Made to measure: smart natural resources management approaches

Coffee-banana intercropping. Photo by P. van Asten, IITA.
Coffee-banana intercropping. Photo by P. van Asten, IITA.

Coffee and banana yields in the East African highlands are often only 10 to 30% of those achieved in commercial farms in Latin America and Asia. This is the result of a mixture of biotic stresses on the crops such as pests, diseases, and weeds, and abiotic constraints such as poor soil quality and drought.

Poor crop management practices that do not sufficiently address these constraints prevent farmers from reaping maximum benefits from their efforts.

However, the importance of these yield-limiting factors differs from region to region. The natural resources management (NRM) approach therefore starts with identifying the gap between the actual, attainable, and potential yields for each location.

Diagnostic surveys and analytical tools such as the boundary line analysis are used to rank and quantify the causes of low yields. This then guides the development of tailor-made measures and actions for farmers.

Smart use of mineral fertilizer and organic matter
Poor soils are one major cause of low yields in the East African highlands. Much of Africa’s soils are old and poor, situated on very old continental plates. Only a few places have soils that still have substantial nutrient stocks, such as those derived from young volcanic material and metamorphic rocks.

Years and years of soil erosion and poor farming methods that mine minerals have worsened the situation.

IITA is working with farmers to combine organic manure and mineral fertilizer to replenish soil nutrients to meet the needs of banana and coffee.

Piet van Asten, IITA systems agronomist based in Uganda, says the approach stresses the judicious use of mineral fertilizer that is moderate in quantity, applied at the right time and in the right way, and combined with locally available organic matter.

“The combination of fertilizers and organic matter provides much-needed additional nutrients that are efficiently used up by the crops. The organic matter helps to retain mineral fertilizers applied in the topsoil and reduces losses from leaching,” he says. “It also improves the soil physical properties which help to retain soil humidity and control the temperature. Plants thrive in such humid and temperate environments as the roots are better able to take up nutrients.”

Poster on banana fertilizer recommendations for Uganda. Courtesy of P. van Asten, IITA.
Poster on banana fertilizer recommendations for Uganda. Courtesy of P. van Asten, IITA.

Sources of local organic matter are mulch, urine, manure, and compost.

Research has shown that adding mineral fertilizers and mulch to both coffee and banana nearly doubles their yields. However, the fertilizer type and dose have to supply the nutrients that are lacking.

Through mapping soil and plant nutrient status, IITA identified the missing nutrients in each region. Subsequently, it developed region-specific recommendations for using fertilizer and organic mulch in parts of Uganda.

Training materials were also developed to teach farmers how to identify nutrient deficiencies in their own farms by observing plant leaves. This should ultimately help them to localize their fertilizer needs down to the farm level.

Halting and preventing soil erosion by placing contour bunds stabilized by forage/mulch grasses and leguminous plants are also important to conserve and improve soil quality.

Smart intercropping systems
IITA has been working on promoting the intercropping of banana/plantain and coffee as research has clearly shown that intercropping works better than monocropping either crop.

Coffee, a shade-loving plant, performs well when grown under banana/plantain. Research findings showed that creating space for the banana/plantain does not reduce the yield of coffee but instead, the farmer gets bonus income from the banana.

Such intercropping systems, says van Asten, spread the socioeconomic risks of farmers as they become less vulnerable to the price fluctuations of a single crop.

“The two intercrops provide farmers with permanent piecemeal harvests from banana and annual or biannual cash booms from coffee,” he said.

Intercropping has other benefits. It leads to sharing of inputs, such as fertilizers purchased through the cash crop system, such as coffee farmers’ cooperatives. It also improves fertilizer-use efficiency, as fertilizer applied to the cash crop also benefits the food crop.

Coffee plants perform better when grown under the shade of banana plants. Photo by P. van Asten, IITA.
Coffee plants perform better when grown under the shade of banana plants. Photo by P. van Asten, IITA.

Intercropping improves the biophysical efficiency of the systems by providing better and more permanent canopy and soil cover that reduce erosion. It improves soil organic carbon stocks (carbon sequestration) through the biomass produced.

Another benefit, says van Asten, is that intercropping can sometimes increase the quality of some crops. For instance, under suboptimal growing conditions, shade-grown coffee is often of better quality and thus could fetch more money on the market.

Linking to input and output markets
In a study of the factors that limit farmers’ usage of mineral fertilizers for their banana plants, Uganda farmers cited lack of access as one constraint. Moreover, they said it was not available in smaller packaging and more affordable sizes. IITA is working to encourage farmer cooperatives that are organized around postharvest handling, sorting, and bulking to organize the supply of inputs such as fertilizer for their members.

According to van Asten, cooperatives have better access to input/output markets and improved powers of negotiation. They have improved access to market information, bulking and storage facilities, savings and credit schemes through collaboration, and agreements with input/output dealers. They can also facilitate the exploration of niche markets through the certification of products in terms of quality, production, and techniques.

Smart extension services
To meet the information needs of farmers, IITA and partners are exploring options to make location-specific information accessible. This includes the use of extension publications, videos, and mobile phone services.

Farmer detrashing banana intercropped with coffee. Photo by P. van Asten, IITA.
Farmer detrashing banana intercropped with coffee. Photo by P. van Asten, IITA.

Together with the Grameen Foundation, IITA is exploring how information can be tailored to the location of the farmer through a (decision-tree) series of questions. The more information a farmer can provide, the more precise the recommendations will be.

The NRM work on coffee and banana shows that there are practical, readily available measures that farmers can use to increase yield and contribute towards the fight against poverty and hunger. However, they have to be region- and crop-specific for maximum impact.

“For all these measures to be successful, they must start with using clean and resistant planting materials. Investing in fertilizers for use on diseased plants is a futile exercise,” concludes van Asten.

Allies in nature

Jean-François Vayssières (j.vayssières@cgiar.org), Appolinaire Adandonon (adanappo@yahoo.fr), Antonio Sinzogan (a.sinzogan@cgiar.org), and Paul van Mele (p.vanmele@cgiar.org)

<em srcset=Bactrocera invadens adults on a mango” width=”250″ height=”188″ />
Bactrocera invadens adults on a mango

Biocontrol has been around for over 2000 years. The most ancient example of biocontrol use recorded was that of Chinese and Southeast Asian fruit growers, who used weaver ants to protect their citrus crops. Farmers in Asia continue to practice this until today.

Weaver ants (one colony of Oecophylla = several nests) live on trees and defend their territories using chemicals or “pheromones” that they leave on leaves, branches, and fruit. Pheromones are chemicals secreted by insects that strongly influence, in the case of ants, the behavior of others of the same species. The release of these pheromones, which is a form of nonverbal communication, can effectively recruit ants to new food sources or trigger warnings as a protection against intruders.

There are two Oecophylla (Hymenoptera Formicidae) species in the world—the Asian species, Oecophylla smaragdina Fabricius, and the African species, O. longinoda Latreille.

Their successful application as an endemic natural enemy is rising in tropical countries. New research started exploring the mechanisms underlying ant protection of plants against arthropods. Apart from direct control mechanisms, including the predation on or deterrence of insect pests during direct encounters, indirect mechanisms have recently been discovered involving the detection of the territories of enemy ants.

Researchers have demonstrated that the Asian Oecophylla species can deter insect herbivores or plant eaters through info-chemical action. A laboratory test showed that a beetle which this ant preys on was more reluctant to feed on leaves sampled within ant territories than on leaves sampled outside.

In Africa, O. longinoda is being used as a biocontrol agent against agricultural pests. This species defends chemically marked territories at both levels, the intraspecific (within species) and interspecific (between species). Due to their pronounced territoriality, permanent surveillance (all year round, day and night), and very efficient recruitment, O. longinoda respond quickly to any increase in prey numbers.

Nest of <em srcset=O. longinoda on a mango tree” width=”250″ height=”188″ />
Nest of O. longinoda on a mango tree

The use of O. longinoda colonies is suitable for perennial cropping systems in sub-Saharan Africa because they are efficient against fruit fly pests, one of the widespread threats, constantly present in tropical agricultural systems.

Two fruit fly (Diptera Tephritidae) species—Bactrocera invadens Drew Tsuruta & White and Ceratitis cosyra (Walker), were found to be the most important species in terms of fly abundance and fruit damage in Bénin during the 2005 and 2006 seasons. The first is an exotic species from Asia, only recently recorded in West Africa, where it has become a huge threat in main fruit crops, such as mango (Mangifera indica) and citrus. The second species is indigenous.

To control fruit flies, growers sometimes resort to pesticides that are registered for cotton production. This control method is not convenient or effective at all.

Because of the economic importance of fruit flies and the lack of appropriate control methods especially in SSA, research efforts on alternative fruit fly control strategies have received greater attention, including the use of endemic biological control agents.

Making more efficient use of natural means of pest control can greatly benefit planters.

At the beginning of 2008, a Regional Control Program of Mango Fruit Flies called “West African Fruit Fly Initiative” (WAFFI) was launched in West Africa by IITA-CIRAD, with World Bank funding. The program was based in IITA-Bénin. Seven West African countries took part in 2008, and another eight in 2009. A part of the WAFFI research focused mostly on the behavior of economically significant fruit flies and their control with biological control agents, such as weaver ants.

In Bénin, we studied the behavior of fruit flies in both laboratory and field tests over several months. These experiments revealed the fundamental role of info-chemicals of ants in repelling females of two fruit fly species during their oviposition or egg-laying period in mango. Interactions were studied on mango between two frugivorous or fruit-eating fly species (B. invadens and C. cosyra) and the African weaver ant (O. longinoda).

Predation of fruit fly larva third larval instar by weaver ants
Predation of fruit fly larva third larval instar by weaver ants

Results showed that: (1) female flies are strongly attracted to the mango fruit at ripening stages for egg laying; (2) without previous passage of ants on the fruit, the oviposition of tephritids (flies) is very important in mango; (3) once weaver ants have “patrolled” on mango fruit, female oviposition is significantly reduced; (4) C. cosyra seemed twice as sensitive as B. invadens about landing on treated fruit vs. untreated fruit; (5) similar results were found for the time spent on mango fruit; (6) ant-treated fruit had six times less damage from B. invadens and four times less damage from C. cosyra than untreated fruits; (7) B. invadens had significantly more pupae per kilogram fruit than C. cosyra in ant-free mango fruit, whereas no significant difference in ant-treated fruit was detected between the native C. cosyra and the exotic B. invadens.

The presence of weaver ants in mango trees reduced the damage caused by the fruit fly family Tephritidae through predation of adult fruit flies (rare), predation of third-stage larvae (quite frequent) and, especially, the effect of pheromones left by the ants on the fruit so that flies are repelled and are discouraged from egg-laying. Weaver ant presence resulted in a marked reduction in fruit damage.

The influence of info-chemicals from predators such as ants on the foraging behavior of fruit insects and more generally on pests could have crucial consequences for future observations and applications on host selection and consequently in host protection against these pests.

Practical information about the use of weaver ants in fruit fly pest control should be made available to all those involved in the fruit industry at every level, particularly local official producers, pickers, and rural advisors.

This work is also a good example of collaboration among IITA, Africa Rice, and CIRAD on a very important issue about high-value products in West Africa.

References
Adandonon A., J.F. Vayssières, A. Sinzogan, and P. Van Mele. 2009. Density of pheromone sources of the weaver ant Oecophylla longinoda affects oviposition behaviour and damage by mango fruit flies (Diptera: Tephritidae). International Journal of Pest Management. 55 (4), 285 – 292.
Dejean A. 1991. Adaptation d’Oecophylla longinoda aux variations spatio-temporelles de la densité en proies. Entomophaga. 36: 29-54.
Dejean A., P.J. Solano, J. Ayrolles, B. Corbara, and J. Orivel. 2005. Arboreal ants build a trap to ambush and capture prey. Nature. 434: 973.
Hölldobler B. and E.O. Wilson. 1978. The multiple recruitment systems of the weaver ant Oecophylla longinoda (Latreille) (Hymenoptera: Formicidae). Behavioral Ecology and Sociobiology, 3: 19-60.
http://www.thefreedictionary.com/. Accessed on 30 Aug 2009.
Offenberg J., M.G. Nielsen, D.J. MacIntosh, S. Havanon, and S. Aksornkoae. 2004. Evidence that insect herbivores are deterred by ant pheromones. Proceedings Royal Society London. 271: 433-435.
Sinzogan A., P. Van Mele, and J.F. Vayssières. 2008. Implications of on-farm research for local knowledge related to fruit flies and the weaver ant Oecophylla longinoda in mango production. International Journal of Pest Management. 54 (3), 241-246
Van Mele P. 2008. A historical review of research on the weaver ant Oecophylla in biological control. Agriculture and Forest Entomology. 10 (1), 13-22.
Van Mele P., J.F. Vayssières, E. Van Tellingen, and J. Vrolijks. 2007. Effects of the African weaver ant Oecophylla longinoda in controlling mango fruit flies (Diptera: Tephritidae). Journal of Economic Entomology. 100 (3), 695-701.
Van Mele P., J.F. Vayssières, A. Adandonon, and A. Sinzogan. 2009. Ant cues affect the oviposition behaviour of fruit flies (Diptera: Tephritidae) in Africa. Physiological Entomology. DOI: 10.1111/j.1365-3032.2009.00685.x
Vayssières J.F., G. Goergen, O. Lokossou, P. Dossa, and C. Akponon. 2005. A new Bactrocera species detected in Bénin among mango fruit flies (Diptera: Tephritidae) species. Fruits. 60 (6), 1-9.
Vayssières J.F. and A. Sinzogan. 2008. The use of weaver ants (Hymenoptera Formicidae) in fruit fly (Diptera: Tephritidae) pest control. Leaflet IITA-CIRAD N°= 5. West African Fruit Fly Initiative (WAFFI). 4 p.
Vayssières J.F., S. Korie, D. Ayegnon, and C. Akponon. 2009. Correlation of fruit fly (Diptera Tephritidae) infestation of major mango cultivars in Borgou (Bénin) with abiotic and biotic factors. Crop Protection. 28, 477-488.
Vayssières J.F., A. Sinzogan, and A. Adandonon. 2009. Principal methods for integrated control of fruit flies in West Africa. Leaflet IITA-CIRAD N°= 6. West African Fruit Fly Initiative (WAFFI). 4 p.

The witch menace

Maize ravaged by Striga
Maize ravaged by Striga. Photo by IITA

The witch’s spell on millions of hectares of cereal crops in sub-Saharan Africa (SSA) will soon be broken. A deadly “potion” using natural enemies is being developed by IITA and its partners to manage the menace.

Striga hermonthica or witchweed, the parasitic weed that attacks cereal crops, such as maize, sorghum, and millet, has caused devastating annual production losses estimated at US$7 billion among small-scale farmers, contributing to hunger, malnutrition, and poverty in SSA.

The sight of the deceptively beautiful pink flowers of Striga spells doom for farmers. The weed grows on the roots of host plants absorbing the plant’s water, photosynthates, and minerals. When the flowers are in bloom, it is already firmly established. Thus, the use of aboveground herbicides is ineffective, since the damage has occurred long before the weed is visible to farmers. Each plant can produce tens of thousands of seeds that are dispersed far and wide by man and nature, and which lie dormant but still potentially active for many years.

Angry farmer with Striga plant
An angry farmer with Striga plant. Photo by IITA

Loss of millions of tons of food
Fen Beed, an IITA plant pathologist, explains that production losses from Striga routinely range from 15 to 90% depending on the crop cultivar, degree of infestation, rainfall pattern, and degree of soil degradation.

Striga infests about 50 million hectares of land in SSA resulting in the loss of over 8 million tons of food annually. The larger areas affected are in Nigeria, Niger, Mali, and Burkina Faso.

Unfortunately, measures developed to control the weed in the developed world, such as soil fumigation, are too costly for the poor subsistence farmers who make up 70 to 80% of farmers in SSA. New management options are thus urgently needed.

One promising, sustainable, and environmentally friendly technology under development is biocontrol using indigenous fungi that are natural enemies of the weed.

Poisoning the witch
A team led by Beed with partners from the University of McGill (Canada) and University of Hohenheim (Germany), and national agricultural research systems (NARS) and universities in West Africa, have identified isolates of a fungus that attacks Striga for use as a bioherbicide.

By studying over hundreds of diseased shoots of Striga in Bénin, Burkina Faso, Ghana, Mali, Niger, and Nigeria, scientists discovered isolates of Fusarium oxysporum f. sp. striga that controlled the weed.

Container trial, Ibadan, Nigeria
Container trial, Ibadan, Nigeria. Photo by IITA

A series of controlled laboratory studies identified the most effective of these as M12-4A, an isolate from Mali, Foxy 2 from Ghana, and PSM-197 from the Nigerian savanna. The isolates attacked Striga in all its growth stages—from seed to germination, from seedling to flowering shoot. They significantly lessened the number of attachments and flowering Striga plants, thus reducing the number of seeds deposited in the soils and limiting the future reappearance of the weed. Furthermore, the isolates were specific to S. hermonthica, had no impact on cereal hosts or any other plants, and did not produce any toxins that harm man or livestock.

Repeated field trials were performed for the first time under West African conditions using Striga-resistant and Striga-susceptible varieties of sorghum and maize in Nigeria, Burkina Faso, and Bénin in partnership with various NARS and universities. The efficacy of the three isolates selected from laboratory studies were compared with other isolates originating from Bénin and Burkina Faso. Amino acids found to disrupt germination of Striga under laboratory conditions were also included but failed to produce significant improvements in weed control under field conditions.

Results showed that PSM-197 and Foxy 2 were the most effective in repressing witchweed, whereas isolate M12-4A was less effective under the range of field conditions tested. Also, there was a 90% reduction in Striga emergence when the biocontrol technology was used in combination with a Striga-resistant maize line.

Two methods were used to apply the fungi: either directly coating the seed using locally available gum arabic or directly adding the fungus in powder formulations of kaolin-based PESTA granules into planting holes. The granular formulation was found to be more efficient, especially for sorghum which has much smaller seeds than maize, where the larger seeds receive more fungal inoculum when applied as a seed coating. However, it is more costly and difficult to distribute to farmers.

Drying sorghum and maize seed coated with PESTA
Drying sorghum and maize seed coated with PESTA. Photo by IITA

Therefore, the seed-coating method offers the most cost-effective method, especially when combined with Striga-resistant germplasm.

Another important finding is that the biocontrol agent works most efficiently when the soil is rich in beneficial (friendly) and not antagonist (nuisance) microorganisms. Container trials at IITA Ibadan showed that the profile of both bacterial and fungal microorganisms was changed when different species of cereals were grown in the same soil—this is because each plant type produces different exudates that are excreted around roots that promote or inhibit the growth of different microorganisms.

Furthermore the profile was changed when different cultivars of the same species of cereal crop (maize or sorghum) were grown. Different fertilizer combinations had similar impacts on microorganism profiles—all of these changes in profiles affect the success of introduced biocontrol agents. This study was done using state of the art PCR-DGGE technology in collaboration with the University of Purdue.

Making the potion available and affordable
Supplying fungal-coated seeds of improved varieties to farmers requires a delivery pathway. Researchers face the challenge of mass producing the biocontrol agents and encouraging farmers to use them. The models being tested for mass production of the F. oxysporum inoculum include on-farm, cottage-industry, small entrepreneur industry, and government initiatives, such as that in Senegal initiated by Foundation Agir pour l’Education et la Santé.

Under the small entrepreneur industry models, one company in Kenya, Real-IPM, has secured funding to register Foxy 2 before mass production using large-scale commercial tanks for liquid culture of the fungus. Another company, Western Seed Company Ltd., has carried out preliminary field tests with support from the Kenya Plant Health Inspectorate Services.

Finding a way to curtail the negative impact of witchweed has been a long journey, but the biocontrol option can provide an important component in an integrated package of strategies for managing this pest.

PESTA granules
PESTA granules. Photo by IITA

“There will never be a silver bullet solution to alleviate the problems faced by farmers from witchweed. It is important to recognize that efficacy and persistence of the biocontrol agent is improved when steps are taken to prevent the soil from being degraded and to enrich it with organic matter,” says Beed.

New techniques are also needed for measuring the extent of losses caused by witchweed and their economic impact. Likewise, control technologies need to be developed and implemented, and their efficacy assessed across the different environments scourged by the pest, he added.

Biocontrol combined with the use of improved cereal cultivars that have increased tolerance/resistance to Striga, and the use of seed-coated herbicides such as imazapyr, in addition to the regular use of trap crops, at last offers small-scale farmers real hope against the “witch”.

Unraveling cassava’s problems

Ivan Ingelbrecht, i.ingelbrecht@cgiar.org

cassava1
Cassava, photo by IITA

Cassava plays an important role in the food security of many developing nations, especially in Africa, and is also an industrial crop in Latin America and Southeast Asia. Native to South America, cassava was introduced to West Africa from Brazil by Portuguese traders in the 16th century. In the 1700s, cassava was independently introduced into East Africa, also by the Portuguese. Because of its high adaptability to low-fertility soils and its ability to withstand erratic and long periods of drought, cassava is a food security crop and a source of cash for resource-poor farmers. Today, over half of the world’s cassava is produced in Africa; Nigeria is the main producer.

As cassava is vegetatively propagated, genetic improvement is arduous because of the high levels of heterozygosity and poor flowering habit in many landraces. Genetic transformation could complement conventional breeding efforts for improving certain traits. Protocols for the genetic modification of model cassava genotypes, such as TMS60444, were first described in 1996 and have now become routine (Taylor et al. 2004).

Despite this progress, the genotypes that are amenable to genetic modification are not grown by farmers or used as genetic stocks in breeding programs, and this limitd their practical utility and impact at the farm level. Because the current transformation methodologies are highly genotype-dependent and limited to these so-called model genotypes, the genetic transformation of farmer-preferred cassava landraces remains a challenge.

State of cassava genetic transformation
Genetic transformation of cassava was first independently described in 1996 by three research groups. Nowadays, there are at least three modes of cassava genetic transformation, all of which rely on the establishment of somatic embryogenesis.

Expression of a reporter gene in leaf, root, and stem of the cultivar Tokunbo, a transgenic African cassava. Photo by IITA
Expression of a reporter gene in leaf, root, and stem of the cultivar Tokunbo, a transgenic African cassava. Photos by IITA

In one, friable embryogenic callus cultures (FEC) are used to transform and regenerate plantlets. This technology works well with TMS60444 and a few South American varieties, such as MCol 1505. This protocol can yield large numbers of transformants and is possibly the most widely used technology to date. Using this method, several new traits have been introduced into cassava. A major international effort involving research organizations in the USA, Europe, Asia, Latin America, and Africa, aims at enhancing the nutrition of cassava through the genetic modification of TMS60444. Although of African origin, this variety is not used by farmers or breeders as it has unfavorable disease and agronomic characteristics. However, over the past decade, this transformation methodology has provided proof of the concept with genes in TMS60444.

A second procedure relies on transformation of embryogenic cells present on immature leaf lobes. Because this technology is faster, it may result in less somaclonal variation than the FEC system since the somatic embryos are not derived from long-term cultures. This methodology also has limitations in terms of farmer-preferred genotypes.

A third methodology uses somatic embryo explants that undergo shoot regeneration (organogenesis). This procedure was first developed by Li et al. (1996) for model cassava genotypes and has been further enhanced by IITA and its collaborator, the University of Copenhagen KU), Denmark, for the African farmer-preferred landraces.

At IITA, somatic embryogenesis has been established for four cultivars: one from West Africa and three from East Africa. These varieties are susceptible to cassava brown streak disease (CBSD) but otherwise have desirable agronomic characteristics. For these four varieties, thousands of somatic embryos (SEs) can be reproducibly generated. Using somatic embryos as source explants, transgenic Tokunbo, a landrace cultivated in several countries in West Africa, has been produced using a beta-glucuronidase (GUS) gene construct.

Transgenic cassava Tokunbo in the greenhouse. Photo by IITA
Transgenic cassava Tokunbo in the greenhouse. Photo by IITA

Enabling technologies
In addition to genetic transformation protocols, a set of other technologies is required for biotechnology-mediated cassava improvement. Since many traits target specific tissues, such as roots, a wider range of promoters or other regulatory elements needs to be investigated and tested in cassava to achieve optimal and stable transgene expression in these tissues or in the whole plant. The aspect of stable gene expression, i.e., the absence of gene silencing, is especially relevant for a vegetatively propagated crop, such as cassava. The international effort to sequence the cassava genome as well as initiatives of several labs, including IITA, to develop functional genomics tools will greatly expand our tool set for biotechnology-mediated cassava improvement, whether through transgenesis or marker-assisted breeding approaches.

Many biotechnologies are protected by intellectual property rights and thus are not always freely accessible, especially to research institutions in the developing world. In this context, a new plasmid was developed at IITA for Agrobacterium-mediated genetic transformation of cassava and other dicotyledonous plants. This plasmid contains a Cassava Vein Mosaic Virus promoter cassette and was tested in tobacco and cassava using a beta-glucuronidase reporter gene. High, constitutive expression was obtained in leaf, stem, and root tissues of both crops, thus showing that the new promoter cassette was fully functional.

A derivative of this plasmid was made by IITA’s partner, the German Resource Centre for Biological Materials (DSMZ) to manipulate gene expression via RNA interference. RNA interference is widely used in animal and plant cells to down-regulate gene expression. In cassava, this approach is currently being used to engineer virus-resistant plants and plants that produce reduced levels of cyanide.

Preparing cassava somatic embryo tissues for shoot induction. Photo by IITA
Preparing cassava somatic embryo tissues for shoot induction. Photo by IITA

Beyond genetic transformation
One of the highlights at the International Congress of Genetics, held in Berlin in 2008, was a report on the use of artificial mini-chromosomes in maize. The methodology makes use of artificial mini-chromosomes that can replicate and are stably transmitted over several generations, in a way similar to natural chromosomes. These mini-chromosomes contain DNA sequences found in centromeres, the chromosomal regions needed for inheritance. Rather than inserting new genes randomly into a plant’s natural chromosomes, as happens in genetic modification, these mini-chromosomes remain separate. One of the advantages of this technique is that it allows multiple genes to be arranged in a defined sequence with their own regulatory sequences, resulting in more consistent gene expression. So far, this technique has been used only in maize but it can potentially be applied to other crops as well, including cassava.

Outlook
Although methods for cassava genetic transformation were developed over a decade ago, genetic transformation of farmer-preferred (African) cultivars has only recently become a research priority. IITA and its partner KU in Denmark have recently produced a first transgenic cassava variety called Tokunbo which is cultivated in Nigeria and some other West African countries.

Cassava plant. Photo by IITA
Cassava plant. Photo by IITA

As a complementary approach to cultivar-specific transformation, the movement of transgenes through conventional breeding should also be considered and, ideally, also artificial mini-chromosomes, the new gene-transfer technology established in maize.

As new and cost-effective DNA sequencing methodologies are becoming mainstream, the amount of genomics and genetic information for cassava will vastly increase in the near future and greatly expand our tool set for biotechnology-mediated improvement. From a technical standpoint, it is realistic to expect that new varieties, in part produced using biotechnology tools, will become available in the near future.

References
Li H-Q, C. Sautter, I. Potrykus, and J. Puonit-Kaerlas. 1996. Genetic transformation of cassava. Nature Biotechnology. 14: 736–738.

Taylor N., P. Chavarriaga, K. Raemakers, D. Siritungaand, and P. Zhang. 2004. Development and application of transgenic technologies in cassava. Plant Molecular Biology 56: 671–688.

Genomic tools for improving African crops

Melaku Gedil, m.gedil@cgiar.org

The increase in genomic techniques in the past few decades has thrown the doors of research wide open to agricultural scientists. Conventional breeding has been augmented by various innovative molecular marker-aided techniques. The first wave of molecular marker technology introduced biochemical markers (isozymes and allozymes).

Digital imaging and microscopy: a tool for research and training. Photo by O. Adebayo, IITA
Digital imaging and microscopy: a tool for research and training. Photo by IITA

These quickly gave way to the first generation DNA-level markers such as Restriction Fragment Length Polymorphism (RFLP, DNA analysis), Randomly Amplified Polymorphic DNA (RAPD), Amplified Fragment Length Polymorphism (AFLP), and simple sequence repeat (SSR)—all mouthfuls to the layperson. Those that lend themselves well to automation and multiplexing (use of simultaneous or more than one set of primers in the reaction mix) prevailed because of their cost-effectiveness.

Advances in sequencing technology enhanced the use of DNA sequence-based markers such as SSR and single neuclotide polymorphism (SNP), allowing the development of automated, high throughput (output) genotyping platforms. In a decade, the cost of genotyping has dramatically declined with various techniques developed that allow flexibility under different circumstances. This emphasized the feasibility of molecular breeding.

New tools
Some of the new molecular biology tools used at IITA include molecular markers for marker-assisted breeding, resistance gene analogs (RGA), Targeting Induced Local Lesions In Genomes (Tilling), DNA chips, application of DArT markers, and bioinformatics.

In IITA, the development of new genomic tools for molecular breeding and gene discovery is under way for the mandate crops. For instance, new markers have been identified, in silico (online), from cassava Expressed Sequence Tags and hundreds of markers validated using a diverse panel of cultivated cassava varieties. After filtering with various criteria, over a hundred new markers were developed, useful for fingerprinting and other molecular genetic applications.

The rapid accumulation of genome sequence data led to the development of an array of functional genomics tools that are being used to understand the complex pathways involved in host plant–pathogen interaction. The RGA technique has applications in cloning, profiling, and host–pathogen interaction.

Photomicroscopy of transformed material, Biotech Lab, IITA. Photo by IITA
Photomicroscopy of transformed material, Biotech Lab, IITA. Photo by IITA

The RGA technique was used in IITA to assess DNA sequence variation in several elite cassava clones, resulting in several novel sequences, some of which were found to be similar to previously reported RGAs. This information is expected to facilitate the identification of gene-targeted markers for molecular breeding and gene discovery in cassava.

Another new tool is Tilling, a popular technique of reverse genetics for detecting mutations in a target gene, followed by the assignment of phenotypes to the gene sequence. It rapidly gained popularity because it is suitable for automation and for screening thousands of samples. Besides being a non-GMO approach for broadening the genetic base, it provides tools for developing markers for marker-assisted breeding for traits that are cumbersome and expensive to measure.

Tilling work to discover induced and natural mutation in cassava was geared towards specific traits that are intractable (or not easily managed or manipulated) using conventional methods. Adaptation of the technique to other IITA mandate crops such as yam, banana, and cowpea entails the selection of target tissue or organ for mutation, and the selection of similar or different target genes. Crops such as maize and soybean have numerous germplasm resources that can be easily adopted and adapted.

Knowledge of the nucleotide sequences of the target genes is a prerequisite for Tilling. The major IITA mandate crops—cassava, yam, and banana—have very limited genomic resources. To date, nucleotide sequence information for a very few, largely chloroplast, genes could be found in Entrez Gene. Investigations in the past decade resulted in the cloning and characterization of expressed cassava genes involved in starch, cyanogen glucosides, and carotenoid biosynthesis. However, even in the absence of a nucleotide sequence for the gene of interest, comparative genomics has been successfully used to identify candidate genes. The completion of the genome sequence of poplar and, more recently, of castor bean, is expected to provide useful genetic tools for identifying candidate genes in cassava. Besides, the ongoing cassava genome sequencing is anticipated to be completed soon, opening a new avenue of research in functional postgenomic studies such as Tilling.

A genome-wide 14K DNA chip for cassava (left) and a scan showing 14,000 different genes (right). Photo by IITA
A genome-wide 14K DNA chip for cassava (left) and a scan showing 14,000 different genes (right). Photo by IITA

DNA chips have also become popular tools for gene discovery and also for diagnostics. They also provide a reverse genetics tool for identifying gene-targeted markers for molecular breeding. A genome-wide DNA microarray for cassava with ~14,000 probes has been developed at IITA. This is the most comprehensive DNA chip for cassava available to date. This microarray has been used for transcriptome analysis of cassava. Candidate genes that are differentially expressed after virus infection have also been identified.

A cassava DArT chip with 735 polymorphic markers was used to fingerprint a diverse cassava population comprising genotypes from Africa, Latin America, Asia, and breeder lines maintained at IITA. Overall reproducibility of the marker set was very high and average call rate was 97%. DArT markers provide reliable and high throughput molecular information for managing biodiversity in germplasm collections and make rapid genome profiles possible for quantitative trait loci (QTL) mapping.

Bioinformatics
Advances in bioscience technologies such as sequencing, synthesis, imaging, and various other nanoscale assays, have dramatically increased the volume of biological data, which in turn, started the concurrent growth of bioinformatics tools. Bioinformatics is broadly defined as the application of computer technology to the storage, retrieval, and analysis of large amounts of biological information.

The major areas of high-end bioinformatics include the development of databases and algorithms for analyzing and annotating various types of microarray platforms, high-density oligonucleotide chips, variety of mass spectrometry, and diverse platforms of new-generation sequencing data. However, the majority of life science scientists and investigators tend to turn to the Internet to seek end-user web tools and resources (software packages). Countless institutions in the West provide a myriad biological data resources and services, including expert-curated databases of nucleic acid and protein sequences, data and text mining tools, genome and transcriptome analysis; protein and other macromolecular structure analysis; networks, pathways, and systems biology; evolution and systems biology tools.

The major tools in the public domain are, however, the development of peer-reviewed, up-to-date, web-accessible databases and web tools (analysis software packages). These resources typically provide an advanced query interface.

User accessing virtual knowledge repository. Photo by J. Oliver
User accessing virtual knowledge repository. Photo by IITA

The explosive growth of web sites has necessitated that users distinguish between inaccurate personal web sites and reliable resources maintained by a consortium of investigators and/or a legitimate institution. The journal Nucleic Acid Research began to publish annually a collection of molecular biology databases and bioinformatics links directory. The most recent updates of molecular biology databases feature over 1000 databases, over 300 of which are on plants, whereas the latest Bioinformatics Links Directory published by the same journal lists over 1200 links.

Another outstanding issue in the use of online bioinformatics tools is that, as the number of such web resources grows astronomically, even learning how to use the interface is becoming cumbersome, prompting the need for one-stop gateway type of tools for integrated querying (e.g., BioMart, OBRC from the University of Pittsburgh; Bioclipse).

One of the advances in bioinformatics is the availability of programming and scripting languages (Perl, Bioperl, Phyton, and Java) for automating complex but routine steps, such as search, retrieval, and parsing (resolving into and examining component parts) search results. While varieties of commercial integrated analysis packages are available, the cost of initial installation and maintenance becomes prohibitive. Developing our capacity for such routine end-user applications is vital to the support of our molecular biology work.

African researchers working on well-studied crops such as rice, wheat, maize, and soybean will have the best genomic resources at their fingertips, provided that they have Internet connection. To take advantage of publicly accessible web resources, including the variety of databases, online software, publications, and multimedia learning materials, African scientists and students need institutional support and considerable internal and external funding. As in other fields of science, bioinformatics lags in SSA due, partly, to poor or nonexistent Internet connection. Fast and broad Internet connection is the key to successful online research.

Research in molecular biology is slowly gaining ground in Africa. Any molecular biology research needs to be augmented by a bioinformatics database and online tools.

There is no shortage of available tools for agricultural research or agricultural information and database management. The challenge is in finding the best ones or combinations that suit institutional needs, resources, or preferences.

IITA will continue to use suitable and affordable conventional and new genomic tools to undertake research on its mandate crops.

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/

Growing banana from “seeds”

Bananas are an important crop for global trade and nutrition where they are intensively cultivated, but few efforts exist to breed superior bananas. One of the reasons for this is that humans have intensively “selected” against seeded bananas and it is difficult or impossible to pollinate many banana varieties and successfully produce seeds.

Finding seeds in breeding plots in Namulonge, Uganda. Photo by IITA

Many of the most important banana varieties are triploid, which means that they carry an extra copy of each chromosome compared to the normal diploid. Being a triploid means that it is difficult for normal chromosome pairing and segregation to make fertile eggs or pollen, which results in most triploids being nearly sterile. Sterile bananas are great for people who don’t like to crack their teeth on banana seeds, but mean that bananas have to be multiplied via vegetative propagation, similar to propagation of potatoes, sweet potatoes, cassava, and selected varieties of other fruit trees or ornamental species.

Gardeners are familiar with “seed potatoes,” small potato tubers that are planted to produce a potato crop. Bananas do not form tubers; new plants derive from “suckers” that emerge from the lower banana stem (corm). These suckers can be uprooted and used to plant new banana plants. Similar to potato tubers, these suckers were a part of the original mother plant, which means that they potentially carry whatever disease pathogens or pests had infested the mother plant. Therefore, banana suckers are one of the main means of transport and spread of certain disease-causing agents, including important fungi, bacteria, and viruses.

Nematodes and pests can also hitchhike on banana suckers to infest the new crop. Not only does such hitchhiking result in early infection/infestation of new banana plants in a farmer’s field, but transporting long distances may help introduce a new disease or pest problem in a new location. This dual hazard of reduced yield potential of already infected planting material that may introduce new pests and diseases emphasizes the need for superior disease-free planting material produced through a “seed system” designed to minimize the risks of spreading pathogens and pests.

Banana bicycle transport, Burundi
Banana bicycle transport, Burundi. Photo by IITA

The traditional means of obtaining banana planting material (“seed”) is to acquire suckers from one’s own banana garden, from a neighbor, or from a more distant source. This method served to spread common varieties around the world and to multiply them in their new locations. This system can be modified to produce more banana suckers or shoots by manipulating banana corms to allow more buds to sprout. One such method that is described here is called macropropagation. A higher tech procedure to rapidly produce many plants in just a few generations of propagation is called tissue culture. In tissue culture, plants are first surface sterilized and then grown in aseptic culture in test tubes using an artificial growth medium based on a gelling agent like agar. The tender tissue-cultured plants can then be planted in the field after rooting and hardening under protected conditions.

Seed systems for producing clean planting material can be operated at various levels of technology and efficiency. In some cases, plant health could be improved by merely raising the awareness of the negative impact of planting “sick” suckers. Where infected plants look visibly different from healthy plants, either because of reduced vigor or visual disease symptoms in infected plants, the propagator could practice negative selection against “sick” plants or positive selection for “healthy” plants (or both). Such plants could be multiplied faster by applying a rapid propagation method such as macropropagation. However, while low-tech and affordable for farmers, such a system does not eliminate problems that cannot be detected by visual observation. Unfortunately, many diseases and pests fall into this category for at least part of their infection cycle.

For crops such as cereals, seed certification systems were developed to assure varietal purity, and later expanded to include freedom from weed seeds and seed-transmitted pathogens. Since most pathogens are seed-transmissible for vegetatively-propagated crops like potato or banana, disease management is the major focus of most seed potato certification programs and banana multiplication programs. Modern technology has provided diagnostic tests to detect significant pathogens. These tests are similar to those used in modern laboratories to diagnose human diseases, and can be expensive. For this reason, it is more efficient to test a small number of plants and multiply those that were negative for all pathogens tested in the battery of diagnostic tests.

It is possible to use tissue culture to efficiently and rapidly multiply plants that tested “clean” in the pathogen testing. Most potatoes eaten in the Western world are just a few field generations removed from tissue-cultured plants used to produce “seed potatoes” in screened glasshouses to start the seed production cycle. Similarly, most dessert bananas in the global export trade are from plants originally propagated in tissue culture from plants that tested clean for known banana diseases. A modified form of tissue culture can also be used to eliminate pathogens from plants that did not test clean, after which they can be propagated to produce “seed” planting material. There is great potential to improve the health of banana plantations in the developing world through increased use of this technology.

Tissue culture is the process of growing plants that have been surface sterilized and planted in test tubes or similar containers in sterile medium that contains all the nutrients they need to grow. This is almost always done in indoor laboratory facilities and the medium also contains the sugars needed to grow, since there isn’t enough light for photosynthesis.

Sanitation is extremely important, since a single mold spore is enough to contaminate a test tube. Tissue-cultured plants are generally tested for pathogens before commencing the multiplication cycle so that infected plants are not multiplied. The small banana plantlets produce small suckers that can be detached and planted as new plants, or an experienced technician can cut sections that contain buds that will grow. Extra shoots can sometimes be induced by cutting through the growing points so that multiple plants develop from single buds. This process can be repeated every 5-8 weeks so that a single plant can produce many new plantlets in a relatively short period of time.Bananas are sometimes unstable in tissue culture and mutant versions can develop. For this reason, most multiplication labs try to limit the number of multiplication cycles before renewing their cultures from field plants observed to have all the correct traits for that variety.

When tissue-cultured plants are rooted in soil, hardened, and then planted back in the field, they can be more susceptible to some pests and diseases than the original plant was. To restore natural levels of resistance, these plants can be reinfected with the endophyte microorganisms that normally coexist with bananas, similar to the gut bacteria that are important for human intestinal health (see related article on endophytes).

Macropropagated banana plant in chamber. Photo by IITA

Macropropagation falls somewhere between tissue culture and traditional systems of distributing suckers. In macropropagation, large suckers from healthy banana plants are removed and the roots and soft stem portion (pseudostem) of the sucker are cut away to expose the buds of the corm (the hard stem portion at the base of the sucker). The bare corms are briefly dipped in boiling water to kill any nematodes (micro-worms) that were not removed when cutting off roots. Small cuts are made through the buds to encourage development of multiple sprouts from each bud. The apical (top) bud is often removed because it can suppress development of lower buds. The corm is then covered with moist wood shavings and incubated in a small plastic-covered chamber for a few weeks to encourage shoot development.

Primary shoots can be rooted and used as planting material, or cut off and the growing point again cut to promote additional shooting. Shoots that develop are broken off with a bit of hard stem and roots attached, placed in small nursery bags in a similar high humidity chamber for a few days to allow root development, and finally moved to a nursery for hardening. Hardened plants can be planted in the field, similar to suckers or hardened plants from tissue culture.

Banana roadside market in rural southwest Uganda. Photo by IITA
Banana roadside market in rural southwest Uganda. Photo by IITA

A major drawback of macropropagation is that rustic or low-tech methods of detecting pathogens have not been developed, so this method can propagate infected plants if they were chosen as mother plants. Both macropropagated plants and tissue-cultured plants have less food reserves than suckers and require more care (compost/manure, watering) after planting than suckers. Careful siting of “mother gardens” established from tissue-cultured plants in clean areas may be the best way to produce suckers for macropropagation.

Traditional seed systems have produced most of the nearly 6 billion banana and plantain plants in Africa currently spread over nearly 4 million hectares of farm and gardens. Many of these are in excellent condition; others have become infected with one or more banana diseases and need to be replaced. Since new banana diseases have been introduced to Africa in the last century, and many diseases have increased in distribution and prevalence, greater care needs to be practiced to multiply “healthy seed”.

Breeding programs are nearly ready to release new varieties with resistance to some of the disease problems.

A combination of new and old seed systems can improve the overall health of new plantings by providing healthy plants of both preferred older varieties and resistant new varieties.

IITA’s research on macropropagation is supported by the Directorate General for Development Cooperation (Belgium) and Agricultural Productivity Enhancement Program (APEP-USAID) Uganda Agricultural Productivity Enhancement Project.

Erostus Njuki Nsubuga: Lessons on partnership

AGT CEO and Managing Director Erostus Nsubuga
AGT CEO and Managing Director Erostus Nsubuga

R4D Review interviewed Erostus Njuki Nsubuga, the chief executive officer and managing director of Agro-Genetic Technologies Ltd (AGT), to get his insights on the IITA-AGT partnership. AGT is the first and only private commercial tissue culture (TC) laboratory in Uganda and so far the biggest single supplier of banana TC planting material in East and Central Africa. It produces up to 8 million tissue culture plantlets per year, of which 1 million are banana plants.

Nsubuga wants to see AGT become well established and profitable by increasing its capacity to provide the region with quality TC planting material at affordable prices and introducing other services such as plant and soil analysis, and produce organic fertilizers. His dream is for AGT to become a one- stop shop that provides total solutions to farmers.

Nsubuga was born in Uganda and spent his early years there. Because of the war, he and his family had to move to other countries in Africa and Europe. He started living on his own at 16, studying in Europe and USA for 24 years to obtain an MSc (Agriculture) and MBA (International Marketing). He worked in international companies and managerial positions for over 20 years but his dream was always to come back, to help his mother who had survived all the wars, to sustain himself and his family, and to contribute to the development of his people and country.

What made you establish AGT?
I had completed one contract and was about to start a new job when I decided to start a TC laboratory at my house. I employed and trained two people to produce TC plants out of my kitchen while I was traveling. At that time (2001–02) banana and coffee wilt diseases were spreading like wildfire in Uganda. It was easy to start with these two crops as there was great demand for disease-free planting material to reduce the spread of diseases and restore healthy plantations. Over time, using my own finances, AGT built a state-of-the-art TC facility and we grew significantly. Our technical team now includes five university graduates and a retired professor. Degree students from Makerere University have been doing their internships at AGT’s laboratory with their programs embedded in our production line.

How did the AGT and IITA partnership come about?
It started when Dr. Thomas Dubois called me out of the blue. IITA was looking for a commercial enterprise to start testing and producing its endophyte-enhanced plants. Under a mutual agreement, AGT and IITA have worked on fine-tuning the enhancement of TC plants with endophytes. We identified and established on-farm trials together, using the same farmers. In the short run, IITA assisted us with laboratory chemicals and AGT also benefited from publicity. In the longer run, production of endophyte-enhanced TC material would be greatly beneficial to AGT and other commercial producers in the region. Now that the original project has expired, we are trying to get this unique product commercialized to supply farmers facing high pest and disease pressure.

Please give some insights on public-private collaboration.
Collaboration can be very important in developing and disseminating research products. For IITA, it has forced them to think commercially from the onset. A good example has been the experimental protocol for endophyte inoculation. After piloting it in my lab, IITA quickly abandoned the use of a nutrient solution in favor of fertilizer-amended soil along the lines of the system used in commercial nurseries. Such partnerships should be developed as early as possible, especially for a technology such as this. This would help AGT to build up its technical, human, and financial capacity to take on the research products once they reach commercialization. Also a very clear agreement has to be drafted and this is sometimes a balancing act.

How could IITA improve its relationship with the private sector?
AGT indicated to IITA that it was open to other research products but wanted to be involved at an early stage. This is what we call a demand-driven research agenda where the consumer is sure of getting research products through private sector involvement. We are now backstopping a socioeconomic study looking at full commercialization of our nurseries in Uganda and Rwanda. At present AGT sells mainly through NGOs and institutions. Direct marketing to farmers would be better.

What lessons have you learned from the partnership with IITA and others?
It is great that research organizations such as IITA have realized the role of private sector involvement in agricultural research and in the product value chain. Such partnerships are relatively new and we still have much to accomplish. Personally, I feel many governments and international research institutions, even IITA, are working too much for the donors, not the farmers. We should tell the donors what needs to be funded. More impact assessment is needed on some research products.

Any suggestions for future collaboration or collaborators?
I hope IITA can do more demand-driven research by including the private sector in the development of research products as early as possible with specific roles for each partner clearly defined.

What do you think makes AGT successful?
I have a professional approach and commitment, with many years of experience in agriculture and entrepreneurship and good relationships, local and international. AGT started when diseases such as banana bacterial wilt and coffee were at their peak, so I was in the right place at the right time.

How else could development organizations and private entities such as AGT help farmers and consumers?
AGT is getting farmers involved in production, distribution, and training by establishing banana nurseries and demonstration gardens owned by local farmers. The farmer then becomes the AGT distributor for that community and the nursery the focal point for training others in modern agricultural practices.

What is your dream for Uganda?
Uganda is the second largest producer but seventy-fifth in banana exports. The Government and all development partners should industrialize this crop and thus lift many out of poverty.

Any thoughts about the world food crisis, food security, GMOs, or development in general?
African countries are the poorest in the world today with many problems. We urgently need biotechnology tools, including GMOs, to address problems. We should not waste time blaming others for creating poverty and hunger but make efforts ourselves to get out of the rut. I still have far to go but I am contributing to the well being of farmers in Uganda and the whole region.