Developing aflasafeTM

Joseph Atehnkeng, j.atehnkeng@cgiar.org, Joao Augusto, Peter J. Cotty, and Ranajit Bandyopadhyay

Aflatoxins are secondary metabolites mainly produced by fungi known as Aspergillus flavus, A. parasiticus, and A. nomius. They are particularly important because of their effects on human health and agricultural trade. Aflatoxins cause liver cancer, suppress the immune system, and retard growth and development of children. Aflatoxin-contaminated feed and food causes a decrease in productivity in humans and animals and sometimes death. Maize and groundnut are particularly susceptible to aflatoxin accumulation, but other crops such as oilseeds, cassava, yam, rice, among others, can be affected as well. Aflatoxin accumulation in crops can lower income of farmers as they may not sell or negotiate better prices for their produce. Because of the high occurrence of aflatoxin in crops, many countries have set standards for acceptable aflatoxin limits in products that are meant for human and animal consumption.

Natural populations of A. flavus consist of toxigenic strains that produce variable amounts of aflatoxin and atoxigenic strains that lack the capability to produce aflatoxin. Carefully selected and widely distributed atoxigenic strains are applied on soil during crop growth to outcompete and exclude toxigenic strains from colonizing the crop. The biocontrol technology has been used extensively in the USA with two products AF36 and afla guard® available commercially. In Africa, aflasafeTM was first developed by IITA in partnership with the United States Department of Agriculture – Agricultural Research Service (USDA-ARS) and the African Agriculture Technology Foundation (AATF). It is currently at different stages of development, adoption, and commercialization in at least nine African countries. Multiyear efficacy trials in farmers’ fields in Nigeria have showed reduced aflatoxin concentration by more than 80%.

Survey to collect and dispatch samples
Product development begins with the collection of crop samples in farmers’ stores across different agroecological zones in each country. Samples collected are mainly maize and groundnut because they are the most susceptible to aflatoxin accumulation at crop maturity, during processing, and storage. Soil samples are collected from fields where these crops were grown to determine the relationship between the Aspergillus composition in the soil and the relative aflatoxin concentration in the crop at maturity.

Import and export permits are required if crop and soil samples are shipped outside a country. The crop samples are analyzed for aflatoxin to obtain baseline information on aflatoxin levels in the region/country and the relative exposure of the population to unacceptable limits of aflatoxin.

Isolation and characterization of Aspergillus species
Aspergillus species are isolated from the crop samples to identify the non-aflatoxin-producing species of A. flavus for further characterization as biocontrol agents. The isolates are identified and grouped into L-strains of A. flavus, SBG, A. parasiticus, and further characterized for their ability to produce aflatoxin by growing them on aflatoxin-free maize grain. Aflatoxin is extracted from the colonized grain using standard protocols to determine isolates that produce aflatoxin (toxigenic) and those that do not produce aflatoxin (atoxigenic). The amount of aflatoxin produced by toxigenic strains is usually quantified to determine the most toxigenic strains that will be useful for competition with atoxigenic strains.

Understanding genetic and molecular diversity
The genetic diversity of the atoxigenic strains is also determined molecularly by examining the presence or absence of the genes responsible for aflatoxin production in each strain. The absence of these genes explains why potential biocontrol isolates would not produce aflatoxin after release into the environment. Amplification of any given marker is taken to mean that the area around that marker is relatively intact, although substitutions and small indels outside the primer binding site may not be detected. Non-amplification could result from deletion of that area, an insertion between the primers that would result in a product too long to amplify by polymerase chain reaction (PCR), or mutations in the priming sites. Non-amplification of adjacent markers is probably best explained by very large deletions.

Identification of vegetative compatible groups
Vegetative compatible group (VCG) is a technique used to determine whether the highly competitive atoxigenic isolates are genetically related to each other. In nature A. flavus species that are genetically related belong to the same VCG or family; those that do not exchange genetic material belong to different VCGs. This is an important criterion for selecting a good biocontrol agent to ensure that the selected biocontrol strains do not “intermate” with aflatoxin-producing strains after field application. With this technique, the distribution of a particular VCG within a country or region is also determined. A VCG that is widely distributed is likely to be a good biocontrol agent because it has the innate ability to survive over years and across different agroecologies. On the contrary, atoxigenic VCGs that have aflatoxin-producing members within the VCG are rejected; atoxigenic VCGs that are restricted to a few locations may also not be selected.

Initial selection of competitive atoxigenic strains
The in-vitro test determines the competitive ability of the atoxigenic isolate to exclude the toxigenic isolate on the same substrate. The competition test is conducted in the laboratory by co-inoculating the most toxigenic isolate with atoxigenic strains on aflatoxin-free maize grains or groundnut kernels. Grains/kernels inoculated with the toxigenic strain or not inoculated at all serve as controls. After incubation and aflatoxin analysis, atoxigenic isolates that reduce aflatoxin by more than 80% in the co-inoculated treatments are selected for unique vegetative compatible grouping.

Selection of candidate atoxigenic strains and multiplication of inocula
aflasafe™ is composed of a mixture of four atoxigenic strains of A. flavus previously selected from crop samples. To select the four aflasafe strains, initially 8-12 elite strains belonging to atoxigenic VCGs are evaluated in large farmers’ fields. Two or three strain mixtures, each with 4-5 elite strains, are released in separate fields by broadcasting at the rate of 10 kg/ha in maize and groundnut at about 30-40 days after planting. The atoxigenic strains colonize organic matter and other plant residues in the soil in place of the aflatoxin-producing strains. Spores of the atoxigenic strains are carried by air and insects from the soil surface to the crop thereby displacing the aflatoxin-producing strains. The four best strains to constitute aflasafeTM are selected based on their ability to exclude and outcompete the toxin-producing isolates in the soil and grain, move from the soil to colonize the maize grains or groundnut kernels in the field, and occur widely and survive longer in the soil across many agroecological zones. The use of strain mixture in aflasafe™ is likely to enhance the stability of the product as more effective atoxigenic strains replace the less effective ones in specific environments. The long-term effect is the replacement of the toxigenic strains with the atoxigenic VCGs over years.

Assessing relative efficacy of aflasafeâ„¢
Field deployment to test efficacy of aflasafeâ„¢ is carried out in collaboration with national partners and most often with the extension services of the Ministry of Agriculture. Awareness is created by organizing seminars with extension agents and farmers. During the meetings presentations are made on the implication of aflatoxin on health and trade thereby increasing their knowledge on the impact of aflatoxins. aflasafeâ„¢ is then introduced as a product that prevents contamination and protects the grains before they are harvested and during storage. Efficacy trials are carried out in fields of farmers who voluntarily agree to test the product. Field demonstrations on the use of aflasafeTM are supervised and managed by the extension agents and farmers. Farmers are trained not only on the biocontrol technology but also on other management practices that enhance better crop quality.

Farmers are also educated on the need to group themselves into cooperatives, aggregate the aflasafeâ„¢-treated grains to find a premium market with companies that value good quality products. Market linkage seminars and workshops are organized between aflasafeâ„¢ farmers, poultry farmers, and the industries to ensure that the farmers get a premium for producing good quality grains and the industries get value for using good quality raw materials for their products.

Ensuring the safety of African food crops

aflasafeâ„¢ team: Ranajit Bandyopadhyay, Joseph Atehnkeng, Charity Mutegi, Joao Augusto, Juliet Akello, Adebowale Akande, Lawrence Kaptoge, Fen Beed, Olaseun Olasupo, Tahirou Abdoulaye, Peter Cotty, Abebe Menkir, and Kola Masha, with several national partners

Ground-breaking research by scientists at IITA and partners is ensuring safe food and health for Africans.

IITA, in collaboration with the United States Department of Agriculture – Agricultural Research Service (USDA-ARS) and the African Agriculture Technology Foundation (AATF), has developed a natural, safe, and cost-effective biocontrol product that drastically cuts aflatoxin contamination in African food crops.

Aflatoxins are highly toxic chemical poisons produced mainly by the fungus Aspergillus flavus in maize and groundnut, and on yam chips, but which also affect other high-value crops such as oilseeds and edible nuts. The fungal chemicals cause liver cancer and also suppress the immune system, retard growth and development, lead to chronic liver disease and cirrhosis, and death in both humans and animals. Livestock are also at risk and poultry are particularly susceptible. Cattle are not so susceptible but if they are fed with contaminated feed the toxin “Aflatoxin M1” passes into the milk.

The biocontrol product – aflasafe™ uses native strains of A. flavus that do not produce aflatoxins (called atoxigenic strains) to “push out” their toxic cousins so that crops become less contaminated in a process called “competitive exclusion”. When appropriately applied before the plants produce flowers these native atoxigenic strains completely exclude the aflatoxin producers.

IITA recommends broadcasting 10 kg/ha aflasafe™ by hand on soil 2–3 weeks before the flowering stage of maize to prevent the aflatoxin- producing fungus from colonizing and contaminating the crop while it remains in the field and subsequently in storage. Even if the grains are not stored properly, or get wet during or after harvest, the product continues to prevent infestation and contamination.

The reduction of aflatoxin in maize fields is greater with the application of aflasafe™ than with the deployment of putative low-aflatoxin maize lines. For example, field studies during 2010 and 2011 in Nigeria established that aflatoxin reduction was 16–72%, due to resistant maize hybrids, 80–92% with aflasafe™, and 80–97% with the combined use of resistance and aflasafe™.

Field testing of aflasafe™ in Nigeria between 2009 and 2012 consistently showed a decrease in contamination in maize and groundnut by 80–90% or more.

In 2009, Nigeria’s National Agency for Food and Drug Administration and Control registered aflasafe™ and permitted treatment of farmers’ fields to generate the data on product efficacy for obtaining full registration. In 2011, IITA distributed about 14 t of aflasafe™ to more than 450 maize and groundnut farms, enabling farmers to achieve an 83% reduction in contamination.

The success of the project has led to the expansion of biocontrol research in Burkina Faso, Ghana, Kenya, Mozambique, Senegal, Tanzania, and Zambia.

Between 2004 and 2006, nearly 200 Kenyans died after consuming aflatoxin-contaminated maize. In 2010 over 2 million bags of maize in Kenya’s Eastern and Central provinces were found to be highly contaminated and were declared as non-tradable.

Research conducted by Leeds University and IITA found that 99% of children at weaning age are exposed to health risks linked to aflatoxin in Bénin and Togo.

Across the world, about US$1.2 billion in commerce is lost annually due to aflatoxin contamination, with African economies losing $450 million each year. Aflatoxins are also non-tariff barriers to international trade since agricultural products are rejected that have more than the permissible levels of contamination (4 ppb for the European Union and 20 ppb for USA).

IITA has identified separate sets of four competitive atoxigenic strains isolated from locally grown maize to constitute a biocontrol product called aflasafe KE01â„¢ in Kenya and aflasafe BF01 in Burkina Faso and aflasafe SN01 in Senegal.

The adoption of this biocontrol technology with other management practices by farmers will reduce contamination by more than 70% in maize and groundnut, increase crop value by at least 5%, and improve the health of children and women.

In 2012, G20 leaders launched a new initiative – AgResults – which included aflasafe™ in Nigeria as one of the first three pilot projects to encourage the adoption of agricultural technologies by smallholder farmers.

IITA’s experience in Nigeria has shown that the cost of biocontrol (about $1.5/kg with a recommended use of 10 kg/ha) is affordable for most farmers in the country.

The biocontrol product aflasafe SN01 can potentially reinstate groundnut exports to the European Union lost by Senegal and The Gambia due to aflatoxin contamination. The World Bank has estimated that in Senegal, an added capital investment cost of $4.1 million and 15% recurring cost would attract a 30% price differential to groundnut oil cake. Exports are expected to increase from 25,000 to 210,000 t. The increased export volume and price would annually add $281 million to groundnut exports. For confectionery groundnut, adherence to good management practices would increase export value by $45 million annually.

Currently, a demonstration-scale manufacturing plant for aflasafeâ„¢ is under construction at IITA with a capacity to produce 5 t/h. Market linkages between aflasafeâ„¢ users, poultry producers, and quality conscious food processors are also being created to promote aflasafeâ„¢ adoption, in collaboration with the private sector.

Costs and benefits
Biocontrol of aflatoxin is one of the most cost-effective control methods, with the potential to offer a long-term solution to aflatoxin problems related to liver cancer in Africa. Cost-effectiveness ratio (CER) of treating all maize fields in Nigeria with aflasafeâ„¢ is between 5.1 and 9.2, rising to between 13.8 and 24.8 if treatments were restricted to maize intended for human consumption. Up to 162,000 disability-adjusted life years (DALYs) can be saved annually by biocontrol in Nigeria.

Initial data from a separate study in Nigeria suggest that farmers will receive a return of from 20 to 60% on investment in aflasafeâ„¢ from the sale of maize harvested from treated fields to poultry feed manufacturers and quality-conscious food processors.

Donor support
Research and development efforts on aflasafe™ have been supported by the following donors: Bill & Melinda Gates Foundation, USAID, USAID-FAS, AATF, Commercial Agriculture Development Project of the Government of Nigeria, The World Bank, Austrian Development Cooperation (ADC), Deutsche Gesellschaft für Internationale Zusammenarbeit, GmbH (GIZ), the European Commission (EC KBBE-2007-222690-2 MYCORED), and Meridian Institute. In addition, IITA has received support from Belgium, Denmark, The German Federal Ministry for Economic Cooperation and Development (GTZ BMZ), Ireland, Norway, Sweden, Switzerland, and the UK Department for International Development (DFID).

References
Atehnkeng, J., P.S. Ojiambo, M. Donner, T. Ikotun, R.A. Sikora, P.J. Cotty, and R. Bandyopadhyay. 2008. Distribution and toxigenicity of Aspergillus species isolated from maize kernels from three agroecological zones in Nigeria. International Journal of Food Microbiology 122 (1-2): 74-84.
Atehnkeng, J., P.S. Ojiambo, T. Ikotun, R.A. Sikora, P.J. Cotty, and R. Bandyopadhyay. 2008. Evaluation of atoxigenic isolates of Aspergillus flavus as potential biocontrol agents for aflatoxin in maize. Food Additives and Contaminants 25 (10): 1266-1273.
Bandyopadhyay, R., M. Kumar, and J.F. Leslie. 2007. Relative severity of aflatoxin contamination of cereal crops in West Africa. Food Additives and Contaminants 24 (10): 1109-1114.
Diedhiou, P.M., R. Bandyopadhyay, J. Atehnkeng, and P.S. Ojiambo. 2011. Aspergillus colonization and aflatoxin contamination of maize and sesame kernels in two agroecological zones in Senegal, Journal of Phytopathology 159 (4): 268-275.
Donner, M., J. Atehnkeng, R.A. Sikora, R. Bandyopadhyay, and P.J. Cotty. 2009. Distribution of Aspergillus section flavi in soils of maize fields in three agroecological zones of Nigeria. Soil Biology and Chemistry 41 (1): 37-44.
Donner, M., J. Atehnkeng, R.A. Sikora, R. Bandyopadhyay, and P.J. Cotty. 2010. Molecular characterization of atoxigenic strains for biological control of aflatoxins in Nigeria. Food Additives 27(5): 576-590.
Egal, S., A. Hounsa, Y.Y. Gong, P.C. Turner, C.P. Wild, A.J. Hall, K. Hell, and K.F. Cardwell. 2005. Dietary exposure to aflatoxin from maize and groundnut in young children from Bénin and Togo, West Africa. International Journal of Food Microbiology 104 (2): 215-224.
Kankolongo, M.A., K. Hell, and I.N. Nawa. 2009. Assessment for fungal, mycotoxin and insect spoilage in maize stored for human consumption in Zambia. Journal of the Science of Food and Agriculture 89 (8): 1366-1375.
Oluwafemi, F., M. Kumar, R. Bandyopadhyay, T. Ogunbanwo, and K.B. Ayanwande. 2010. Bio-detoxification of aflatoxin B1 in artificially contaminated maize grains using lactic acid bacteria. Toxin Reviews 29 (3-4): 115-122.
Wu, F. and Khlangwiset, P. 2010. Health and economic impacts and cost-effectiveness of aflatoxin-reduction strategies in Africa: case studies in biocontrol and postharvest interventions. Food Additives and Contaminants: Part A, 27: 496-509.

Initiative tackles killer aflatoxin

IITA and partners recently launched a project that will provide farmers in Nigeria and Kenya with a natural, safe, and cost-effective solution to prevent the contamination of maize and groundnut by a cancer-causing poison, aflatoxin. It is funded by the Bill & Melinda Gates Foundation.

Maize cobs attacked by fungi. Photo by IITA.
Maize cobs attacked by fungi. Photo by IITA.

Aflatoxin is produced by a fungus (Aspergillus flavus). It damages human health and is a barrier to trade and economic growth. The toxin, however, is not produced in all strains of the fungus. The project’s biocontrol technology introduces nontoxic strains of the fungus in the affected fields. These “good guys” overpower and reduce the “bad guys,” the population of toxic strains, drastically reducing the rate of contamination.

During the launching of the project, Wilson Songa, Agricultural Secretary in Kenya’s Ministry of Agriculture, said that Kenya welcomed the initiative after recent losses of lives and millions of tons of maize to aflatoxin contamination.

“Kenya has become a hotspot of aflatoxin contamination. Since 2004, nearly 150 people have died after eating contaminated maize,” he said.

IITA had worked with the United States Department of Agriculture to develop a biocontrol solution for aflatoxin, testing it in many fields in Nigeria. The project will take the biocontrol product, commercialize it, and make it available to farmers.

Ranajit Bandyopadhyay, IITA’s plant pathologist, says the project is adding value to previous investments in biocontrol. It will support the final stage of commercialization of aflasafe™ in Nigeria and selection of the most effective strains, development of a biocontrol product, and gathering of data on efficacy in Kenya.

The Nigerian government has joined forces with IITA and the World Bank to help contain the contamination of food crops by aflatoxins.

The collaboration will make aflasafeâ„¢ available to farmers to greatly reduce the aflatoxin menace.

The new approach is part of the Commercial Agriculture Development Program supported by the World Bank and implemented in Kano, Kaduna, Enugu, Cross River, and Lagos States in Nigeria.

In Nigeria, produce from resource-poor maize farmers faces rejection from the premium food market because of aflatoxin contamination.

In on-farm research trials in Kaduna State—north-central Nigeria—during 2009 and 2010, farmers who treated their fields with aflasafe™ were able to reduce the levels of contamination by 80 to 90%.

Related website

Aflatoxin management website – www.aflasafe.com

aflasafeâ„¢: a winning formula

Biological control of aflatoxins using aflasafeâ„¢ is providing hope for African farmers battling with crop contamination and opening doors for the private sector looking to invest on a winning formula in the agricultural sector.

Scientists have developed a cost-effective, safe, and natural method to prevent aflatoxin formation in maize while in the field. Aflatoxin causes liver cancer and suppresses the immune system, endangering both humans and animals. It also retards growth and development of children. This colorless chemical is invisible and its presence and contamination levels can only be confirmed by laboratory tests.

The biocontrol technology works by introducing native (local) strains of the fungus Aspergillus flavus that do not produce the aflatoxin (the ‘good guys’) in the affected fields. This good fungus boxes out and drastically reduces the population of the poison-producing strains (the ‘bad guys’).

The aflasafeâ„¢ technology has the potential to provide relief to millions of maize farmers in sub-Saharan Africa depending on agriculture as a source of livelihood.

According to Ranajit Bandyopadhyay, IITA Plant Pathologist, a single application of this biopesticide 2-3 weeks before maize flowering is sufficient to prevent aflatoxin contamination throughout and beyond a cropping season and even when the grains are in storage.

With an initial investment outlay of US$1−3 million in an aflasafe™ manufacturing plant, investors are likely to reap about $1.33 million annually. Bandyopadhyay said that investing in an aflasafe™ manufacturing plant in Nigeria would pay off considering the huge demand for quality maize in the country. His estimates showed that over 60% of harvested maize in Nigeria currently has high levels of aflatoxins and are prone to being rejected by the feed industry.

Institutions involved in the initiative include IITA, Agriculture Research Service of the United States Department of Agriculture, AATF, and local partners.

Related website

Aflatoxin management website – www.aflasafe.com

Ranajit Bandyopadhyay: Ending aflatoxin contamination in Africa

Developing countries lose billions of dollars in trade annually to aflatoxin contamination in foods. Worst still, the contamination endangers the health of millions of people in the region. But the good news is that IITA has developed a biocontrol product (aflasafe) to tackle this problem. Ranajit Bandyophadhay speaks to Godwin Atser on the journey that led to the development of aflasafe and other issues.

Ranajit (left) explains to partners how aflasafe works
Ranajit (left) explains to partners how aflasafe works

Tell us about your work at IITA
I am a plant pathologist, and one of my main responsibilities is how to manage plant disease. The other is to ensure food safety.

What about your work on biocontrol?
This is one the most exciting projects that I have ever had. The work on biocontrol is on a toxin found on maize and peanuts called aflatoxins. The toxin causes a lot of harm to people’s health and also makes farmers sell their products at lower prices. So, the losses are both in terms of health and trade. What I am trying to do is to manage the aflatoxins using a holistic approach, such as using resistant varieties, better crop management practices, and also the biological control method.

What is unique about your biocontrol work?
One thing that is unique is that we are using the natural resources from Nigeria to manage an economic and medical problem. We are making use of nontoxic fungi to eliminate the harmful fungi (aflatoxins).

Why are aflatoxins important?
Aflatoxins are harmful chemicals that are produced by a fungus called Aspergillus flavus.

The fungi produce toxins in maize, peanuts, and generally grains. When people eat them, it harms them and causes diseases such as liver cancer and kwashiorkor, among others. Worst still, farmers cannot sell their products at the premium price.

What makes you keen about biocontrol?
It is one of the strongest components of the holistic approach. If we can have the biocontrol approach adopted by farmers, most of the problems concerning aflatoxins which they face during postharvest will be greatly reduced.

Do you see IITA in the position to offer the biocontrol option to farmers?
Absolutely yes. The reason is that we actually started with good science and that science has given birth to a new product which the farmers are willing to use.

Happy farmer with aflasafe
Happy farmer with aflasafe

Tell us about this product
The product is “aflasafe”. We coined the name aflasafe; when farmers use the product on their farms, they would produce grains that are free from aflatoxins.

Did you face any challenge in developing the product?
The first challenge was developing the product itself. The fresh challenge now is how to get a large manufacturing firm to begin massive production, advocacy, and awareness so that it gets to the farmers.

Any interest so far?
We made a presentation to the Minister of Health, Prof. Babatunde Osotimehin, and he was so excited about the product. We also did a field deployment and the farmers were also very happy about it.

Who were your partners in this work?
Many organizations and people were involved in this work. They include the AATF, National Agency for Food and Drug Administration and Control, farmers, United States Department of Agriculture, US Agency for International Development, Prof Peter Cotty, Dr Joseph Atehnkeng, and several others.

How were you able to handle these partners?
Every partner is a unique entity but one thing important is to build trust. Once that is done, the partnership gets smooth.

Research wise, what are you future plans?
My future plan is to get this product used on at least one million hectares. I intend to put all my efforts to see that this product is used for the benefit of the farmers in general and women and children who are more vulnerable to aflatoxins.

Related website

Aflatoxin management website – www.aflasafe.com