HomeInternational Rice Research Notesvol. 22 no. 2 (1997)

DNA Fingerprinting of Bacterial Blight Pathogen Directly from Infected Leaves

M L. George | M Villamayor | C M. Vera Cruz | T W. Mew | R J. Nelson

Discipline: Agriculture

 

Abstract:

In this study, we shortened the time required to obtain DNA fingerprints by using pathogen DNA prepared directly from cells leached from infected leaves. Leaves of IR24 plants grown in the greenhouse were inoculated (clip method) with six isolates of Xanthomonas oryzae pv. oryzae belonging to different lineages, and infected leaves were collected 14 d later. To ‘ooze’ bacteria from the leaves, 2-cm-long leaf fragments containing the advancing tip of lesions were cut crosswise into 10 small pieces with flame-sterilized scissors, and then soaked in 400 μL of sterile distilled water for at least 1 h.

 

The leaf pieces were discarded and the cells concentrated by spinning the suspension for 2 min in a microcentrifuge. Most of the water was discarded, leaving about 25 μL, in which the cells were resuspended. The cells were lysed in 200 μL of 70% ethanol (final concentration) and then spun for 10 s to pellet the cell debris. The supernatant was transferred to a new tube and spun for 5 min to precipitate DNA. The DNA was air-dried, dissolved in 10 μL of sterile water and 5 μL served as template for polymerase chain reaction (PCR). To check the consistency of the fingerprint patterns, purified DNA from the isolates used for inoculation served as positive controls. Standard PCR protocols were followed using JEL1 /JEL2 primers and with the repPCR primers BOX, ERIC, and REP using DNA from bacterial exudates from lesions and using DNA from cultured cells.