Harnessing Rimocidins-Producing Streptomyces sp. JCK-6116 as a Sustainable Fungicide for Biocontrol of Cucumber Soil-Borne Diseases

Abstract

Fusarium oxysporum f. sp. cucumerinum, the causal agent of cucumber fusarium wilt, along with Rhizoctonia solani AG-4 and Pythium ultimum-that causes cucumber damping-off-are soil-borne fungal and Oomycetes pathogens responsible for significant economic losses in agriculture. In this study, the culture filtrate of Streptomyces sp. JCK-6116, isolated from soil, exhibited strong inhibitory activity against the mycelial growth of multiple phytopathogenic fungi in a 96-well microtiter plate assay. In the dual culture assay, JCK-6116 inhibited the growth of 20 species of plant pathogenic fungi and Oomycetes, suggesting a wide antifungal spectrum. Three active compounds-rimocidin A, B, and C-were isolated from JCK-6116 and identified. These rimocidins exhibited antifungal effects against fungi by binding to ergosterol in the fungal membrane. However, none of the compounds exhibited anti-oomycete activity against the tested Oomycetes strains. Among the three compounds, rimocidin A demonstrated the strongest antifungal activity with minimum inhibitory concentration values ranging from 1.25-10 μg/ml. Furthermore, the culture broth of JCK-6116, at 10-fold dilution, effectively suppressed fusarium wilt and the two damping-off diseases in cucumber. Its butanol extract was also effective against the two fungal diseases but showed no activity against P. ultimum damping-off disease. These findings indicate that the culture broth contains metabolites with anti-oomycete activity. This study demonstrates that Streptomyces sp. JCK-6116 has significant potential as a biological control agent for managing soil-borne diseases caused by fungi and Oomycetes.

Keywords: Antifungal activity; Streptomyces; biocontrol; damping-off disease; fusarium wilt; rimocidins.

Fig. 1. Growth and morphology of Streptomyces sp. JCK-6116 on ISP2 agar.

(A) Top view and (B) bottom view of colonies grown on TSA plates for 7 days at 28°C; (C) Neighbor-joining phylogenetic tree based on 16S gene sequence of strain JCK-6116 and related Streptomyces species. Arthrobacter globiformis NBRC 12137^T was used as the outgroup. Bootstrap values (≥ 50%) from 1,000 replications are indicated at branch nodes. The scale bar indicates the number of nucleotide substitutions per site. TSA, tryptic soy agar.

Fig. 2. Antifungal activity of Streptomyces sp. JCK-6116 against 20 phytopathogenic fungi and Oomycetes on (A) dual culture assay and (B) percentage of pathogen growth inhibition.

Fig. 3. LC-ESI-TOF-MS spectra in negative and positive ion mode of rimocidin C (A-B), rimocidin B (C-D), rimocidin A (E-F) and chemical structure of rimocidins (G).

Fig. 4. Disease control efficacy of culture broth and crude extract against plant diseases.

(A–B) Fusarium wilt caused by Fusarium oxysporum f. sp. cucummerinum, (C–D) Damping-off caused by Rhizoctonia solani, (E–F) Damping-off caused by Pythium ultimum. CB: Culture broth tested at 10- and 100-fold dilutions. Bu: Butanol extract tested at 50 and 300 μg/ml. Commercial fungicides Kajiran and Jalrokend served as positive controls. Water-treated plants served as untreated controls.