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.

Molecular diagnostic tools for plant health protection

Lava Kumar (L.kumar@cgiar.org)
Head of IITA’s Germplasm Health Unit and Virologist, Ibadan, Nigeria

Molecular tools in disease diagnosis
Rapid advancements in biotechnologies have led to the development of a myriad of molecular diagnostic tools in the past decade1. These tools, either based on the properties of nucleic acid (DNA or RNA) or proteins of the target agents, have improved the efficacy, accuracy, and speed of detection and identification of disease-causing agents and characterization of the diversity of pathogens and pests.

Researcher observing mouse hybridoma cell lines under microscope in the Virology and Molecular Diagnostics Unit, IITA, Ibadan, Nigeria. Photo by IITA.
Researcher observing mouse hybridoma cell lines under microscope in the Virology and Molecular Diagnostics Unit, IITA, Ibadan, Nigeria. Photo by IITA.
Most popular protein detection methods depend on antigen-antibody interactions. Polyclonal or monoclonal antibodies produced against the proteins of interest are used as probes to detect the target proteins by techniques such as enzyme-linked immunosorbent assay (ELISA), Western immunoblotting, dot immunobinding assay, and a number of variants of these techniques, Meanwhile, nucleic acid-based diagnostic tools are based on the hybridization of homologous nucleotides, size of the DNA fragments generated by restriction enzyme treatment, order of nucleotide arrangement, or a combination of more than one of these approaches. Polymerase chain reaction (PCR), developed in the mid-1980s, has led to the development of several new and simplified techniques, fast established as a mainstay of applied molecular biology and molecular diagnostics.

Platform for development of molecular diagnostics
The objective of the molecular diagnostics research in IITA is to develop tools and technologies for better understanding, diagnosis, and monitoring of biological systems. This program emphasizes the development of simple and accurate tools and procedures for rapid identification of pathogens and pests affecting the food and horticultural crops in sub-Saharan Africa (SSA). Both protein and nucleic-acid based diagnostic tools have been developed against target agents (viruses, fungi, bacteria, phytoplasma, insect pests, and mycotoxins). These tools are critical to several programs on crop improvement and crop protection, including evaluation of germplasm for host resistance, breeding for pest and disease resistance, surveillance surveys, and monitoring programs.

ELISA-based diagnostics are preferred for the identification of plant viruses. It is simple, reliable, cost-effective, and easy to adopt in minimally-equipped labs. Backed with facilities for purifying proteins, and production of polyclonal and monoclonal antibodies, ELISA-based diagnostics were established for about 20 economically important viruses affecting IITA’s mandate crops in SSA (e.g., Maize streak virus, cassava mosaic begomoviruses, Cowpea mottle virus, Southern bean mosaic virus, and more). Antibodies were also produced against nonviral targets such as mycotoxins. Polyclonal antibodies produced against aflatoxin B1 were used to develop the ‘Afla-ELISA’ test for quantitative estimation of aflatoxins in maize and other commodities (see companion article on Afla-ELISA). Monoclonal antibodies are usually produced for discriminating closely related virus species or strains (e.g., African cassava mosaic virus and East African cassava mosaic virus). The production of monoclonal antibodies is expensive and tedious, but it offers the advantage of perpetual production of antibodies from mouse hybridoma cell lines. Because of this, IITA has placed increasing emphasis on producing monoclonals for all important pathogens.

PCR-based diagnostics are developed as an alternative tool or to overcome the limitations of ELISA in detecting viroids, viral satellites, and to discriminate strains and closely related species. Oligonucleotide primers have been developed based on the genomic data generated from our research programs and those available in the public database for the specific detection of targets in PCR assays. Procedures were also established to simplify PCR application. For instance, a procedure established for direct detection of viruses in leaf sap bypasses the need for nucleic extraction2. Emphasis is placed on the development of multiplex PCR assays for the simultaneous detection of more than one virus in a single reaction. A multiplex PCR method has been developed for the simultaneous detection of African cassava mosaic virus and East African cassava mosaic like-viruses responsible for cassava mosaic disease in SSA2. This test was further improved to detect cassava brown streak viruses that have emerged as a major threat to cassava in East Africa, thereby making it a one-stop test for detecting all the major viruses infecting cassava in SSA.

Similar efforts are being devised to detect all viruses infecting yam. Real-time PCR using TaqmanTM probes are being developed to quantify virus concentrations within the plants to characterize host response to virus inoculation. Presently, specific and generic diagnostic tools for the detection of almost all the pathogens that affect major food staples in SSA have been established at IITA.

Pathogen diversity and DNA barcodes
Detailed knowledge of pathogen diversity is a prerequisite to developing unambiguous diagnostic tools. Pathogen populations are characterized by sequencing the specific genes and the data generated is used to interpret origin and spread of the pathogen, taxonomy, and phylogeny. For diversity assessment, gene targets are selected based on the pathogen that comprise, ribosomal Internal Transcribed Sequence (ITS), mitochondrial cytochrome oxidase-I (COI), histone, virus coat protein, etc. This approach has been used for assessing the diversity of Colletotrichum gloeosporioides responsible for anthracnose of yam, Cercospora spp. causing gray leaf spot of maize, cassava brown streak virus, banana bunchy top virus, and several others agents. Information generated from these studies have provided valuable clues to understand the origin and drivers of spread, identification of previously uncharacterized pathogens3,4 and identification of unique markers known as “DNA barcodes” for use as genetic markers for identifying pathogens and pests5.

Workflow in development of protein biomarkers. Source M. Cilia, Cornell University.
Workflow in development of protein biomarkers. Source M. Cilia, Cornell University.
Biomarkers for insect vectors
Recently a new initiative was started in collaboration with Cornell University to identify protein biomarkers to rapidly identify variation in vectoring potential of aphid and whitefly vector populations. Diagnostic tools developed in this program will aid in better understanding the virus-vector interactions, disease epidemiology, and improved management of insect vector-borne virus diseases.

Training in application of molecular diagnostics
In addition to technology development, efforts are made to transfer technology, products, and skills to stakeholders in national research and extension services. This is done through collaborative activities and organization of training courses at regular intervals in collaboration with national organizations such as the Nigerian Institute of Science Laboratory Technology (NISLT). During the training courses, specific emphasis is placed on the application of diagnostics in monitoring and surveillance programs. Standard diagnostic protocols are compiled into a cook-book style laboratory manual6 and distributed during the training courses.

End note
Molecular diagnostics development programs in IITA consider the latest knowledge and state-of-the-art technologies in establishing simple and robust tools that are relevant to end-users, are low-cost, and conducive for adoption in minimally equipped labs. We are adding new tools, such as, loop-mediated isothermal amplification reaction (LAMP) assay and deep sequencing approaches to broaden the knowledge on pathogens occurring in our mandate crops to increase the repertoire of available tools.

Molecular diagnostic tools are routinely used in germplasm indexing, phenotypic evaluation of germplasm, disease surveillance, and monitoring programs in SSA. They are also used in collecting baseline information and monitoring shifts in pathogen and pest dynamics due to changes in agriculture systems and climate change effect. These tools are already proving useful in rapid detection and identification of new and emerging pathogens and pests [e.g., Paracoccus marginatus (papaya mealybug) in Nigeria; Phytophthora colocasiae causing taro leaf blight in Nigeria and Ghana; 16srII group phytoplasma responsible for witches’ broom disease of soybean in Southern Africa; and Banana bunchy top virus in Benin].

References
1 Benali, S., et al. 2011. Eur. J. Sci. Res. 50:110–123.
2 Alabi, O.J., et al. 2008. J. Virol. Methods 154:111–120.
3 Alabi, O.J., et al. 2010. Arch. Virol. 155:643–656.
4 Sharma, K., et al., 2010. Phytopathology 100 (6): S117.
5 Kumar, P.L. and K. Sharma. 2010. DNA barcodes for pathogens of African food crops. R4D Review 4: 51–53. www.R4DReview.org.
6 Kumar, P.L. (ed.). 2009. Methods for diagnosis of plant virus diseases: a laboratory manual. IITA, Ibadan, Nigeria. 90 pp.

Transgenic banana for Africa

Leena Tripathi, l.tripathi@cgiar.org

Banana (Musa spp.) are one of the most important food crops after maize, rice, wheat, and cassava. Annual production in the world is estimated at 130 million t, nearly one-third of it grown in sub-Saharan Africa, where the crop provides more than 25% of the food energy requirements for over 100 million people. East Africa is the region that produces and consumes the most banana in Africa. Uganda is the world’s second largest producer after India, with a total of about 10 million t.

Banana plantation damaged by Xanthomonas wilt. Photo by IITA.
Banana plantation damaged by Xanthomonas wilt. Photo by IITA.

The banana Xanthomonas wilt (BXW) disease caused by the bacterium Xanthomonas campestris pv. musacearum (Xcm) was first reported about 40 years ago in Ethiopia on Ensete spp., a close relative of banana. Outside Ethiopia, BXW was first identified in Uganda in 2001, subsequently in the DR Congo, Rwanda, Kenya, Tanzania, and Burundi. The disease is highly contagious and is spread plant-to-plant through the use of contaminated agricultural implements. It is also carried by insects that feed on male buds, and is present on plant material, including infected debris. The rapid spread of the disease has endangered the livelihoods of millions of farmers who rely on banana for staple food and cash.

Infection by Xcm results in the yellowing and wilting of leaves, uneven and premature ripening of fruits, and yellowish and dark brown scars in the pulp. Infected plants eventually wither and die. The pathogen infects all varieties, including East African Highland Banana (EAHB) and exotic types, resulting in annual losses of over US$500 million across East and Central Africa.

Options for BXW control using chemicals, biocontrol agents, or resistant cultivars are not available. Although BXW can be managed by following phytosaniary practices, including cutting and burying infected plants, restricting the movement of banana materials from BXW-affected areas, decapitating male buds, and using “clean” tools, the adoption of such practices has been inconsistent. They are labor-intensive and farmers believe that debudding affects the fruit quality.

The use of disease-resistant cultivars has been an effective and economically viable strategy for managing plant diseases. However, resistance to BXW has not been found in any banana cultivar. Even if resistant germplasm is identified, conventional banana breeding to transfer resistance to farmer-preferred cultivars is a difficult and lengthy process because of the sterility of most cultivars and also the long generation times.

Transgenic technologies that facilitate the transfer of useful genes across species have been shown to offer numerous advantages to avoid the natural delays and problems in breeding banana. They provide a cost-effective method to develop varieties resistant to BXW. Transgenic plants expressing the Hypersensitive Response Assisting Protein (Hrap) or Plant Ferredoxin Like Protein (Pflp) gene originating from sweet pepper (Capsicum annuum) has been shown to offer effective resistance to related Xanthomonas strains.

Plants established in confined field trial 5 months after planting. Source: L. Tripathi, IITA.
Plants established in confined field trial 5 months after planting. Source: L. Tripathi, IITA.

IITA, in partnership with the National Agricultural Research Organization (NARO)-Uganda and the African Agriculture Technology Foundation (AATF), has developed transgenic banana expressing the Hrap or Pflp gene using embryogenic cell suspensions or meristematic tissues of four banana cultivars, Sukali Ndiizi, Mpologoma, Nakinyika, and Pisang Awak. More than 300 putatively transformed plants were regenerated and validated via PCR assay and Southern blot. Of these, 65 transgenic plants have exhibited strong resistance to BXW in the laboratory and screenhouse tests. The plants did not exhibit any differences from their nontransformed controls, suggesting that the constitutive expression of these genes has no effect on plant physiology or other agronomic traits.

The 65 resistant lines were planted in a confined field trial in October 2010 at the National Agriculture Research Laboratories (NARL), Kawanda, Uganda, after approval was obtained from the National Biosafety Committee. These transgenic lines are under evaluation for disease resistance and agronomic performance in field conditions. The transgenic lines are slated for environmental and food safety assessment in compliance with Uganda’s biosafety regulations, and procedures for risk assessment and management, and seed registration and release. After completing the necessary biosafety validation and receiving approval from the Biosafety Committee, the Xcm-resistant cultivars are expected to be deregulated for cultivation in farmers’ fields in Uganda.

We plan to stack the Pflp and Hrap genes in the same cultivars to enhance the durability of resistance against Xcm. We have developed more than 500 transgenic lines with the double genes construct (pBI-HRAP-PFLP) which are being evaluated for disease resistance under contained screenhouse conditions.

This technology may also provide effective control of other bacterial diseases such as moko or blood disease, of banana occurring in other parts of the world. The elicitor-induced resistance could be a very useful strategy for developing broad-spectrum resistance. The elicitor is a protein secreted by pathogens that induce resistance. The transgenic banana carrying these genes may also display resistance to fungal diseases such as black sigatoka and Fusarium wilt. Experiments on this are being conducted in our lab in Uganda.

Confined field trial of banana plants. Source: L. Tripathi, IITA.
Confined field trial of banana plants. Source: L. Tripathi, IITA.

We are also planning to stack genes for resistance to Xcm and nematodes into one line to produce cultivars with dual resistance that would tackle two of the most important production constraints in Eastern Africa.

The development of Xcm-resistant banana using the transgenic approach is a significant technological advance that will increase the available arsenal of weapons to fight the BXW epidemic and save livelihoods in Africa. It can become a high-value product for farmers.

This research is supported by the Gatsby Charitable Foundation, AATF, and USAID.

Note: The Pflp and Hrap genes are owned by Taiwan’s Academia Sinica, the patent holder. IITA has negotiated a royalty-free license through the AATF for access to these genes for use in the commercial production of BXW-resistant banana varieties in sub-Saharan Africa.