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.

Why manage noncrop biodiversity

Muris Korkaric, m.korkaric@iita-uganda.org and Fen Beed, f.beed@cgiar.org

When it comes to the diversity of nonplant taxa, the numbers alone are highly impressive. There are an estimated 5–30 million species of microorganisms globally but only two million have been formally described. In 1 g of soil, over a billion bacteria cells can be found, but fewer than 5% of the species have been named or can be grown on artificial media. For fungi, about 1.5 million species are estimated to exist and yet only 5% have been characterized taxonomically.

Disease symptom caused by Colletotrichum fuscum on lettuce leaf. Photo by F. Beed, IITA.
Disease symptom caused by Colletotrichum fuscum on lettuce leaf. Photo by F. Beed, IITA.

Nematodes remain particularly poorly described with only a fraction of the suspected half million found in nature known to man. For insects, arachnids, and myriapods only 1.1 million have been named from a potential 9 million. These numbers compare with an estimated 420,000 seed plants of which most have been described.

Knowledge of biodiversity is uneven, with strong biases towards the species level, large animals, temperate systems, and the components of biodiversity used by people. Although biodiversity underlies all ecosystem processes, modern agriculture is based on a very limited genetic pool of crops and an even more limited exploitation of the genetic resources of nonplant taxa.

This is surprising, considering that as a consequence of their diversity microorganisms and insects play pivotal roles across ecosystems that far exceed those of plants. They provide critical functions and services for food and agriculture. They are indivisibly connected with ecosystem resilience, crop health, soil fertility, and the productivity and quality of food. Modern agriculture in the developed and especially the developing world uses only a small fraction from this rich pool of genetic resources.

Conserving and using nonplant taxa
One of the vital pillars in the work of the Consultative Group on International Agricultural Research (CGIAR) is the conservation and use of agrobiodiversity and related knowledge. Over 650,000 accessions of crop, forage, and agroforestry genetic resources are stored and maintained through the centers’ genebank system and distributed to researchers and breeders throughout the world.

However, scientists from different CGIAR centers are also involved in collection, conservation, and sustainable use of insects and mites, fungi, bacteria, viruses, and nematodes that are either beneficial or antagonistic to crops. These research collections are used in two main areas: (1) crop health and productivity, where the collection supports screening for resistance in breeding programs, pathogen diagnostics, and the development of biological control technologies, and (2) soil health, fertility and ecosystem resilience where for example, collections support the development of biofertilizers.

IITA’s main collections of nonplant taxa are housed at the stations in Ibadan (Nigeria) and Cotonou (Bénin). At the headquarters in Ibadan, the collection and study of plant pathogenic fungi, bacteria, and viruses of important crops are coordinated and collections are maintained. Examples are those for yam and cassava anthracnose, cassava bacterial blight, and soybean rust pathogens.

Aflatoxin-producing fungus Aspergillus flavus growing out of maize grains in a culture medium. Photo by J. Atehnkeng.
Aflatoxin-producing fungus Aspergillus flavus growing out of maize grains in a culture medium. Photo by J. Atehnkeng.

Some of the collections contain large numbers of isolates of the same species which are often unique, not being found elsewhere in the world. International repositories might hold many different species, but tend to store fewer isolates per species and rarely prospect across the developing world. A diverse range of isolates gives a more complete representation of the genetic diversity which can be crucial for understanding evolutionary patterns, pathogen variation, and population dynamics. It helps breeding programs to identify targets for resistance selection.

Collections of isolates of the same species can be used to develop appropriate biocontrol technologies. One such example is IITA’s collection of Aspergillus flavus, a fungus that normally produces aflatoxin, a compound that is toxic to humans and animals. Over 4,500 strains have been collected from Nigeria alone and screened for toxin production and their ability to outcompete other strains when found simultaneously on foodstuffs. The atoxigenic and most competitive strains have been used to formulate aflasafe®, a biocontrol product (see R4D Review September 2009 issue).

Also in Ibadan, collections of beneficial soil microorganisms are studied and maintained. These organisms (such as Rhizobia spp. and mycorrhizae) enhance the nutrient uptake of leguminous crops and can be used as biofertilizers.

At IITA-Bénin, microorganisms and arthropods have been characterized and preserved for use in biological control programs to manage invasive crop pests and weeds. Plant pathogens have been identified and stored since the deployment of appropriate control measures first requires definitive identification of the causal agent of the disease. The biodiversity center maintains over 360,000 insect and mite specimens and is one of the largest reference collections in West Africa (see R4D Review September 2009).

Other IITA stations keep smaller working collections of nonplant taxa. At IITA-Uganda, collections of nematodes, bacteria, and fungi are maintained—mainly those associated with banana production. Certain Fusarium strains, for example, are used for endophyte-improved banana tissue culture for enhanced pest and disease resistance.

Looking like strung beads, these are part of a sample of insects received by the IITA biodiversity center in 12 months. Photo by G. Goergen, IITA.
Looking like strung beads, these are part of a sample of insects received by the IITA biodiversity center in 12 months. Photo by G. Goergen, IITA.

IITA is a lead organization for the conservation and use of nonplant taxa across sub-Saharan Africa. It is now characterizing nonplant taxa collections across the CGIAR as part of the World Bank-funded GPG2 project (Phase II of the Collective Action for the Rehabilitation of Global Public Goods in the CGIAR Genetic Resources System). This is the first system-wide inventory and collation of the existing global, nonplant taxa collections. The aim is to provide a coordinated and harmonized service for research and use of noncrop taxa to support durable farming systems in the developing world.

Future challenges and opportunities
There is a growing appreciation of the fact that farming occurs in an ecological context with complex interactions between crop and nonplant taxa that can be beneficial or antagonistic. There is also increasing demand for sustainable and environment-friendly solutions to manage pests and diseases, with the expectation that the biopesticide market share will increase to over 4.2% by 2010 and, for the first time, reach a market of over US$1 billion. Due to the rate of population increase the World Bank estimates that the global demand for food will double within the next 50 years. At the same time, the amount of arable land is decreasing from pressure from nonfarming activities and the unsustainable farming practices that are causing losses in soil fertility. This scenario is exacerbated by the fact that 40% of what is grown in the world is lost to weeds, pests, and diseases. In developing countries it is common for up to 70% of the yield to be lost due to attacks from insects and microbial diseases.

Therefore, agricultural production needs to be intensified and more marginal land used to produce sufficient food. This requires the deployment of improved land management techniques combined with the selection and distribution of appropriate crop and noncrop germplasm to exploit interactions with beneficial nonplant taxa and resist increased pressure from antagonistic nonplant taxa. Other factors such as climate change are likely to add new layers of complexity to these challenges. To predict risk and develop appropriate adaptation strategies, CGIAR and governments will become increasingly reliant on knowledge of and access to nonplant taxa genetic resources for food and agriculture. This will be used for research, training, or direct use in agriculture and originate, or be found, in a range of countries or centers.

Collections form the mechanism through which information and access to nonplant taxa can be obtained, but the survival of these collections is under threat from funding constraints. Appropriate policies, investments, and collaborations among CGIAR centers and with international collections are urgently needed to recognize noncrop taxa as global public goods. This would facilitate the conservation of collections, increase their visibility, and maximize their use for the benefit of sustainable farming systems. Especially in Africa, where the biodiversity is high, but the taxonomic and technological capacity is limited, work is needed to manage the full potential of nonplant taxa for food and agriculture.