Insights into Antifungal Mechanisms of Bacillus velezensis S141 against Cercospora Leaf Spot in Mungbean (V. radiata).

Cercospora leaf spot (CLS) is caused by Cercospora canescens and is one of the most important diseases of mungbean (Vigna radiata). Cercospora leaf spot may result in economic loss in production areas. The present study investigated the potential of Bacillus velezensis S141 as a biocontrol agent for C. canescens PAK1 growth on culture plates. Cell-free secretions from a dual culture of S141+PAK1 inhibited fungal growth more than those from a single culture of S141. The biocontrol efficiency of S141 against Cercospora leaf spot on mungbean was then evaluated by spraying. The disease severity of Cercospora leaf spot was significantly reduced in plants treated with S141, with a control efficiency of 83% after 2 days of infection. Comparative transcriptomics and qRT-PCR ana-lyses of S141 during C. canescens inhibition were performed to elucidate the antifungal mechanisms underlying its antifungal activity against Cercospora leaf spot. According to the differentially expressed genes, most up-regulated genes involved in the biosynthetic genes encoding enzymatic hydrolases, including protease, ß-glucanase, and N-acyl glucosaminase, were detected in strain S141 following its interaction. Moreover, genes related to secondary metabolites (surfactin, bacilysin, and bacillomycin D) were up-regulated. Collectively, these results suggest that S141 exhibited strong antifungal activity against C. canescens due to multiple enzymatic hydrolases and secondary metabolites. Therefore, the present study provides insights into the biological network responsible for the antifungal activity of B. velezensis S141 against C. canescens.

A Secondary Metabolite of Cercospora sp., Associated with Rosa damascena Mill., Inhibits Proliferation, Biofilm Production, Ergosterol Synthesis and Other Virulence Factors in Candida albicans.

Here we describe the antimicrobial potential of secondary metabolites, fulvic acid (F.A.) and anhydrofulvic acid (AFA), produced by RDE147, an endophyte of Rosa damascena Mill. The endophyte was identified as Cercospora piaropi by ITS and ß-tubulin-based phylogenetic analyses, while chemoprofiling of the endophyte by column chromatography and spectroscopy yielded two pure compounds, F.A. and AFA. The compounds demonstrated different antimicrobial profiles, with AFA suppressing the growth of C. albicans at 7.3 µg ml-1 IC50. Further studies revealed that AFA strongly restricted the biofilm production and hyphae formation in C. albicans by down-regulating several biofilm and morphogenesis-related genes. The time-kill assays confirmed the fungicidal activity of AFA against C. albicans, killing 83.6% of the pathogen cells in 24 h at the MIC concentration, and the post-antibiotic effect (PAE) experiments established the suppression of C. albicans growth for extended time periods. The compound acted synergistically with amphotericin B and nystatin and reduced ergosterol biosynthesis by the pathogen, confirmed by ergosterol estimation and comparative expression profiling of selected genes and molecular docking of AFA with C. albicans squalene epoxidase. AFA also suppressed the expression of several other virulence genes of the fungal pathogen. The study determines the anti-C. albicans potential of AFA and its impact on the biology of the pathogen. It also indicates that Cercospora species may yield potential bioactive molecules, especially fulvic acid derivatives. However, it is imperative to conduct in vivo studies to explore this molecule's therapeutic potential further.

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.

Exploiting Potential of Trichoderma harzianum and Glomus versiforme in Mitigating Cercospora Leaf Spot Disease and Improving Cowpea Growth.

BACKGROUND AND OBJECTIVE: Trichoderma species are of utmost importance in agro-biotechnological applications because, in their interactions with plant hosts, they out-compete most pathogenic microorganisms. This study aimed at exploiting the potential of Trichoderma harzianum together with Glomus versiforme and its mutants, in inhibiting cowpea leaf spot rot induced due to Cercospora canescens infestation and improving agronomic growth parameter in a screen house experiment. MATERIALS AND METHODS: The experiment was designed using single and co-inoculation of the bioagents: in all, eleven treatments were applied, consisting of Glom_verwild, Glom_ver30, Glom_ver60, Glom_ver90, Trich_h, Glom_verwild+Trich_h, Glom_ver30+Trich_h, Glom_ver60+Trich_h, Glom_ver90+Trich_h, Pathogen alone and control. Cowpea growth yield parameters and disease severity were assessed after 7 weeks. RESULTS: The deployed treatments improved agronomic growth parameters substantially (p<0.05) relative to control. Glom_ver 60+Trich_h treatment exerted the highest agronomic growth improvement yield. In addition, the best reduction in the incidence and severity of cowpea leaf spot disease was obtained using Glom_ver 60+Trich_h. A significantly higher germination rate in seeding, confirms both inhibitory and growth improvement potency of the bio inoculants treatment. CONCLUSION: This study's findings confirmed the beneficial impacts of the treatment of seed and soil with dual T. harzianum and G. versiforme, in improving the immunity of cowpea to Cercospora canescens leaf spot infection and improve cowpea growth.

Comparative transcriptome analysis of two Cercospora sojina strains reveals differences in virulence under nitrogen starvation stress.

BACKGROUND: Cercospora sojina is a fungal pathogen that causes frogeye leaf spot in soybean-producing regions, leading to severe yield losses worldwide. It exhibits variations in virulence due to race differentiation between strains. However, the candidate virulence-related genes are unknown because the infection process is slow, making it difficult to collect transcriptome samples. RESULTS: In this study, virulence-related differentially expressed genes (DEGs) were obtained from the highly virulent Race 15 strain and mildly virulent Race1 strain under nitrogen starvation stress, which mimics the physiology of the pathogen during infection. Weighted gene co-expression network analysis (WGCNA) was then used to find co-expressed gene modules and assess the relationship between gene networks and phenotypes. Upon comparison of the transcriptomic differences in virulence between the strains, a total of 378 and 124 DEGs were upregulated, while 294 and 220 were downregulated in Race 1 and Race 15, respectively. Annotation of these DEGs revealed that many were associated with virulence differences, including scytalone dehydratase, 1,3,8-trihydroxynaphthalene reductase, and ß-1,3-glucanase. In addition, two modules highly correlated with the highly virulent strain Race 15 and 36 virulence-related DEGs were found to contain mostly ß-1,4-glucanase, ß-1,4-xylanas, and cellobiose dehydrogenase. CONCLUSIONS: These important nitrogen starvation-responsive DEGs are frequently involved in the synthesis of melanin, polyphosphate storage in the vacuole, lignocellulose degradation, and cellulose degradation during fungal development and differentiation. Transcriptome analysis indicated unique gene expression patterns, providing further insight into pathogenesis.