Identification of Stalk Rot Bacteria of Corn by Multilocus Sequence Analysis

Abstract

Samples of bacterial stalk rot of corn were collected from Saraburi and Kanchanaburi provinces. The Isolated bacteria were pathogenicity tested on corn seedlings of the varieties SM2678 and ST0390 by toothpick inoculating method. Eighteen bacterial isolates that fulfilled Koch’s postulates on pathogenicity were further characterized by Gram’s staining, physiological, biochemical testing, and by PCR with specific primer sets designed for Dickeya spp. Results revealed that all of 18 bacterial isolates were Gram negative, rod–shaped, 0.25–0.67 x 1.07–5.69 μm,occurred mostly alone or in pairs, but sometimes in short chains,motile, facultative anaerobic, can be grown at 36 ºC, did not grow in 5% NaCl, produced catalase, gelatinase and indole,did not produce oxidase and urease, reduced nitrate, catabolized citrate, sucrose, lactose and D–(–)–arabinose, but not D–sorbitol, D–(+)–maltose and L–arginine. All these phenotypesindicatedthatthe isolates resembled to Dickeya spp. Positive PCR detection with specific primer sets to Dickeya spp. were also obtained. Sequence comparative analysis of 16S rRNA and recA genes from four isolates, DZ15SB01, DZ15SB06, DZ15KB01 and DZ15KB05 grouped into Dickeya spp. Further investigation based on multilocus sequence analysis (MLSA) by using concatenated phylogenies produced from multiple loci of house–keeping genes,dnaJ,dnaX, gyrB, recA, recN and rpoBthese four bacterial isolates were resemble of Dickeya zeae.