Development of DNA based detection and monitoring technique for Xanthomonas oryzae pv. oryzicola (Fang et al.) Swings

Repetitive-sequence based amplification (rep-PCR) of genomic DNA templates of Xanthomonas oryzae pv. oryzicola, which causes bacterial leaf streak in rice, generated complex DNA fingerprints. Primers corresponding to enterobacterial intergenic consensus (ERIC), repetitive extragenic palindromic (REP) and BOX sequences differentiated X. oryzae pv. oryzicola from X. oryzae pv. oryzae. Attempts to clone rep-PCR generated DNA fragments unique in X. oryzae pv. oryzicola strains for sequencing and synthesis of specific primers failed. However, unique fragments generated from X. oryzae pv. oryzicola BLS 305 by primers based on a X. oryzae pv. oryzicola repetitive sequence (R41) were cloned. Based on the sequence information of two cloned inserts, 2R41-3e and 2R41-1j, a total of 11 forward and reverse primers were synthesized and tested in genomic DNA of Philippine and foreign strains of X. oryzae pv. oryzicola, X. oryzae pv. oryzae, other xanthomonads, some phytopathogenic bacteria, soil bacteria, rice seed bacteria and rice lead saprophytes. None of these primers gave specific amplification to X. oryzae pv. oryzicola. Amplification products were also detected in X. oryzae pv. oryzae, other xanthomonads and other bacteria. Applicability of the primers for X. oryzae pv. Opryzicola detection would therefore rely on the size of the amplification product. The single primer PR41-1R yielded an intense 378 bp fragment in X. oryzae pv. oryzicola and X. oryzae pv. oryzae strains with the exception of the American and African strains of X. oryzae pv. oryzae. The 378 bp fragment was also detected in X. campestris pv. secalis and in X. Brown Blotch, another pathogen of rice, but which can be differentiated from X. oryzae using primers PR41-3F2/PR41-3R or PR41-3F4/PR41-3R. Using Primer PR41-1F1, X. oryzae pv. oryzicola strains were differentiated from X. oryzae pv. oryzae strains by their characteristic patterns