Specific disease surveillance sheath brown rot of rice in highland rice plantation of Chiang Rai province and characterization of bacterial pathogen

Abstract

In 2016, specific disease surveillance survey on bacterial pathogens associated with sheath brown rot and grain discoloration was conducted in highland rice plantation of Chiang Rai province. King's medium B (KMB) was used for specific bacterial isolation of Pseudomonas fluorescens. Two hundred and fourteen bacterial isolates with fluorescent pigmentation on KMB was obtained from diseased samples and seventy - seven isolates from representing the collecting districts were subjected for hypersensitivity reaction (HR) test in tobacco plants. Eight isolates were found to be HR positive, and they were detected by PCR using 16S rRNA primers specific to P. fuscovaginae. Results from subsequence cloning and nucleotide sequencing comparison of the 16S rRNA of these eight isolates to the GenBank database, four isolates namely 59PFCRMSO2-7 59PFCRMSO2-8 59PFCRMSO2-10 and 59PFCRMU4-8 were in the same cluster with P. fuscovaginae. These four isolates caused the sheath brown rot symptom on inoculated rice seedlings. After cultivated on KMB at 30ºC for 48 hr, white cream to cream colour, opaque, circular, convex, entire, colonies size 0.3 to 0.8 mm in diameter with green fluorescent viewed under UV light were observed. They are aerobic, Gram-negative with rod-rounded end cells (0.5-0.8×1.9-3.3 µm). The incubation for four to five days at 28ºC on Nutrient agar, induced white cream colonies with a diameter of 4-6 mm. The biochemical test result was positive for oxidase, gelatinase and arginine dehydrolase, but negative for sucrose, arabinose, trehalose, 2-ketogluconate, inositol, sorbitol and adonitol, and not reduce nitrate, which was similar to P. fuscovaginae, except that growth at 37ºC was observed. Multilocus sequencing analysis (MLSA) of the concatenated nucleotides sequences of acsA, aroE, dnaE, guaA, gyrB, mutL, ppsA, pyrC, recA and rpoB housekeeping genes revealed these four isolates were similar to those of P. fuscovaginae Philippines strains, IRRI 6609 IRRI 7007 and S-E1, at the similarity level above 99%. Further confirmation was done by whole-genome nucleotides alignment against rice infecting Pseudomonas draft genomes which showed the similarity level above 90% to P. fuscovaginae IRRI 6609. The emergency action measures were immediately implemented to stop disease spread by The Rice Department and the Department of Agriculture (DOA) of Thailand. All rice plants in the infected areas were eradicated.