Enhanced cercosporin production by co-culturing Cercospora sp. JNU001 with leaf-spot-disease-related endophytic bacteria.

BACKGROUND: Owing to the excellent properties of photosensitization, cercosporin, one of naturally occurring perylenequinonoid pigments, has been widely used in photodynamic therapy, or as an antimicrobial agent and an organophotocatalyst. However, because of low efficiency of total chemical synthesis and low yield of current microbial fermentation, the limited production restricts its broad applications. Thus, the strategies to improve the production of cercosporin were highly desired. Besides traditional optimization methods, here we screened leaf-spot-disease-related endophytic bacteria to co-culture with our previous identified Cercospora sp. JNU001 to increase cercosporin production. RESULTS: Bacillus velezensis B04 and Lysinibacillus sp. B15 isolated from leaves with leaf spot diseases were found to facilitate cercosporin secretion into the broth and then enhance the production of cercosporin. After 4 days of co-culture, Bacillus velezensis B04 allowed to increase the production of cercosporin from 128.2 mg/L to 984.4 mg/L, which was 7.68-fold of the previously reported one. Lysinibacillus sp. B15 could also enhance the production of cercosporin with a yield of 626.3 mg/L, which was 4.89-fold higher than the starting condition. More importantly, we found that bacteria B04 and B15 employed two different mechanisms to improve the production of cercosporin, in which B04 facilitated cercosporin secretion into the broth by loosening and damaging the hyphae surface of Cercospora sp. JNU001 while B15 could adsorb cercosporin to improve its secretion. CONCLUSIONS: We here established a novel and effective co-culture method to improve the production of cercosporin by increasing its secretion ability from Cercospora sp. JNU001, allowing to develop more potential applications of cercosporin.

Identification and characterization of Cercospora beticola necrosis-inducing effector CbNip1.

Cercospora beticola is a hemibiotrophic fungus that causes cercospora leaf spot disease of sugar beet (Beta vulgaris). After an initial symptomless biotrophic phase of colonization, necrotic lesions appear on host leaves as the fungus switches to a necrotrophic lifestyle. The phytotoxic secondary metabolite cercosporin has been shown to facilitate fungal virulence for several Cercospora spp. However, because cercosporin production and subsequent cercosporin-initiated formation of reactive oxygen species is light-dependent, cell death evocation by this toxin is only fully ensured during a period of light. Here, we report the discovery of the effector protein CbNip1 secreted by C. beticola that causes enhanced necrosis in the absence of light and, therefore, may complement light-dependent necrosis formation by cercosporin. Infiltration of CbNip1 protein into sugar beet leaves revealed that darkness is essential for full CbNip1-triggered necrosis, as light exposure delayed CbNip1-triggered host cell death. Gene expression analysis during host infection shows that CbNip1 expression is correlated with symptom development in planta. Targeted gene replacement of CbNip1 leads to a significant reduction in virulence, indicating the importance of CbNip1 during colonization. Analysis of 89 C. beticola genomes revealed that CbNip1 resides in a region that recently underwent a selective sweep, suggesting selection pressure exists to maintain a beneficial variant of the gene. Taken together, CbNip1 is a crucial effector during the C. beticola-sugar beet disease process.