Establishing the pathogenicity of four Cercospora species associated with Cercospora leaf blight of soybean

Cercospora leaf blight (CLB) of soybean is caused by multiple Cercospora species globally, presenting significant challenges to disease management. The lack of standardized methods for inoculating and inducing CLB symptoms in controlled environments inhibits experiments on pathogen biology and host resistance. Consequently, limited understanding of the biology and epidemiology of these pathogens prohibits the development of tailored management strategies. Inducing sporulation of Cercospora species associated with CLB on amended media has been challenging, and the recommendations in the literature are inconsistent. To address these issues, standardized protocols were developed for inducing sporulation and reproducing CLB symptoms in controlled environments. Eleven culture media were tested for Cercospora cf. flagellaris conidia production, with V8A being the most effective. Five V8A concentrations were tested, and the conidia were harvested between 3 to 7 days with the highest conidial production (4.3 x 105 per ml) occurring on day 4 with 30% V8A. Peak sporulation varied, with C. cf. flagellaris peaking on day 4 and Cercospora cf. sigesbeckiae, Cercospora kikuchii, and Cercospora iranica peaking on day 3. Pathogenicity tests under controlled conditions fulfilled Koch's postulates by reproducing symptoms for all species tested, confirming their role in CLB development. These protocols provide the first standardized, efficient, and reproducible methods for Cercospora sporulation and inoculation. These methods offer insights for fundamental research into Cercospora biology and their interactions with soybeans, ultimately improving CLB management.