Identification of Bacterial Pathogens Causing Panicle Blight and Dirty Panicle of Rice by Multilocus Sequence Analysis

Abstract

Forty–six isolates of putative panicle blight bacterium were collected during the years 2011–2013. All were Gram negative, rod–shaped ranging in size from 0.53–0.57x 2–2.5 μm, accumulated poly–β–hydroxybutyrate (PHB) and formed gray–white colonies. The bacteria produced water–soluble pigment on King’s medium B, required oxygen for growth, growth on pH 4, 8 and 9 media, 3% NaCl contained media and appeared as type A or type B colony on the selective medium (S–PG) for Burkholderia glumae. Positive for oxidase, gelatinase and negative for arginine dihydrolase, urease and starch hydrolysis were observed. Pathogenicity tests were performed by inoculation of bacterial suspension on two month–old rice seedlings. All of 46 isolates caused the sheath rot symptom. Comparative analysis of 16S rRNA sequences of these isolates grouped them into the genus Burkholderia with 45 isolates closely related to B. glumae and one isolate to B. gladioli group. This was confirmed by the species–specific PCR of 16S–23S rDNA internal transcribed spacer region. Due to the unavailability of bacterial type strain for DNA–DNA hybridization, polyphasic taxonomic study based on classical identification and multilocus sequencing analysis (MLSA) of 16S rRNA, atpD, gltB, lepA and recA were employed for bacterial identification. Results clearly indicated that bacteria isolates 1BGRE5–1, 1BGSP3–4, 1BGCR2–2, 2BGCN4–1, 2BGCN5–2, 3BGNP1–4, 3BGNP2–3, 3BGST 2–1, 3BGNY1–1 and 3BGNY2–4 which caused of bacterial panicle blight of rice were resembled of B. glumae.