Fungichromin Production by Streptomyces padanus PMS-702 for Controlling Cucumber Downy Mildew

Abstract

Streptomyces padanus PMS-702 strain produces a polyene macrolide antibiotic fungichromin and displays antagonistic activities against many phytopathogenic fungi. In the present study, experimental formulations were assessed to improve the production of fungichromin, the efficacy of PMS-702 on the suppression of sporangial germination, and the reduction of cucumber downy mildew caused by Pseudoperonospora cubensis. PMS-702 strain cultured in a soybean meal-glucose (SMG) medium led to low levels of fungichromin accumulation and sporangial germination suppression. Increasing medium compositions and adding plant oils (noticeably coconut oil) in SMG significantly increased fungichromin production from 68 to $1,999.6{\mu}g/ml$. Microscopic examination reveals that the resultant suspensions significantly reduced sporangial germination and caused cytoplasmic aggregation. Greenhouse trials reveal that the application of PMS-702 cultural suspensions reduced downy mildew severity considerably. The addition of Tween 80 into the synthetic medium while culturing PMS-702 further increased the suppressive efficacy of downy mildew severity, particularly when applied at 24 h before inoculation or co-applied with inoculum. Fungichromin at $50{\mu}g/ml$ induced phytotoxicity showing minor necrosis surrounded with light yellowish halos on cucumber leaves. The concentration that leads to 90% inhibition (IC90) of sporangial germination was estimated to be around $10{\mu}g/ml$. The results provide a strong possibility of using the S. padanus PMS-702 strain as a biocontrol agent to control other plant pathogens.

Fig. 1. Efficacy of cultural suspensions of S. padanus PMS-702 on the germination of sporangia produced by P. cubensis. (A) Dilution effects on sporangial germination. PMS-702 was cultured in soybean meal-glucose (SMG) broth for 3, 5 or 7 days. Cultural suspensions were mixed (1:1, v/v) with sporangia (10⁴ sporangia/ml) to make 10×, 20× or 50× solution, and incubated at 20℃ in the dark for 2 h. Mock controls were treated with SMG only. (B) Effects of plant oils (1%) on sporangial germination. (C) Concentration effects of corn oil and coconut oil on sporangial germination. PMS-702 was cultured in SMG amended with or without oil for 5 days and mixed with sporangia. Cultural suspensions at a 100-fold dilution were mixed with sporangia. In each treatment, 10 μl of suspensions was placed on a glass depression slide and observed microscopically. For each treatment, 50 randomly selected sporangia were examined. Data are means of three biological replicates. Means indicated by different letters were significantly different according to the Fisher’s least significance difference test (P = 0.05).

Fig. 2. Sticky substances formed by S. padanus PMS-702 increase the suppressive efficacy of germination of sporangia produced by P. cubensis. (A) In-vitro assays of sporangial germination. PMS-702 was cultured in soybean meal-glucose (SMG), SMG amended with 1% coconut oil (designated SMG-C-1), or SMG-C-1 containing sticky substances (SMG-C-2) for 5 days. Cultural suspensions were mixed (1:1, v/v) with sporangia (10⁴ sporangia/ml) to make 100× or 200× solution, and incubated at 20℃ in the dark for 2 h. (B) In-planta assays of sporangial propagation on cucumber leaves. Spornagia treated with water (mock control) propagate quickly on cucumber leaves incrreasing 12-fold after a 5-day incubation. Sporangia treated with PMS-702 suspensions cultured in SMG-C-2 (200×) propagate much slower than mock control after 5 dai. Data are means of three biological replicates. Means indicated by different letters were significantly different according to the Fisher’s least significance difference test (P = 0.05). (C) Microscopic images of a sporangium treated with PMS-702 in SMG-C-2 for 24 h and stained with cotton blue, showing cytoplasmic aggregation (indicated by an arrow). (D) A germinating zoospore after released from a sporangium treated with water. Only reprenestatives are shown.

Fig. 3. Improvement of fungichromin production by S. padanus PMS-702 through different formulations. PMS-702 was cultured in soybean meal-glucose (SMG) or modified media (see below for details) as indicated at 30℃ for 5 days. Fungichromin was extracted from cultural filtrates with ethyl acetate, separated by HPLC, and quantified using a regression line generated from a commercially available standard. Chemical structure of fungichromin is also shown. SMG: 0.5% soybean meal, 0.5% glucose and 0.04% CaCO₃ SMG-S: SMG + sticky substances SMG-M: 1.12% soybean meal, 1.12% glucose and 0.046% CaCO₃ SMG-C-2: SMG-S + 1% coconut oil SMG-M-S-C: SMG-M + sticky substances + 1% coconut oil

Fig. 4. Tween 80 improves the efficacy of S. padanus PMS-702 in reducing downy mildew severity on cucumber. (A) Detached leaf assays. (B) Whole plant assays. PMS-702 was cultured in soybean meal-glucose amended with 1% coconut oil and sticky substances (SMG-C-2) amended with different concentrations of Tween 80 for 5 days and sprayed onto detached cucumber leaves, which were immediately inoculated with sporangial suspensions (10⁴ sporangia/ml) of P. cubensis. Leaves treated with water were used as mock controls. The treated leaves were kept in a moist petri dish and the whole plants were bagged for 2 days and maintained in a greenhouse after bag removal. Downy mildew severity was examined 7 dai. Data are means of three biological replicates and treatment means separated by nonlinear regression.

Fig. 5. Spray timing of cell suspensions of S. padanus PMS-702 for controlling downy mildew severity on cucumber leaves. (A) Cucumber leaves showing downy mildew symptoms after treated with PMS-702 suspensions cultured in SMG-C-2 amended with Twee 80 or water (control). (B) Quantification of downy mildew severity on cucumber leaves. PMS-702 was cultured in soybean meal-glucose amended with 1% coconut oil and sticky substances (SMG-C-2), with or without 2% Tween 80 for 5 days and sprayed onto 3-week-old cucumber seedlings 24 h before inoculation (hbi), post inoculation (hpi) or co-application (0 hbi) with sporangial suspensions (10⁴ sporangia/ml) of P. cubensis. Plants sprayed with water were used as mock controls. The treated plants were bagged for 2 days and maintained in a greenhouse after bag removal. Downy mildew severity was examined 7 dai. Data are means of three biological replicates. Means indicated by different letters were significantly different according to the Fisher’s least significance difference test (P = 0.05).

Fig. 6. Phytotoxicity of fungichromin on cucumber leaves and its effects on sporangial germination and downy mildew severity. (A) Phytotoxicity of fungichromin on detached cucumber leaves. (B) Fungichromin increases the inhibitory percentage of sporangial germination and decreases downy mildew severity on cucumber leaves. Fungichromin was dissolved in DMSO to make different concentrations and 10 μl of each was placed onto cucumber leaves. To test the effect of fungichromin on downy mildew severity, cucumber leaves were sprayed with fungichromin and inoculated with sporangia. The treated leaves or plants were incubated in a moist incubator. Disease incidence was recorded 7 dai. For sporangial germination, sporangia (10⁴ sporangia/ml) were mixed with fungichromin and incubated at 20℃ in the dark. Sporangia treated with DMSO were used as mock controls. After 2-h incubation, 50 randomly selected sporangia from each of the treatments were examined microscopically.