PCR-mediated identification and characterization of rice bacterial blight pathogen (Xanthomonas oryzae pv. Oryzae) isolates collected from Sindh province of Pakistan
Muhammad Tahir Khan, Javed Asghar Tariq, Muhammad Arif, Shafquat Yasmeen And Imtiaz Ahmed Khan
Bacterial leaf blight (BLB) is one of the most devastating diseases of rice crop. Its severe incidence has been reported in the Sindh province of Pakistan in recent years. It can cause up to 50% crop losses, whereas in favorable conditions, the disease can result in premature plant death. This study was conducted to identify and characterize the BLB causing pathogen Xanthomonas oryzae pv. oryzae (Xoo) in Sindh province through molecular approaches. Pure cultures of 120 bacterial isolates, obtained from various locations of the province, were developed using the streaking method. The purified cultures were grown overnight in LB medium until saturation. Cultures of the bacterial isolates were then subjected to DNA isolation employing the CTAB method. The extracted bacterial DNA was subjected to PCR reaction. XOR primer pair [XOR-F (5'-GCATGACGTCATCGTCCTGT-3') and XOR-R2 (5'-CTCGGAGCTATATGCCGTGC-3')] was employed for amplification of 16S-23S rDNA spacer region-specific to Xoo bacterium. The PCR product was run in 1.2% gel electrophoresis in TBE buffer. The targeted band of 470 bp was observed in a total of 34 isolates analyzed through PCR. The identified Xoo isolates were further subjected to RAPD-PCR, which yielded various polymorphic bands. The binary data collected from RAPD analysis was used for investigating genetic relatedness and diversity of the Xoo isolated using the un-weighted pair-group (UPGMA) method. The UPGMA analysis showed extensive genetic variability among the studied isolates as the isolates were observed to cluster into seven clusters. This is the first report of PCR-based confirmation of Xoo in Sindh province, as per our knowledge. Rapid, reliable, and accurate identification of Xoo is very important to adopt disease management strategies to minimize crop losses. Moreover, the RAPD based genetic analysis of these isolates may serve as a platform study for refined characterization of new isolates for which racial classification has not been determined in Sindh province.
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