Molecular diagnostic tools for plant health protection

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

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

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

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

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

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

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

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

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

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

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

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

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

Transgenic banana for Africa

Leena Tripathi, l.tripathi@cgiar.org

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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