Establishing the Pathogenicity of Four <i>Cercospora</i> Species Associated With Cercospora Leaf Blight of Soybean

Cercospora leaf blight (CLB) of soybean is caused by multiple <i>Cercospora</i> species globally, presenting significant challenges to disease management<b>.</b> 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 <i>Cercospora</i> 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 <i>Cercospora</i> cf. <i>flagellaris</i> conidial production, with V8 agar (V8A) being the most effective. Five V8A concentrations were tested, and the conidia were harvested between 3 and 7 days with the highest conidial production (4.3 × 10⁵ per ml) occurring on day 4 with 30% V8A. Peak sporulation varied, with <i>C</i>. cf. <i>flagellaris</i> peaking on day 4 and <i>Cercospora</i> cf. <i>sigesbeckiae</i>, <i>Cercospora kikuchii</i>, and <i>Cercospora iranica</i> 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 <i>Cercospora</i> sporulation and inoculation. These methods offer insights for fundamental research into <i>Cercospora</i> biology and their interactions with soybeans, ultimately improving CLB management.