biocontrol for use in Burundi to reduce aflatoxin contamination throughout the value chain.

Impact of frequency of application on the long-term efficacy of the biocontrol product Aflasafe in reducing aflatoxin contamination in maize.

Aflatoxins, produced by several Aspergillus section Flavi species in various crops, are a significant public health risk and a barrier to trade and development. In sub-Saharan Africa, maize and groundnut are particularly vulnerable to aflatoxin contamination. Aflasafe, a registered aflatoxin biocontrol product, utilizes atoxigenic A. flavus genotypes native to Nigeria to displace aflatoxin producers and mitigate aflatoxin contamination. Aflasafe was evaluated in farmers' fields for 3 years, under various regimens, to quantify carry-over of the biocontrol active ingredient genotypes. Nine maize fields were each treated either continuously for 3 years, the first two successive years, in year 1 and year 3, or once during the first year. For each treated field, a nearby untreated field was monitored. Aflatoxins were quantified in grain at harvest and after simulated poor storage. Biocontrol efficacy and frequencies of the active ingredient genotypes decreased in the absence of annual treatment. Maize treated consecutively for 2 or 3 years had significantly (p < 0.05) less aflatoxin (92% less) in grain at harvest than untreated maize. Maize grain from treated fields subjected to simulated poor storage had significantly less (p < 0.05) aflatoxin than grain from untreated fields, regardless of application regimen. Active ingredients occurred at higher frequencies in soil and grain from treated fields than from untreated fields. The incidence of active ingredients recovered in soil was significantly correlated (r = 0.898; p < 0.001) with the incidence of active ingredients in grain, which in turn was also significantly correlated (r = -0.621, p = 0.02) with aflatoxin concentration. Although there were carry-over effects, caution should be taken when drawing recommendations about discontinuing biocontrol use. Cost-benefit analyses of single season and carry-over influences are needed to optimize use by communities of smallholder farmers in sub-Saharan Africa.

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.

Degradation of Aflatoxins B1 by Atoxigenic Aspergillus flavus Biocontrol Agents.

Aflatoxins are potent Aspergillus mycotoxins that contaminate food and feed, thereby impacting health and trade. Biopesticides with atoxigenic Aspergillus flavus isolates as active ingredients are used to reduce aflatoxin contamination in crops. The mechanism of aflatoxin biocontrol is primarily attributed to competitive exclusion but, sometimes, aflatoxin is reduced by greater amounts than can be explained by displacement of aflatoxin-producing fungi on the crop. Objectives of this study were to (i) evaluate the ability of atoxigenic A. flavus genotypes to degrade aflatoxin B1 (AFB1) and (ii) characterize impacts of temperature, time, and nutrient availability on AFB1 degradation by atoxigenic A. flavus. Aflatoxin-contaminated maize was inoculated with atoxigenic isolates in three separate experiments that included different atoxigenic genotypes, temperature, and time as variables. Atoxigenic genotypes varied in aflatoxin degradation but all degraded AFB1 >44% after 7 days at 30°C. The optimum temperature for AFB1 degradation was 25 to 30°C, which is similar to the optimum range for AFB1 production. In a time-course experiment, atoxigenics degraded 40% of AFB1 within 3 days, and 80% of aflatoxin was degraded by day 21. Atoxigenic isolates were able to degrade and utilize AFB1 as a sole carbon source in a chemically defined medium but quantities of AFB1 degraded declined as glucose concentrations increased. Degradation may be an additional mechanism through which atoxigenic A. flavus biocontrol products reduce aflatoxin contamination pre- or postharvest. Thus, selection of optimal atoxigenic active ingredients can include assessment of both competitive ability in agricultural fields and their ability to degrade aflatoxins.

Prevalence of Aflatoxin- and Fumonisin-Producing Fungi Associated with Cereal Crops Grown in Zimbabwe and Their Associated Risks in a Climate Change Scenario.

In most sub-Saharan African countries, staple cereal grains harbor many fungi and some produce mycotoxins that negatively impact health and trade. Maize and three small grain cereals (sorghum, pearl millet, and finger millet) produced by smallholder farmers in Zimbabwe during 2016 and 2017 were examined for fungal community structure, and total aflatoxin (AF) and fumonisin (FM) content. A total of 800 maize and 180 small grain samples were collected at harvest and during storage from four agroecological zones. Fusarium spp. dominated the fungi associated with maize. Across crops, Aspergillusflavus constituted the main Aspergillus spp. Small grain cereals were less susceptible to both AF and FM. AF (52%) and FM (89%) prevalence was higher in maize than in small grains (13-25% for AF and 0-32% for FM). Less than 2% of small grain samples exceeded the EU regulatory limit for AF (4 µg/kg), while <10% exceeded the EU regulatory limit for FM (1000 µg/kg). For maize, 28% and 54% of samples exceeded AF and FM Codex guidance limits, respectively. Higher AF contamination occurred in the drier and hotter areas while more FM occurred in the wetter year. AF exposure risk assessment revealed that small grain consumption posed low health risks (≤0.02 liver cancer cases/100,000 persons/year) while maize consumption potentially caused higher liver cancer rates of up to 9.2 cases/100,000 persons/year depending on the locality. Additionally, FM hazard quotients from maize consumption among children and adults were high in both years, but more so in a wet year than a dry year. Adoption of AF and FM management practices throughout the maize value chain coupled with policies supporting dietary diversification are needed to protect maize consumers in Zimbabwe from AF- and FM-associated health effects. The higher risk of health burden from diseases associated with elevated concentration of mycotoxins in preferred maize during climate change events can be relieved by increased consumption of small grains.

Identification of early and extra-early maturing tropical maize inbred lines resistant to Exserohilum turcicum in sub-Saharan Africa.

Northern corn leaf blight (NCLB) incited by the fungus Exserohilum turcicum is a foliar disease that significantly limits maize production and productivity in West and Central Africa (WCA), particularly in the mid-altitudes but during the last decade it has become a menace in lowland agro-ecologies. The most economical and environmentally friendly disease management strategy is the cultivation of maize varieties resistant or tolerant to NCLB. However, no early maturing (EM) and extra-early maturing (EEM) NCLB resistant varieties are commercially available in WCA. One hundred inbred lines each of EM and EEM derived from tropical maize germplasm were inoculated with a virulent isolate of E. turcicum at five locations in Nigeria during the 2017 and 2018 growing seasons. The objective of the study was to identify promising NCLB resistant lines and to investigate inter-relationships among the traits. Analysis of variance revealed highly significant genotype and genotype by environment (G × E) interactions for disease severity, grain yield (GYLD), and other agronomic traits. The average disease severity (TURC) values ranged from 1.9 to 5.8 and 2.9 to 5.7 for the EM and EEM inbred lines, respectively. The levels of reaction of the inbred lines to NCLB ranged from highly resistant to highly susceptible. Stepwise regression analysis showed that ears per plant, ear and plant aspects were significantly influenced by the disease scores. Ears per plant, ear and plant aspects, TURC and GYLD traits were employed to develop a base index (BI) for selecting NCLB resistant inbred lines for hybrid development. TZEI 135 and TZEEI 1 were outstanding in GYLD and also had the highest positive BI values in the EM and EEM inbred lines, respectively. The identification of NCLB resistant lines in this study has set the premise for development of NCLB resistant hybrids for WCA as well as the improvement of tropical maize breeding populations for NCLB resistance.

Temperature Influences on Interactions Among Aflatoxigenic Species of Aspergillus Section Flavi During Maize Colonization.

Fungal species within Aspergillus section Flavi contaminate food and feed with aflatoxins. These toxic fungal metabolites compromise human and animal health and disrupt trade. Genotypically and phenotypically diverse species co-infect crops, but temporal and spatial variation in frequencies of different lineages suggests that environmental factors such as temperature may influence structure of aflatoxin-producing fungal communities. Furthermore, though most species within Aspergillus section Flavi produce sclerotia, divergent sclerotial morphologies (small or S-type sclerotia vs. large or L-type sclerotia) and differences in types and quantities of aflatoxins produced suggest lineages are adapted to different life strategies. Temperature is a key parameter influencing pre- and post-harvest aflatoxin contamination of crops. We tested the hypothesis that species of aflatoxin-producing fungi that differ in sclerotial morphology will vary in competitive ability and that outcomes of competition and aflatoxin production will be modulated by temperature. Paired competition experiments between highly aflatoxigenic S-type species (A. aflatoxiformans and Lethal Aflatoxicosis Fungus) and L-type species (A. flavus L morphotype and A. parasiticus) were conducted on maize kernels at 25 and 30°C. Proportions of each isolate growing within and sporulating on kernels were measured using quantitative pyrosequencing. At 30°C, S-type fungi were more effective at host colonization compared to L-type isolates. Total aflatoxins and the proportion of B vs. G aflatoxins were greater at 30°C compared to 25°C. Sporulation by L-type isolates was reduced during competition with S-type fungi at 30°C, while relative quantities of conidia produced by S-type species either increased or did not change during competition. Results indicate that both species interactions and temperature can shape population structure of Aspergillus section Flavi, with warmer temperatures favoring growth and dispersal of highly toxigenic species with S-type sclerotia.

Distribution of active ingredients of a commercial aflatoxin biocontrol product in naturally occurring fungal communities across Kenya.

Human populations in Kenya are repeatedly exposed to dangerous aflatoxin levels through consumption of contaminated crops. Biocontrol with atoxigenic Aspergillus flavus is an effective method for preventing aflatoxin in crops. Although four atoxigenic A. flavus isolates (C6E, E63I, R7H and R7K) recovered from maize produced in Kenya are registered as active ingredients for a biocontrol product (Aflasafe KE01) directed at preventing contamination, natural distributions of these four genotypes prior to initiation of commercial use have not been reported. Distributions of the active ingredients of KE01 based on haplotypes at 17 SSR loci are reported. Incidences of the active ingredients and closely related haplotypes were determined in soil collected from 629 maize fields in consecutive long and short rains seasons of 2012. The four KE01 haplotypes were among the top ten most frequent. Haplotype H-1467 of active ingredient R7K was the most frequent and widespread haplotype in both seasons and was detected in the most soils (3.8%). The four KE01 haplotypes each belonged to large clonal groups containing 27-46 unique haplotypes distributed across multiple areas and in 21% of soils. Each of the KE01 haplotypes belonged to a distinct vegetative compatibility group (VCG), and all A. flavus with haplotypes matching a KE01 active ingredient belonged to the same VCG as the matching active ingredient as did all A. flavus haplotypes differing at only one SSR locus. Persistence of the KE01 active ingredients in Kenyan agroecosystems is demonstrated by detection of identical SSR haplotypes six years after initial isolation. The data provide baselines for assessing long-term influences of biocontrol applications in highly vulnerable production areas of Kenya.

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.

Field efficacy of two atoxigenic biocontrol products for mitigation of aflatoxin contamination in maize and groundnut in Ghana.

Biological control is one of the recommended methods for aflatoxin mitigation. Biocontrol products must be developed, and their efficacy demonstrated before widespread use. Efficacy of two aflatoxin biocontrol products, Aflasafe GH01 and Aflasafe GH02, were evaluated in 800 maize and groundnut farmers' fields during 2015 and 2016 in the Ashanti, Brong Ahafo, Northern, Upper East, and Upper West regions of Ghana. Both products were developed after an extensive examination of fungi associated with maize and groundnut in Ghana. Each product contains as active ingredient fungi four Aspergillus flavus isolates belonging to atoxigenic African Aspergillus Vegetative Compatibility Groups (AAVs) widely distributed across Ghana. An untreated field was maintained for each treated field to determine product efficacy. Proportions of atoxigenic AAVs composing each product were assessed in soils before product application, and soils and grains at harvest. Significant (P < 0.05) displacement of toxigenic fungi occurred in both crops during both years, in all five regions. Biocontrol-treated crops consistently had significantly (P < 0.05) less aflatoxins (range = 76% to 100% less; average = 99% less) than untreated crops. Results indicate that both biocontrol products are highly efficient, cost-effective, environmentally safe tools for aflatoxin mitigation. Most crops from treated fields could have been sold in both local and international food and feed premium markets. Adoption and use of biocontrol products have the potential to improve the health of Ghanaians, and both income and trade opportunities of farmers, aggregators, distributors, and traders.

Assessment of willingness-to-pay for Aflasafe KE01, a native biological control product for aflatoxin management in Kenya.

Contamination of key staples with aflatoxins compromises the quality of food and feed, impedes trade, and negatively affects the health of consumers whereas acute exposure can be fatal. This study used the Contingent Valuation Method (CVM) on a sample of 480 farmers in counties prone to aflatoxin contamination to assess the willingness to pay (WTP) by farmers for Aflasafe KE01, a promising biological control product for the management of aflatoxin contamination of key staples in Kenya, compare its cost with that of a similar product in use in Nigeria, and determine factors likely to affect its adoption. Four hundred and eighty households from four counties identified as aflatoxin hotspots in Kenya were purposively selected and interviewed using a semi-structured questionnaire. The mean WTP per kilogram of Aflasafe KE01, using Contingent Valuation Method in the four counties ranged from Kenya Shillings (Ksh) 113 to 152/kg compared to a cost of Ksh. 130/kg, the price of a similar product, AflasafeTM, in Nigeria. Factors that positively influenced farmers' WTP included information from crop extension services and access to credit. To facilitate the adoption of Aflasafe KE01 or any other biocontrol product in Kenya and elsewhere, there is a need for increased education efforts through extension services to farmers about aflatoxins. Strategies to ensure that the biocontrol product is integrated into the credit scheme of the technological packages to farmers need to be considered.

Functional Biology and Molecular Mechanisms of Host-Pathogen Interactions for Aflatoxin Contamination in Groundnut (Arachis hypogaea L.) and Maize (Zea mays L.).

Aflatoxins are secondary metabolites produced by soilborne saprophytic fungus Aspergillus flavus and closely related species that infect several agricultural commodities including groundnut and maize. The consumption of contaminated commodities adversely affects the health of humans and livestock. Aflatoxin contamination also causes significant economic and financial losses to producers. Research efforts and significant progress have been made in the past three decades to understand the genetic behavior, molecular mechanisms, as well as the detailed biology of host-pathogen interactions. A range of omics approaches have facilitated better understanding of the resistance mechanisms and identified pathways involved during host-pathogen interactions. Most of such studies were however undertaken in groundnut and maize. Current efforts are geared toward harnessing knowledge on host-pathogen interactions and crop resistant factors that control aflatoxin contamination. This study provides a summary of the recent progress made in enhancing the understanding of the functional biology and molecular mechanisms associated with host-pathogen interactions during aflatoxin contamination in groundnut and maize.

Aflatoxin-producing fungi associated with pre-harvest maize contamination in Uganda.

Maize is an important staple crop for the majority of the population in Uganda. However, in tropical and subtropical climates, maize is frequently contaminated with aflatoxins, a group of cancer-causing and immuno-suppressive mycotoxins produced by Aspergillus section Flavi fungi. In Uganda, there is limited knowledge about the causal agents of aflatoxin contamination. The current study determined both the aflatoxin levels in pre-harvest maize across Uganda and the structures of communities of aflatoxin-producing fungi associated with the maize. A total of 256 pre-harvest maize samples were collected from 23 major maize-growing districts in eight agro-ecological zones (AEZ). Maize aflatoxin content ranged from 0 to 3760 ng/g although only around 5% for Ugandan thresholds. For EU it is about 16% of the samples contained aflatoxin concentrations above tolerance thresholds. A total of 3105 Aspergillus section Flavi isolates were recovered and these were dominated by the A. flavus L morphotype (89.4%). Densities of aflatoxin-producing fungi were negatively correlated with elevation. Farming systems and climatic conditions of the AEZ are thought to have influenced communities' structure composition. Fungi from different AEZ varied significantly in aflatoxin-producing abilities and several atoxigenic genotypes were identified. The extremely high aflatoxin concentrations detected in some of the studied regions indicate that management strategies should be urgently designed for use at the pre-harvest stage. Atoxigenic genotypes detected across Uganda could serve as aflatoxin biocontrol agents to reduce crop contamination from fields conditions and throughout the maize value chain.

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.

"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.

Draft Genome Sequences of Three Isolates of Coniothyrium glycines, Causal Agent of Red Leaf Blotch of Soybean.

Coniothyrium glycines, the causal agent of red leaf blotch in soybeans, is considered a high-consequence biological agent. With limited genomic information known, there are no molecular genotyping or detection methods available. We report the draft genome sequences of three C. glycines isolates, greatly enhancing our knowledge of this species.