Kumar, P.L.

Kumar, P. Lava

CGSpace

Special 45th Anniversary EditionR4D Review1967-201245Edition 8March 2012contentsMind the gap...2 EDITOR’S NOTEBoosting yam productivity in Ghana and Nigeria  Pro-vitamin A cassava released  Multi-CGIAR center initiative launched  Plant Virology Symposium slated in 20134IITA R4D Review Issue 8, March 2012Editors Lava Kumar and Katherine Lopez   Production team Copy editor: Rose Umelo Creative production: Godson Bright        Adegboyega Juba, and      Clement Ono-Raphael  Editorial board  Robert Asiedu, David Chikoye, and  Victor ManyongThe IITA R4D Review is published by IITA, 		solutions for hunger and poverty. Copyright  IITA holds the copyright to its publications but encourages duplication of these materials for noncommercial purposes. 		of these materials is prohibited. Permission to make digital or hard copies of part or all of this work for personal or classroom use is hereby granted without fee and without a formal request provided that copies are not 	advantage and that copies bear this notice 	for components not owned by IITA must be honored and permission pursued with 	permission is required to copy otherwise, to republish, to post on servers, or to redistribute to lists.   Disclaimer We do not endorse, approve, or give any warranty on the content of this publication. All liability for loss and damage arising from your use of our content is excluded (except where death or personal injury arises from our negligence or loss or damage arises from any fraud on our part).  ISSN 2071-3681 Maize genetic improvement for enhanced productivity gains     6A success tale on improving two legume crops in Africa    11Breeding superior banana hybrids    16Cassava improvement in the era  of “agrigenomics”    21Yam breeding at IITA    27 Genomics for yam breeding   31A ‘Green Revolution’ in the West African  cocoa belt   356 FEATURESTOOL BOX45!"#$%		&		'	 Nteranya Sanginga:  “Science can solve agriculture's problems”52 LOOKING IN Valerie Bemo:  ‘‘Collaboration required for major breakthroughs  in African agriculture’’"Agrigenomics" for crop improvement    55 Transgenics in crop improvement research at IITA   58Molecular diagnostic tools for plant health protection  61Partnerships as relationships for agricultural development 42 BEST PRACTICEF R O N T I E R S5548    WHO’S WHO	collecting tassels for hand pollination.  Photo by C. Ono-Raphael.61Molecular 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 			and speed of detection 		disease-causing agents and characterization of the diversity of pathogens and pests. 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 several 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 fast established as a mainstay of applied molecular biology and molecular diagnostics. Molecular diagnostic tools for plant health protection Lava Kumar (L.kumar@cgiar.org)Researcher observing mouse hybridoma cell lines under microscope in the Virology and Molecular Diagnostics Unit, IITA, Ibadan, Nigeria. Photo by IITA.L. Kumar, Head of Germplasm Health Unit and Virologist, IITA, Ibadan, Nigeria.62Platform for developing molecular diagnostics 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 		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 		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 				were used to develop !"	#$%&'"*quantitative estimation 			+and other commodities (see article on page 45). 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 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 			threat to cassava in East Africa, thereby making it a one-stop test for 		viruses infecting cassava in SSA. 63Similar 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, 	diagnostic tools for the detection of almost all the pathogens that 			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 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 other agents. Information generated from these studies have provided valuable clues to understand the origin and drivers of 		of previously uncharacterized pathogens3,4 and 		//6	7:;"barcodes” for use as genetic markers for identifying pathogens and pests5. 			 Source M. Cilia, Cornell University<=>	?='	/to transmission and all other isoforms in populations=:&G$		6. Targeted proteomics assays to quantify peptides using a  triple quadrupole mass spectrometer and validate # samplesCorrelation Analysis5. Import sequences into Skyline to identify best peptides in vector populations to develop biomarker assay4. DNA sequencing to  identify polymorphisms linked to transmission phenotype 'INTSHVEYDTASRHY 'INTSHVEYDTASRHY 'INTSHVEYDTELRHY64Biomarkers 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 		6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. During the 	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 			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 agricultural systems and climate change effect. These tools are already proving useful in rapid detection 		6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 1Benali, S. et al. 2011. Advances of molecular markers application in plant pathology research. European Journal of '^	=50:110–123.2Alabi, O.J. et al. 2008. Multiplex PCR method for the detection of African cassava mosaic virus and East African cassava mosaic Cameroon virus in cassava. Journal of Virology Methods. 154:111–120.3Alabi, O.J. et al. 2010. Two new ‘legumoviruses’ (genus Begomovirus) naturally infecting soybean in Nigeria. Archives of Virology. 155:643–656.4Sharma, K. et al. 2010. Genetically distinct Cercospora species cause grey leaf spot of maize (Zea mays L.) in Nigeria. Phytopathology 100 (6): S117.5Kumar, P.L. and K. Sharma. 2010. DNA barcodes for pathogens of African food crops. R4D Review 4: 51–53. www.R4DReview.org.6Kumar, P.L. (ed.). 2009. Methods for diagnosis of plant virus diseases: a laboratory manual. IITA, Ibadan, Nigeria. 90 pp. Join the discussions online atwww.r4dreview.orgISSN: 2071-3681 

Molecular diagnostic tools for plant health protection

https://cgspace.cgiar.org/bitstream/10568/77431/1/U12ArtKumarMolecularNothomNodev.pdf

Molecular diagnostic tools for plant health protection

Abstract

Similar works

Full text

Available Versions

CGSpace