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Simultaneous Detection of Xanthomonas oryzae pv. oryzae and X. oryzae pv. oryzicola in Rice Seed Using a Padlock Probe-Based Assay

October 2014 , Volume 104 , Number  10
Pages  1,130 - 1,137

Yanli Tian, Yuqiang Zhao, Rui Xu, Fengquan Liu, Baishi Hu, and R. R. Walcott

First, third, fourth, and fifth authors: College of Plant Protection and Key Laboratory of Integrated Management of Crop Diseases and Pests, Ministry of Education, Nanjing Agricultural University, Nanjing 210095, China; second author: Shanghai Agricultural Technology Extension and Service Center; Shanghai 201103, China; fifth author: National Engineering Research Center for Cucurbits, Changji 831100, China; and sixth author: Department of Plant Pathology, University of Georgia, Athens 30602

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Accepted for publication 19 March 2014.

Based on 16S-23S internal transcribed spacer ribosomal DNA sequence data, two padlock probes (PLPs), P-Xoo and P-Xoc, were designed and tested to detect Xanthomonas oryzae pv. oryzae and X. oryzae pv. oryzicola, respectively. These PLPs were combined with dot-blot hybridization to detect X. oryzae pv. oryzae and X. oryzae pv. oryzicola individually in rice seed. Using this technique, a detection sensitivity of 1 pg of X. oryzae pv. oryzae genomic DNA was observed. The technique also facilitated the detection of X. oryzae pv. oryzae in rice seedlots with 2% artificially infested seed. With regards to X. oryzae pv. oryzicola a detection threshold of 1 pg genomic DNA was observed and the pathogen was successful detected in rice seedlots with 0.2% artificially infested seed. The PLP assays detected X. oryzae pv. oryzae and X. oryzae pv. oryzicola in 39.3% (13 of 33) and 21.3% (10 of 47) of naturally infested commercial rice seedlots, respectively. In contrast, conventional polymerase chain reaction using OSF1/OSR1 and XoocF/XoocR primers sets detected X. oryzae pv. oryzae and X. oryzae pv. oryzicola in 9.1% (3 of 33) and 8.5% (4 of 47) of the same rice seedlots, respectively. We also detected both pathogens simultaneously in two seedlots, which successfully proved that PLPs (P-Xoo and P-Xoc) combined with reverse dotblot hybridization can be used to simultaneously detect multiple pathogens in naturally infested commercial rice seedlots. This approach has the potential to be an important tool for detecting multiple pathogens in seed and thereby preventing the spread of important pathogens.

© 2014 The American Phytopathological Society