Developing aflasafeâ„¢

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.

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

Jacob Hodeba Mignouna: Leading the way in science

Mignouna
Jacob Hodeba Mignouna

Jacob Mignouna is a molecular biologist/biotechnologist with an MSc degree in chemical engineering and a PhD in molecular biology and genetics, both from the Catholic University of Louvain, Belgium.

He joined IITA in 1992, as a research scientist-biotechnologist. He led the Biotechnology Unit and developed and implemented a research program on the use of molecular genetic tools to improve food crops and efficiently manage crop genetic resources.

He was a distinguished Frosty Hill Research fellow and had worked as visiting scientist at the Institute for Genomic Diversity, Cornell University, Ithaca, New York; Research Associate Professor of Biotechnology and co-Director of USAID’s Farmer-to-Farmer program in East Africa at Virginia State University, Petersburg, Virginia; and Biosafety Consultant for the USAID Program for Biosafety Systems (PBS), International Food Policy Research Institute (IFPRI), Washington D.C., USA.

As Technical Operations Manager at African Agriculture Technology Foundation (AATF), he identifies opportunities for agricultural technology interventions, assesses the feasibility and probability of success of project concepts, identifies sources of appropriate technologies, negotiates their access and deployment, and provides overall leadership in the implementation of AATF’s project portfolio.

farmers-meetingPlease describe AATF’s work and your work.
AATF is a not-for-profit organization that facilitates and promotes public-private partnerships for the access and delivery of appropriate proprietary agricultural technologies for use by resource-poor smallholder farmers in sub-Saharan Africa (SSA).

The Foundation is a one-stop-shop that provides expertise and know-how that facilitates the identification, access, development, delivery, and use of proprietary agricultural technologies.

AATF works toward food security and poverty reduction in SSA, and its structure and operations draw upon the best practices and resources of both the public and private sectors.

It also contributes to capacity building in Africa by engaging African institutions to work in partnership with others.

AATF strives to achieve sustainable impact at the farm level through innovative partnerships that bring together players all along the food value chain—from smallholder farmers to national agricultural systems, regional and international research organizations, and technology developers.

Currently, AATF is working on biotechnology projects focusing on maize, cowpea, banana, rice, and sorghum—all important crops in Africa. We are also looking at ways to address aflatoxin contamination in peanuts and cereals and processing of cassava.

How did the AATF and IITA partnership come about?
In 2004, IITA approached AATF seeking to access candidate genes conferring resistance against banana bacterial wilt (BXW). IITA had already established contact with Academia Sinica, Taiwan, which held patents to the technology and wanted AATF to negotiate for a license to the ferrodoxin-like protein (pflp) and hypersensitive response assisting protein (hrap) genes from the institute.

In August 2005, IITA, Uganda’s National Agricultural Research Organisation (NARO), and AATF convened a two-day consultative meeting at which stakeholders, including other national research institutes from the Great Lakes region, including IRAZ and other NARS in the region, drafted a project concept note on developing banana bacterial wilt-resistant germplasm.

Soon after, AATF approached Academia Sinica, Taiwan, to license the pflp and hrap genes to it on humanitarian basis.

The initiative has since grown into a full-fledged project designed to enable smallholder farmers in Africa have access to disease-resistant high-yielding banana developed from East African highland varieties.

The project has two components. One focuses on developing transgenic varieties using the acquired technology and the other on improving the capacity of institutions in the region to produce high-quality disease-free planting materials using tissue culture technique.

AATF coordinates the project, including providing support in management of intellectual property rights and regulatory issues, while IITA leads the research, working with Academia Sinica and various institutions, including NARO-Uganda and IRAZ (the national research institution of Burundi), and public and private tissue culture laboratories in Kenya, Tanzania, Uganda, Burundi, Rwanda, and DR Congo.

Through the collaborative research, five banana cultivars—Kayinja, Nakitembe, Mpologoma, Sukali Ndizi, and Nakinyika—have been transformed using an Agrobacterium-mediated system. Several transgenic lines have been produced and tested in vitro by artificial inoculation with the pure Xanthomonas campestris pv. musacearum (Xcm) bacterial culture. Some of the promising lines showed no bacterial wilt symptoms. These plants were further analyzed and confirmed to have the transgene pflp integrated into the banana genome.

With progress on banana transformation well under way, AATF will soon commission a biosafety study. The findings will inform stakeholders as they develop a roadmap for the various processes required for regulatory approvals as the project progresses through the product development pipeline.

Farmers preparing cassava leaves for silage. Photo by S. Kolijn
Farmers preparing cassava leaves for silage. Photo by S. Kolijn, IITA

Please share your insights on collaboration and partnership.
First, collaboration works well if there is a clearly articulated and shared need for joint effort.

Secondly, such partnerships work best if roles and responsibilities are well defined. Work in the banana project is governed by a Memorandum of Association that recognizes the capacities of the partner organizations and facilitates each to contribute optimally to the project.

Also important is the need to bring on board potential partners early enough so that they can provide their input into the project design right from the concept stage. In this project, and generally in all AATF initiatives, we have found comprehensive consultations with a wide range of stakeholders, especially at the formative stage to be a critical success factor.

Third is information flow. Building a communication strategy into the project design ensures that the information needs of partners and external stakeholders are adequately met.

Capacity building is core to all AATF partnerships because of the key role it plays in moving the technologies through the entire food value chain, including scaling up of technologies. In this project, the hub of banana transformation work is at Kawanda, where IITA researchers are working with scientists from national research systems and jointly carrying out the transformation work. This kind of collaboration ensures that staff of national agricultural research institutes in the target countries provide continuity of work in their home country.

Another important aspect of partnership is focus on the smallholder farmer. We have found that having a shared commitment to improve the livelihoods of resource-poor farmers—a clear statement about the ultimate focus of our work—enhances stakeholders’ commitment to project activities.

Then, of course there is the need to have clear negotiated ways to deal with conflict, ensure accountability, and other governance issues.

How did AATF handle the licensing agreement for using the genes for developing resistance to Xanthomonas wilt in bananas?
AATF typically follows a strategy in which it takes the role of the principal and “responsible party” in facilitating public-private partnerships. AATF has entered into licensing agreements to access and hold proprietary technologies and to ensure freedom to operate (FTO) for all the components of the technologies. The Foundation then sublicenses partner institutions to carry out research and adapt technologies for regulatory compliance, and to produce and distribute the technologies. After signing the relevant agreements allowing use of the technology, AATF and partners are guided by a business plan that spells out the roles of each partner and how the technology will be used.

As the principal party, AATF monitors compliance with the requirements of sublicenses to minimize the risk of technology failure, and facilitates the work of appropriate partner institutions to ensure that links in the value chain are connected and result in technology products that reach smallholder farmers.

How would this research impact on banana producers and consumers in Africa?
Millions of people across the East African highlands depend on banana for their livelihoods, directly for food and smallholder producers for the market or as traders and other players in the crop’s value chain. Since banana Xanthomonas wilt broke out in the region, it has caused losses estimated at over US$500 million in Uganda, eastern DR Congo, Rwanda, Kenya, and Tanzania.

In parts of Uganda, where the crop is a staple, some families reported that their banana production had decreased by up to 80%. Given the severity of losses caused by BXW and the fact that the effectiveness of existing remedies is limited, development of disease-resistant varieties will have a huge impact on livelihoods. The benefits can be multiplied many times over by making available clean planting materials to enable farmers to rapidly expand their production.

Increased production will lead to higher incomes for families from sale of the crop, including to the vastly untapped European and American markets, now dominated by South American countries, which account for 60% of the global banana trade.

Scientists inteviewing cassava and maize farmers. Photo by K. Lopez
Scientists inteviewing cassava and maize farmers. Photo by K. Lopez, IITA

What are some of the biggest constraints to adoption of biotechnological tools or products in Africa?
I believe that properly applied agricultural biotechnology holds the key to food security in Africa. Molecular genetics tools should be used not only to improve crops but also to create a better understanding of the abundant diversity of African genetic resources for food, feed, medicine, etc. The biggest constraints to adoption of biotech tools include limited resources—both infrastructural and in terms of trained scientists and other personnel.

Some African countries also lack a regulatory environment conducive to biotech research and development. Although there have been positive changes over the past couple of years, a lot more needs to be done in these areas, including developing regulations to operationalize biosafety laws.

What could be done to take advantage of opportunities that current agricultural technologies provide and harness them for the development of African agriculture or the improvement of food security in SSA?
There are various ways but a key one is by building partnerships, such as those AATF facilitates, that can help access needed technologies, move them from product development and into the hands of farmers. This means different organizations working together to identify and resolve farmer constraints through pooling of available resources where necessary.

We also need to rapidly enhance our capacity to use biotech research. African governments and institutions need to come together and harness their various strengths to develop biotech infrastructure on the continent.

This means training more high-level scientists, equipping laboratories that can serve as centers of excellence and strengthening collaboration among African institutions and between them and research centers and universities abroad.

Lack of awareness about biotech is a major challenge. There is a need for well-designed communication campaigns not only to increase awareness and knowledge of biotechnology, but to increase public acceptance and use of technologies.

You used to head the Biotech Unit at IITA. Please tell R4D Review about your experiences in using biotechnology tools then.
The focus of the Biotechnology Unit, which comprised seven scientists and 45 support staff, was to use the tools that were then available for improving IITA mandate crops. Our work was mainly in two areas. One was developing genetic markers for the characterization of genetic resources, molecular breeding for pests and disease characterization, and exchange of germplasm. The other area was genetic engineering, where we applied tools to address intractable pests and diseases, such as insects that affect cowpea, viral and fungal diseases affecting plantain and banana and cassava mosaic disease. We also addressed diseases and pests in yam, another important food.

What are your aspirations for Africa?
My vision is to see Africa embrace all available tools, including biotechnology and develop the capacity to use them to produce enough food and improve the livelihoods of communities across Africa.

Contact: h.mignouna@aatf-africa.org