BP Singh

Detection of Bacterial Pathogens and Salvaging of Infected Germplasm 151campestris causing black rot in crucifers are detected by thismethod.(c) Agar plating method : The first agar plating method fordetection of X. campestris pv. campestris in Brassica seeds wasdeveloped by Lundsgaard (1973). The method has been modifiedand used for the detection of many seed borne bacteria in differentcrops. The bacterium in seeds may be detected as given below:(i) Direct plating: X. campestris pv. campestris in crucifer seeds;X. campestris pv. phaseoli, P. syringae pv. phaseolicola bean;Clavibacter michiganensis subsp. michiganensis in tomato can bedetected by direct plating the seeds on the agar medium.Randhawa and Schaad (1984) have developed semi-selective agarmedia (BSCAA) and successfully detected X. campestris pv.campestris in crucifer seeds on this medium.Plating with seed washing: X. campestris pv. campestris(ii)causes black rot in crucifers, X. campestris pv. carotae causesbacterial blight in carrot and other pathogens can be detected bythis method. For detection of X. campestrispv. campestris in cruciferseeds, 10000 seeds (about 40 g) are soaked in 0.85% NaCI solution(3-5°C) containing 0.02% Tween-20,S mg benlate 50% WP and 0.5m1 of a 1:10 dilution of Bravo 500. The contents are shaken on arotary shaker for 1.5 hr at 3-5°C, then filtered through sterile cheesecloth and rinsed with 25 ml sterile water. Seeds are discarded andthe liquid is centrifuged at 12,000 rpm for 10 min. at 2°C to pelletthe bacteria. The supernatant is discarded and each pellet is.suspended in 3 ml of sterile 0.85% NaCl (saline). The suspensionis diluted serially to 1:10, 1:100, 1:1000 and 0.1 m1 of each dill,ltionis plated individually in plates of semi-selective media (Randhawaand Schaad, 1984). The plates are incubated at 30°C and observedfor suspected bacterial colonies after 3 and 5 days.(iii) Plating after seed maceration: Bacterial pathogens can bedetected by seed maceration technique. The surface sterilizedseeds are soaked in sterile buffer water or distilled water at room.temperature for 3-4 hr and then crushed with pestle and mortar.The paste is then transferred to sterile water columns (quantity ofwater depends on the quantity and size of the seed), stirred wellon shaker or in centrifuge at 12,000 rpm and allowed to settledown. The supe:rnatant is discarded. The pellet is re-suspended