Detection of Xanthomonas sp., the Causal Agent of Onion Bacterial Blight, in Onion Seeds Using a Newly Developed Semi-selective Isolation Medium

Abstract

Onion bacterial blight, caused by Xanthomonas sp., is a potentially severe disease in several tropical and subtropical areas. Although little research has been undertaken on this pathosystem, seed transmission of the pathogen has been hypothesized. Because of an important bacterial microflora naturally associated with onion seeds, detection of the pathogen is difficult using non-selective agar media. A new semi-selective medium, whose selectivity was obtained by a combination of four antibiotics, was developed. The new NCTM1 medium contained (per liter) yeast extract 7 g, peptone 7 g, glucose 7 g, agar 15 g, neomycin 10 mg, cephalexin 30 mg, trimethoprime 3 mg, pivmecillinam 100 mg and propiconazole 20 mg. Plating efficiencies, using 16 pure cultures of the pathogen, ranged from 79% to 142%, with an average of 110% compared to the basal medium. All onion Xanthomonas sp. strains from several countries grew on NCTM1 medium. The pathogen was repeatedly isolated using this medium from seed samples containing approximately 10⁶ saprophytic bacteria per gram, as well as from symptomless plant material. Xanthomonas sp. was detected only in seeds originating from one infected seed production site. This is the first report of selective isolation of Xanthomonas sp. from onion seeds. NCTM1 medium should be a valuable tool to study the ecology and epidemiology of Xanthomonas sp. causing onion bacterial blight.