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.

Increasing capacity for plant healthcare

Plants, like people, need healthcare. But in Africa, where agriculture is dominated by smallholders, farmers do not have access to reliable plant health advice and management services.

Many farmers rely on extension workers and researchers from national and international organizations for such needs. And such help is not always readily or quickly available.

Bunchy top virus-affected banana in Rusizi Valley, DRC, Rwanda, and Burundi. Photo by IITA
Bunchy top virus-affected banana in Rusizi Valley, DRC, Rwanda, and Burundi. Photo by IITA

This is why IITA and its partners are developing the capacity of national agricultural research and extension systems (NARES) in research, disease surveillance, diagnostics, and deployment of control options. A good example is in banana: when national partners at the L’Institut des Sciences Agronomiques du Burundi (ISABU) in Central Africa needed help in diagnosing and culturing the pathogen that was attacking banana, they turned to IITA for assistance. ISABU wanted to develop local capacity to independently make diagnoses, culture Banana Xanthomonas Wilt (BXW) from diseased banana plant samples, and provide treatment advice.

At that time, IITA was already working on BXW in Burundi under the Crop Crisis Control Project (C3P), managed by the Catholic Relief Services (CRS). IITA and CRS liaised closely to develop a regional training course, for national partners from Burundi, Rwanda, and Democratic Republic of Congo (DRC) to learn new techniques, while encouraging greater collaboration among scientists.

Thus, IITA and partners that include CABI UK, Central Science Laboratory (CSL), CRS, and the Consortium for Improving Agriculture-based Livelihoods in Central Africa (CIALCA) conducted a Training Course on Surveillance and Vigilance for Plant Diseases in Burundi early this year. It is a pilot effort to kick-start a series of capacity building initiatives in the banana-growing countries in the region.

The training was attended by participants from extension and research, universities, and a regional organization. Trainers came from IITA, CABI and Global Plant Clinic (GPC, see box), and Central Science Laboratory (CSL).

Training covered new methods for surveillance and vigilance of all banana diseases. Feedback from the participants highlighted the need for sustained training and the importance of introducing a system of mobile plant clinics to effectively link farmers and transfer knowledge.

The mobile plant clinics initiative was developed by CABI UK as part of GPC, led by Eric Boa and has been tried and tested across the world. Under the umbrella of Mobile Plant Clinics and GPC, IITA had collaborated on initiating clinics in Rwanda, Cameroon, Sierra Leone, and Benin and providing training in diagnostics and surveillance in Uganda, DRC, and Burundi.

“Training, however, is just the tip of the iceberg. It is important to consolidate capacity building in diagnostic techniques and to ensure that people adopt new methods with confidence and then use them regularly,” said Fen Beed, IITA’s plant pathologist based in Uganda. “Isolating and identifying plant bacteria require practice as does the conduct of participatory disease surveys. When such methods are reliably deployed, the national programs could significantly improve the reliable detection of BXW and other disease outbreaks.”

Training participants look at Banana Xanthomonas Wilt chart. Photo by IITA

Knowing where a disease occurs allows extension staff to target particular areas and plan control programs. This requires careful organization and marshalling of resources. Although IITA already has effective recommendations for managing BXW, it lacks mechanisms for presenting them to farmers and monitoring their uptake. Further effort is needed to implement training that emphasizes direct action to help farmers.

In their after-training report, Beed and colleagues said that “Effective extension depends on sound intelligence about disease distribution and the damage it causes. National governments need to understand the risks posed to new areas and the actions required to control disease through sound research planning and identification of best management strategies.”

Beed and colleagues forwarded this blueprint for managing risk and reducing banana disease losses to ensure success of a plant healthcare service managed by national programs.

Surveillance
It is important to undertake systematic and comprehensive surveys of banana growing areas to get an update on the distribution of BXW and control strategies being used by growers. The surveys provide the opportunity to determine spread and identify reasons why control strategies may not have been adopted. Where control methods have been deployed their socioeconomic impact can be quantified.

The extensive surveys will assess incidence and severity of BXW and other banana pests and diseases.
Systematic and quantitative surveillance of banana-growing areas begin with participatory surveys, a promising technique for assessing large numbers of growers quickly. Survey results can identify sites where permanent sample plots (PSP) would be established for more intensive assessments. PSP sites should be regularly monitored for disease incidence, severity, and efficacy of control methods. Data produced can determine disease spread and help to evaluate socioeconomic impact and deployment of control options.

The C3P project made huge strides towards developing databases on the spread of BXW and the influence of farmers’ practices to control this disease. These databases can be further updated with information from the surveys and with data generated from pilot sites.

The databases could be linked to regional databases of climate, growing conditions, topography, farmer demographics, and agricultural practices (e.g., produced by the CIALCA project and many others). This allows use of the databases for predicting spread and risk due to disease at various geographic scales.

Vigilance
The next step is to establish and operate an extensive system of mobile plant clinics in targeted areas. Training courses for plant doctors are available and both DR Congo and Rwanda already have some experience in running clinics. The clinics concentrate on giving advice and gathering “intelligence” about banana problems, providing information on disease control, and offering services for other crops and diseases. This is important since farmers rarely grow bananas in isolation of other crops.

Once clinics are established and their benefits realized they can be self-sustaining and can provide a routine service to farmers and extension officers.

Upgrading facilities
There is a need to ensure that participating laboratories can isolate and confirm the presence of pathogens that cause BXW and other diseases of banana. Field staff should learn how to collect diseased plant samples for sending to diagnostic centers. Diagnostic centers will be established in the region and linkages developed with advanced research institutes (ARI) to provide technical backstopping for disease diagnostics using, for example, molecular techniques.

Banana field trials in Rwanda. Photo by IITA
Banana field trials in Rwanda. Photo by IITA

In addition, for BXW, rapid diagnostic field-based kits will be fully tested for accuracy to confirm the presence of the disease. Standard operating procedures for laboratory methods should be introduced to ensure consistent results and interpretation of results. The responsibilities of staff from national, regional, and ARI laboratories should be identified and links among them strengthened to create and nurture a network of expertise available to all.

Awareness raising
Data produced from the three activities can be used to publish new disease reports and develop pest risk analysis (PRA) documents for each banana disease in the region. PRA documents are crucial as they summarize all current information and increase awareness of disease recognition, distribution, control and risks. They must be routinely updated with new information and shared across the region to alert stakeholders of potential risks. This can lead to the deployment of preemptive disease control strategies before a disease epidemic breaks out.

Monitoring and evaluation
Detailed assessment of the progress and linkages should be undertaken. The increased capacity in laboratory and field techniques should be shared by project members through training. The support of IITA and the GPC in diagnostics, surveillance, and vigilance techniques encourages national and regional cooperation and use of new methodologies. Empowering scientists and extension staff and making them accountable for their actions is a powerful way to encourage sustainable development and to promote trade.

Linkages
The benefit of creating a knowledge network for banana diseases in the region is clear. This network can be expanded through linkages with scientists and the private sector and key extension, research, and government staff from Burundi, DRC, Rwanda, and regional organizations.

The International Plant Diagnostic Network (IPDN) was set up in response to NARES’ surveys that highlighted the lack of diagnostic capacity in much of Africa and in recognition that this directly hindered the adoption of appropriate and effective integrated pest management programs and therefore international trade. IPDN has been established in collaboration with IITA in East and West Africa to increase communication and data sharing. Software for digital imaging and diagnosis, information management, and access to disease management recommendations provides a platform for enhanced diagnosis and communication between laboratory staff and experts across the world. Improved diagnostics tools and protocols have been developed and tested. This has been combined with training programs to enhance technical capacity and increase networking among diagnosticians in East and West Africa.

Initiatives such as IPDN can benefit by collaboration with similar internet-based initiatives in Africa such as the East Africa Phytosanitary Information Committee (EAPIC). EAPIC is linked to FAO’s International Plant Portal to provide posting of plant pests for each respective country, which now includes Kenya, Tanzania, Uganda, and Zambia. The plant pest list helps in developing harmonized border inspection protocols, which support capacity building efforts in plant pest survey, identification, and communication systems, such as IPDN.

A follow-on project with these components that combines good science, effective surveillance, and proven advisory services could strengthen the contribution of extension and research to increase food security, income generation, and improved trade in Africa. It also highlights support required from national and regional organizations, governments, and donors. These include local training for diagnostic techniques and expansion of participatory disease surveys and strengthening of disease vigilance through the establishment of mobile plant clinics.

Figure 1. Disease management scenario (fire fighting vs. preemptive control)
Figure 1. Disease management scenario (fire fighting vs. preemptive control)

”Addressing all these considerations will contribute significantly towards providing a service to support farmers and trade that would move away from the current scenario of ‘fire-fighting’ diseases to providing preemptive control (see Figure 1),” concluded Beed.

Global Plant Clinics

The CABI Bioscience Global Plant Clinic (GPC) provides a comprehensive diagnostic and advisory service for disease problems on all tropical crops. The Service is unique in its global operation and the range of plant diseases it handles. CABI Bioscience has been identifying plant diseases for over 90 years and other key partners in GPC include Rothamstead Research and Central Science Laboratories. The Global Plant Clinic gives expert advice on the interpretation and application of diagnostic results drawing on extensive international experience in a wide range of crops and information from CAB International’s award-winning Crop Protection Compendium.

Mobile plant clinic in Butembo, DR Congo. Photo by IITA

The GPC has initiated a series of mobile plant health clinics that offer regular and reliable advice on all plant health problems affecting any crop. These clinics are run by plant “doctors”, many of whom are agronomists or extension workers, who work for existing, grassroots organizations.

The clinics are not a technology but an advisory service. They link diagnostic labs with extension workers (plant doctors) and provide regulatory bodies in plant health with up-to-date information on current priorities by clinic ‘area of influence’. Such clinics have little direct expense. In the long term they need public investment and private support (from farmers or input suppliers such as those responsible for improved varieties or even fertilizer).

According to GPC head Dr Eric Boa, “Farmers benefit from advice at clinics: they preempt new problems and avert losses by quick action; reduce pesticide use; and reduce losses and save money by giving good or better recommendations for managing a problem. On vigilance/surveillance, clinics identify current problems affecting priority problems in an area.”

In banana, the most recent disease outbreak due to banana Xanthomonas wilt (BXW) was first reported to move from Ethiopia to Uganda by regional scientists and was subsequently confirmed by the GPC in Uganda in 2001. As the disease spread within Uganda and relentlessly across the region research programs led by CRS, IITA, and other national scientists tracked its movement into Burundi, DRC, Rwanda, Tanzania, Kenya, and the causal agent was confirmed by GPC.

BXW is one of several damaging diseases in East Africa and the demand for better surveillance and vigilance through mobile plant clinics has been widely expressed. The deployment of control options through clinics was based upon methods used to control a similar disease of banana caused by another bacterium. These primarily consist of the use of disease-free planting material and farmers tools and the removal of male flower buds to prevent infection from insect vectors.

http://194.203.77.76/globalplantclinic/

The future of African bananas

The use of genetic engineering has transformed agriculture, and food production and development by providing options and solutions where none existed before—to the benefit of billions of the world’s inhabitants.

Tripathi discusses work with staff at Namuloge, IITA-Uganda. Photo by IITA

IITA and its partners have been using genetic transformation as a crop improvement tool to help produce more and better food staples. The Institute—with partners such as the National Agricultural Research Organization (NARO) of Uganda, Academia Sinica (Taiwan), and the African Agricultural Technology Foundation (AATF) in Kenya—is at the forefront of research “designing” a genetically modified banana that is resistant to the worst bacterial disease so far—Banana Xanthomonas Wilt (BXW). Entire banana fields can be destroyed, especially those planted to Pisang awak, a susceptible exotic variety widely grown to make banana beer.

Bananas are a major staple in East Africa produced mostly by smallholder subsistence farmers. Uganda is the world’s second leading grower with a total annual production of about 10.5 million tons. It is Africa’s biggest producer and consumer of bananas and plantains.

Most growers cannot afford costly chemicals to control the many pests and diseases that affect banana cultivation. As diseases continue to spread, demand grows for new improved varieties.

Bacterial wilt caused by Xanthomonas campestris pv. musacearum is threatening banana production and the livelihoods of these smallholder growers, and solutions have to be found fast before it could destabilize food security in the region.

Work on developing a GMO banana has been ongoing since BXW was first reported in 2001. The disease has been identified in the Eastern Democratic Republic of Congo, Rwanda, Kenya, and Tanzania, and is widespread in Uganda. It attacks almost all varieties of bananas, causing these countries an annual loss of over US$500 million. These can be reduced bunch weights or absolute yield loss or clean planting material is unobtainable for new plantations.

Banana Xanthomonas wilt-infected plants. Photo by IITA

“Developing resistant varieties is a long-term but more sustainable way to control pests and diseases. Improving the plant’s defense mechanism against BXW through genetic engineering is still the best line of defense because of its many advantages,” commented molecular geneticist Leena Tripathi based in IITA-Uganda, Kampala. “Farmers are reluctant to employ labor-intensive disease control measures.”

“Genetic engineering offers many opportunities for improving existing elite varieties not amenable to conventional cross-breeding, such as bananas. It allows breeders to develop new varieties quickly through the introduction of cloned genes into commercial varieties.”

Transgenic bananas possess a gene or genes that have been transferred from another plant species. The term “transgenic plants” refers to plants created in a laboratory using recombinant DNA technology.
Tripathi said that the development of stable and reproducible transformation and regeneration technologies has opened new horizons in banana and plantain breeding. The development of transgenic banana and plantain has been reported by several groups, but a commercial transgenic banana variety is yet to be released.

There are no cross-fertile wild relatives in many banana-producing areas. Most edible bananas and plantains are male and female sterile. The clonal mode of propagation makes the risk of gene flow from banana to another crop species not an issue.

IITA’s in vitro screening method for early evaluation of resistance to BXW uses small tissue culture-grown plantlets. This method can be used by breeders for screening Musa germplasm with larger numbers of cultivars for resistance to BXW and other bacterial diseases.

Currently, most transformation protocols for banana use cell suspensions, Tripathi said. Establishing cell suspensions is a lengthy process and cultivar dependent. At present, the major barrier in transforming East African Highland Bananas (EAHB), a cooking banana from Uganda, is the limited success in producing embryogenic cell suspension cultures from a wide range of cultivars. IITA scientists in collaboration with NARO have developed a rapid and efficient protocol using a cultivar-independent transformation system for improving Musa species including EAHB. This new technique has paved the way for the development of a transgenic banana using transgenes from sweet pepper that confer resistance against BXW.

Tripathi explains how the technology works: the ferredoxin-like amphipathic protein (pflp) and hypersensitive response-assisting protein (hrap), were isolated from the sweet pepper, Capsicum annuum. These are novel plant proteins that intensify the harpinPSS-mediated hypersensitive response (HR). These proteins have a dual function: iron depletion antibiotic action and harpin-triggered HR enhancement. The transgenes were shown to delay the hypersensitive response induced by various pathogens in nonhost plants through the release of the proteinaceous elicitor, harpinPss in various crops including dicots such as tobacco, potato, tomato, broccoli, orchids and monocots such as rice. Elicitor-induced resistance is not specific against particular pathogens, hence it is a very useful strategy.

The pflp genes encode for ferrodoxin, which exists in all organisms, and is therefore common in human diets. This protein is safe for human consumption and the environment. 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 the pflp and hrap genes for use in the production of BXW-resistant varieties in sub-Saharan Africa.

Hundreds of transformed lines of various banana cultivars have already been generated, and are under screening for disease resistance under laboratory conditions. The most promising will be evaluated for efficacy against BXW in confined field trials under different farming systems by national partners with IITA. The transgenic lines will be tested for environmental and food safety, in compliance with target country biosafety regulations, risk assessment and management, and seed registration and release procedures. The project will also study public perceptions, consumer preferences, and the acceptability of transgenic banana in Africa to guide commercialization and wide use.

“Wide-scale deployment of genetically modified, farmer-preferred banana varieties in African countries would succeed only with effective interinstitutional partnerships, particularly with advanced research institutions, AATF, national committees on biosafety, nongovernmental organizations, and private tissue culture companies,” explained Tripathi. “This project will enhance the capacity of partners from the national agricultural research and extension systems in genetic transformation of banana, molecular biology, and biosafety. High-yielding BXW-resistant banana will bring greater productivity for smallholder farmers in East Africa and improved food security.”