Biotechnology 9 (2): 89-105, 2010 ISSN 1682-296X © 2010 Asian Network for Scientific Information Corresponding Author: Y. Sere, Plant Pathology Unit, Africa Rice Center (AfricaRice), 01 BP 2031, Cotonou, Benin Tel: +22921350188 Fax: +22921350556 89 Development of a Combined Molecular Diagnostic and DNA Fingerprinting Technique for Rice Bacteria Pathogens in Africa A. Onasanya, A. Basso, E. Somado, E.R. Gasore, F.E. Nwilene, I. Ingelbrecht, J. Lamo, K. Wydra, 1,9 1,2 1 3 4 5 6 7,8 M.M. Ekperigin, M. Langa, O. Oyelakin, Y. Sere, S. Winter and R.O. Onasanya 9 10 5 1 11 9 Africa Rice Center (AfricaRice), 01 BP 2031, Cotonou, Benin Republic 1 Institut national de la Recherche Agronomique du Niger (INRAN), BP. 60 Kollo, Niger 2 Rwanda Agricultural Research Institute, BP 138 Butare, Rwanda 3 Africa Rice Center (AfricaRice), PMB 5320, Ibadan, Nigeria 4 Central Biotechnology Laboratory, IITA, PMB 5320, Ibadan, Nigeria 5 National Crop Resources Research Institute (NaCRRI), P.O. Box 7084, Kampala, Uganda 6 Institute of Plant Diseases and Plant Protection Leibniz Universität Hannover Herrenhäuser Str. 2, 7 30419 Hannover, Germany Centre for Tropical and Subtropical Agriculture and Forestry (CeTSAF), Tropenzentrum Georg, 8 August Universität Goettingen Buesgenweg 1, D-37077 Goettingen, Germany Federal University of Technology Akure, PMB 704, Akure, Nigeria 9 Institute of Agricultural Research of Mozambique – DARN, Av. das FPLM No 2698, 10 Caixa Postal 3658, Mavalene, Maputo, Mozambique Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) GmbH, 11 Inhoffenstraße 7 B, 38124 Braunschweig, Germany Abstract: A combined molecular diagnostic and DNA fingerprinting PCR technique for Xanthomonas oryzae pv. oryzae (Xoo), Xanthomonas oryzae pv. oryzicola (Xoc), Pseudomonas fuscovaginae (Pf) and Pseudomonas syringae pv. syringae (Pss) pathogens from rice has been developed in Africa by this study, using four primer pairs designed from Xoo (NC_007705.1), Xoc (NZ_AAQN01000001.1), Pf (AB021381.1) and Pss (NC_007005.1) complete genome sequence. Molecular PCR diagnostic showed that the presence of at least a band indicates positive detection of a bacterial pathogen and absence of a band indicates negative for no bacterial pathogen detected, while in the same PCR assay the presence of one or more band at different position revealed the DNA fingerprint of a bacterial pathogen. Out of 95 bacterial cultured isolates analyzed, 84 contained Xoo, 50 contained Xoc, 19 contained Pf and 16 contained Pss pathogens. DNA fingerprinting of the 84 Xoo pathogens revealed seven Xoo genotypes, four Xoc genotypes were identified from 50 Xoc pathogens and 19 Pf pathogens produced three Pf genotypes while 16 Pss pathogens formed three Pss genotypes. Development of a reliable molecular technique for Xoo, Xoc, Pf and Pss identification and differentiation is a prerequisite into understanding the genetics of Xoo, Xoc, Pf and Pss population structure in Africa and deployment of durable resistance cultivars. Key words: Xanthomonas oryzae pv. oryzae, Xanthomonas oryzae pv. oryzicola, Pseudomonas fuscovaginae, Pseudomonas syringae pv. syringae, molecular diagnostic, DNA fingerprinting, PCR technique, genotype, Africa INTRODUCTION (Kennedy et al., 2002). More than 70 diseases caused by Rice (Oryza sativa L.) is the primary food grain recorded on rice. Xanthomonas oryzae pv. oryzae (Xoo), consumed by almost half of the world’s population, Xanthomonas oryzae pv. oryzicola (Xoc), Pseudomonas making it the most important food crop currently fuscovaginae (Pf) and Pseudomonas syringae pv. produced (Cottyn et al., 2001). It provides 27% of energy syringae (Pss) are bacterial pathogens capable of causing and 20% of proteins in developing countries disease on different rice cultivars. Bacterial Leaf Blight fungi, bacteria, viruses and nematodes have been