Leena Tripathi

Cassava Virus Diseases: Biology, Epidemiology, and Management

Cassava (Manihot esculenta Crantz.) is the most important vegetatively propagated food staple in Africa and a prominent industrial crop in Latin America and Asia. Its vegetative propagation through stem cuttings has many advantages, but deleteriously it means that pathogens are passed from one generation to the next and can easily accumulate, threatening cassava production. Cassava-growing continents are characterized by specific suites of viruses that affect cassava and pose particular threats. Of major concern, causing large and increasing economic impact in Africa and Asia are the cassava mosaic geminiviruses that cause cassava mosaic disease in Africa and Asia and cassava brown streak viruses causing cassava brown streak disease in Africa. Latin America, the center of origin and domestication of the crop, hosts a diverse set of virus species, of which the most economically important give rise to cassava frog skin disease syndrome. Here, we review current knowledge on the biology, epidemiology, and control of the most economically important groups of viruses in relation to both farming and cultural practices. Components of virus control strategies examined include: diagnostics and surveillance, prevention and control of infection using phytosanitation, and control of disease through the breeding and promotion of varieties that inhibit virus replication and/or movement. We highlight areas that need further research attention and conclude by examining the likely future global outlook for virus disease management in cassava.

A rapid technique for screening banana cultivars for resistance to Xanthomonas wilt

The banana Xanthomonas wilt disease (BXW) has threatened the livelihood of millions of farmers in East Africa. Use of resistant varieties is the most cost-effective method of managing this bacterial disease. A reliable and rapid screening method is needed to select resistant banana varieties. An in vitro screening method was developed for early evaluation of Xanthomonas wilt resistance using small tissue culture-grown plantlets. Eight cultivars of banana were screened with sixteen isolates of Xanthomonas campestris pv. musacearum using this method. There were significant differences (P < 0.0001) in susceptibility among the various banana cultivars tested, whereas no significant difference (P = 0.92) in pathogenicity was observed between the pathogen isolates. The cv. Pisang Awak (Kayinja) was found to be highly susceptible and Musa balbisiana resistant. Nakitembe was found to be moderately resistant while cvs Mpologoma, Mbwazirume, Sukali Ndiizi, FHIA-17 and FHIA-25 were susceptible. The susceptibility of these cultivars was further tested in vivo by artificial inoculation of potted plants with similar results. This study shows that an in vitro screening test can serve as a convenient, cheap and rapid screening technique to discriminate BXW-resistant from BXW-susceptible banana cultivars.

Cassava Virus Diseases

Cassava (Manihot esculenta Crantz.) is the most important vegetatively propagated food staple in Africa and a prominent industrial crop in Latin America and Asia. Its vegetative propagation through stem cuttings has many advantages, but deleteriously it means that pathogens are passed from one generation to the next and can easily accumulate, threatening cassava production. Cassava-growing continents are characterized by specific suites of viruses that affect cassava and pose particular threats. Of major concern, causing large and increasing economic impact in Africa and Asia are the cassava mosaic geminiviruses that cause cassava mosaic disease in Africa and Asia and cassava brown streak viruses causing cassava brown streak disease in Africa. Latin America, the center of origin and domestication of the crop, hosts a diverse set of virus species, of which the most economically important give rise to cassava frog skin disease syndrome. Here, we review current knowledge on the biology, epidemiology, and control of the most economically important groups of viruses in relation to both farming and cultural practices. Components of virus control strategies examined include: diagnostics and surveillance, prevention and control of infection using phytosanitation, and control of disease through the breeding and promotion of varieties that inhibit virus replication and/or movement. We highlight areas that need further research attention and conclude by examining the likely future global outlook for virus disease management in cassava.

Haploids: Constraints and opportunities in plant breeding

The discovery of haploids in higher plants led to the use of doubled haploid (DH) technology in plant breeding. This article provides the state of the art on DH technology including the induction and identification of haploids, what factors influence haploid induction, molecular basis of microspore embryogenesis, the genetics underpinnings of haploid induction and its use in plant breeding, particularly to fix traits and unlock genetic variation. Both in vitro and in vivo methods have been used to induce haploids that are thereafter chromosome doubled to produce DH. Various heritable factors contribute to the successful induction of haploids, whose genetics is that of a quantitative trait. Genomic regions associated with in vitro and in vivo DH production were noted in various crops with the aid of DNA markers. It seems that F2 plants are the most suitable for the induction of DH lines than F1 plants. Identifying putative haploids is a key issue in haploid breeding. DH technology in Brassicas and cereals, such as barley, maize, rice, rye and wheat, has been improved and used routinely in cultivar development, while in other food staples such as pulses and root crops the technology has not reached to the stage leading to its application in plant breeding. The centromere-mediated haploid induction system has been used in Arabidopsis, but not yet in crops. Most food staples are derived from genomic resources-rich crops, including those with sequenced reference genomes. The integration of genomic resources with DH technology provides new opportunities for the improving selection methods, maximizing selection gains and accelerate cultivar development. Marker-aided breeding and DH technology have been used to improve host plant resistance in barley, rice, and wheat. Multinational seed companies are using DH technology in large-scale production of inbred lines for further development of hybrid cultivars, particularly in maize. The public sector provides support to national programs or small-medium private seed for the exploitation of DH technology in plant breeding.

Generation of transgenic plantain (Musa spp.) with resistance to plant pathogenic nematodes

Plant parasitic nematodes impose a severe constraint on plantain and banana productivity; however, the sterile nature of many cultivars precludes conventional breeding for resistance. Transgenic plantain cv. Gonja manjaya (Musa AAB) plants, expressing a maize cystatin that inhibits nematode digestive cysteine proteinases and a synthetic peptide that disrupts nematode chemoreception, were assessed for their ability to resist nematode infection. Lines were generated that expressed each gene singly or both together in a stacked defence. Nematode challenge with a single species or a mixed population identified 10 lines with significant resistance. The best level of resistance achieved against the major pest species Radopholus similis was 84% ± 8% for the cystatin, 66% ± 14% for the peptide and 70% ± 6% for the dual defence. In the mixed population, trial resistance was also demonstrated to Helicotylenchus multicinctus. A fluorescently labelled form of the chemodisruptive peptide underwent retrograde transport along certain sensory dendrites of R. similis as required to disrupt chemoreception. The peptide was degraded after 30 min in simulated intestinal fluid or boiling water and after 1 h in nonsterile soil. In silico sequence analysis suggests that the peptide is not a mammalian antigen. This work establishes the mode of action of a novel nematode defence, develops the evidence for its safe and effective deployment against multiple nematode species and identifies transgenic plantain lines with a high level of resistance for a proposed field trial.

Genetic engineering for improvement of Musa production in Africa

Bananas and plantains (Musa sp.) are the most important staple food and source of carbohydrates in many countries of Africa. The production is often constrained by many pests and diseases. In order to augment conventional breeding and to avoid constraints imposed by some pests and pathogens, transgenic approaches are being considered. The development of transgenic Musa plants has been achieved recently using the microprojectile bombardment procedure or Agrobacterium-mediated transformation. The transgenic approach shows potential for the genetic improvement of the crop using a wide set of transgenes currently available which may confer resistance to nematode pests, fungal, bacterial and viral diseases. This article discusses the applications of genetic engineering for the enhancement of Musa production.

Field trial of Xanthomonas wilt disease-resistant bananas in East Africa

volume 32 NumBeR 9 SePTemBeR 2014 nature biotechnology to intensified production of active oxygen species and activation of the hypersensitive response when plants are challenged with bacterial pathogens4. Overexpression of Pflp has been shown to provide resistance against various bacterial pathogens, such as Erwinia, Pseudomonas, Ralstonia and Xanthomonas spp., in transgenic tobacco4, tomato5, orchids6, calla lily7 and rice8. However, none of these transgenic plants was tested for disease resistance under field conditions. In previous work, transgenic plants of two banana cultivars ‘Sukali ndiizi’ (apple banana; AAB group) and ‘Nakinyika’ (EAHBs; AAA group) were generated by constitutively expressing the Hrap9 or Pflp10 gene. Here we evaluated the best 65 resistant lines (40 lines expressing Hrap gene and 25 lines with Pflp gene) in a confined field trial at National Agriculture Research Laboratories, Kawanda, Uganda, against X. campestris pv. musacearum for two successive crop cycles (see Supplementary Methods) (Fig. 1). To the Editor: Banana is a major staple crop in East Africa produced mostly by smallholder subsistence farmers. More bananas are produced and consumed in East Africa than in any region of the world. Uganda is the world’s second foremost grower with a total annual production of about 10.5 million tons. The average daily per capita consumption in Uganda ranges from 0.61 to over 1.6 kg, one of the highest in the world. In this Correspondence, we report preliminary results from a confined field trial in Uganda of transgenic bananas resistant to the deadly banana Xanthomonas wilt (BXW) disease. BXW caused by Xanthomonas campestris pv. musacearum is threatening banana production, the livelihoods of the smallholder growers in East and Central Africa1, and the stability of food security in the region. The disease has caused estimated economic losses of about

Unlocking the potential of tropical root crop biotechnology in east Africa by establishing a genetic transformation platform for local farmer-preferred cassava cultivars

2–8 billion over the past decade and substantial reductions in production have resulted in major price increases1. BXW was originally reported1 in Ethiopia and first identified in Uganda in 2001 and subsequently in the Democratic Republic of Congo, Rwanda, Kenya, Tanzania and Burundi. The disease is very destructive, infecting all banana varieties, including both East African Highland bananas (EAHBs) and exotic dessert and beer bananas. The economic impact of the disease is potentially disastrous because it destroys whole plants leading to complete yield loss. Once these pathogens have become established in smallholder plantations, disease control is very difficult. As diseases continue to spread, demand grows for new improved varieties. There are currently no commercial pesticides, biocontrol agents or resistant cultivars available to control BXW1. Although BXW can be managed by following phytosanitary practices, the adoption of such practices has been inconsistent as they are labor intensive1. Given the rapid spread and devastation of BXW across Africa, the lack of known genetic resistance in banana against X. campestris pv. musacearum, and the difficulties associated with conventional breeding of this highly sterile crop, genetic transformation through the use of modern biotech tools offers an effective and viable way to develop resistant varieties. Two potential transgenes for controlling BXW are those encoding hypersensitive response-assisting protein (Hrap) and plant ferredoxin-like protein (Pflp) from sweet pepper (Capsicum annuum). Both have been proven effective against related bacterial pathogens, such as Erwinia, Pseudomonas, Ralstonia and Xanthomonas spp., in other crops2–8. Hrap is one of the important hypersensitive cell death–associated genes that could be used to protect plants from bacterial pathogen attack2. HRAP has been reported to intensify the hypersensitive response mediated by harpinPSS (harpin derived from Pseudomonas syringae pv. syringae), by dissociating multimeric forms of the hairpin into dimers and monomers that trigger stronger hypersensitive cell death necrosis. Constitutive expression of Hrap genes in transgenic tobacco2 and Arabidopsis3 has been shown to confer enhanced resistance against virulent pathogens under laboratory and glasshouse conditions. Resistance resulting from overexpression of the Pflp gene in transgenic plants is due Field trial of Xanthomonas wilt disease-resistant bananas in East Africa

Field resistance of transgenic plantain to nematodes has potential for future African food security

Cassava genetic transformation capacity is still mostly restricted to advanced laboratories in the USA, Europe and China; and its implementation and maintenance in African laboratories has remained scarce. The impact of transgenic technologies for genetic improvement of cassava will depend largely on the transfer of such capabilities to researchers in Africa, where cassava has an important socioeconomic niche. A major constraint to the development of genetic transformation technologies for cassava improvement has been the lack of an efficient and robust transformation and regeneration system. Despite the success achieved in genetic modification of few cassava cultivars, including the model cultivar 60444, transgenic cassava production remains difficult for farmer-preferred cultivars. In this study, a protocol for cultivar 60444 developed at ETH Zurich was successfully implemented and optimized to establish transformation of farmer-preferred cassava cultivars popular in east Africa. The conditions for production and proliferation of friable embryogenic calli (FEC) and Agrobacterium-mediated transformation were optimized for three east African farmer-preferred cultivars (Ebwanatereka, Kibandameno and Serere). Our results demonstrated transformation efficiencies of about 14–22 independent transgenic lines per 100 mg of FEC for farmer-preferred cultivars in comparison to 28 lines per 100 mg of the model cultivar 60444. The presence, integration and expression of the transgenes were confirmed by PCR, Southern blot analysis and histochemical GUS assay. This study reports the establishment of a cassava transformation platform at International Institute of Tropical Agriculture (IITA) hosted by Biosciences eastern and central Africa (BecA) hub in Kenya and provides the basis for transferring important traits such as virus resistance and prolonged shelf-life to farmer-preferred cultivars in east Africa. We anticipate that such platform will also be instrumental to transfer technologies to national agricultural research systems (NARS) in sub-Saharan Africa.

Fine-tuning banana Xanthomonas wilt control options over the past decade in East and Central Africa

Plant parasitic nematodes impose losses of up to 70% on plantains and cooking bananas in Africa. Application of nematicides is inappropriate and resistant cultivars are unavailable. Where grown, demand for plantain is more than for other staple crops. Confined field testing demonstrated that transgenic expression of a biosafe, anti-feedant cysteine proteinase inhibitor and an anti-root invasion, non-lethal synthetic peptide confers resistance to plantain against the key nematode pests Radopholus similis and Helicotylenchus multicinctus. The best peptide transgenic line showed improved agronomic performance relative to non-transgenic controls and provided about 99% nematode resistance at harvest of the mother crop. Its yield was about 186% in comparison with the nematode challenged control non-transgenic plants based on larger bunches and diminished plant toppling in storms, due to less root damage. This is strong evidence for utilizing this resistance to support the future food security of 70 million, mainly poor Africans that depend upon plantain as a staple food.