Partial characterization of different isolates of Xanthomonas albilineans and production of a polyclonal antibody for rapid disease detection

Seventeen potential isolates of the leaf scald bacterium were isolated from sugarcane leaves collected from Iloilo and Victorias City, Negros Occidental. Pure cultures of each isolate maintained in Modified Wilbrinks Medium (MWM) plants were inoculated on sugarcane plants, (VMC 98-79) for pathogenicity. Polymerase Chain Reaction (PCR) using specific primers was done to confirm the identity of the bacterium. The cultural and physiological characteristics of the different isolates were determined. All isolates were gram-negative, rod-shaped, aerobic, able to grow at 28 deg C, utilized glucose, mannose and glycerol but not arabinose. Pure culture was used for antiserum production in a 3-month old white rabbit. The 4th bleed was found to yield the highest titer and was considered as the best bleed. This was obtained one weed after the 7th injection. An absorbance reading of 3.165 was obtained when tested against 8'10sup7 CFU/ml of bacterial suspension and 1:1000 antiserum, dilution. A dilution of the produced polyclonal antiserum to 1:1000 v/v in antibody buffer yielded an absorbance reading of 1.283 at 405 nm using a 8'10sup3 dilution of bacterial suspension in carbonate coating buffer. A 1:1000 dilution of the produced antiserum reacted positively to the 14 yellow bacteria isolated from sugarcane even when diluted at 8'10sup3 CFU/ml. Non-sugarcane Xanthomonas spp such as Xanthomonas oryzae pv. oryzae, Xanthomonas oryzae pv. oryzicola and Xanthomonas axonopodis pv. poinsetticola yielded negative reactions against the antiserum in indirect ELISA. Indexing of introduced varieties of sugarcane in Post-Entry Quarantine Greenhouse, IPB, UPLB showed that 12 out of 170 sugarcane leaf samples, (9 symptomatic and 3 asymptomatic) were positive by ELISA using the developed antibody against Xanthomonas albilineans.