Antifungal and antiaflatoxigenic mechanism activity of freeze-dried culture filtrate of Streptomyces philanthi RL-1-178 on the two aflatoxigenic fungi and identification of its active component.

AIMS: The study reports the antifungal and antiaflatoxigenic mechanism activity of freeze-dried culture filtrate of Streptomyces philanthi RL-1-178 (DCF RL-1-178) against two aflatoxigenic strains (Aspergillus parasiticus and A. flavus) and identification of its active component. METHODS AND RESULTS: Significant inhibition in ergosterol biosynthesis by the DCF RL-1-178 appeared on the plasma membrane. Moreover, the DCF RL-1-178 showed dose-dependent inhibition of methylglyoxal (MG) (an aflatoxin inducer) biosynthesis and exhibited a novel antiaflatoxigenic action mechanism. Significant impairments in enzymatic [superoxide dismutase (SOD) and catalase (CAT)] and nonenzymatic [oxidized and reduced glutathione (GSH) and ratio of oxidized and reduced glutathione (GSSG)] anti-oxidative defense molecules were observed in the two aflatoxigenic cells. The active component of the DCF RL-1-178 was identified as natamycin. The natamycin exhibited against A. parasiticus and A. flavus with the minimum inhibitory concentration (MIC) values of 0.5 and 1.0 µg ml-1, respectively, while the minimum fungicidal concentration values were the same (4.0 µg ml-1). CONCLUSIONS: The DCF RL-1-178 containing natamycin exhibited the following effects: (1) inhibition of cellular ergosterol biosynthesis on plasma membrane, (2) reduction in MG (aflatoxin inducer) confirmed novel antiaflatoxigenic mechanism of action, and (3) caused remarkable debasement in antioxidant defense enzymes (SOD and CAT) and nonenzymatic defense molecules (GSH and GSSG) revealing biochemical mechanism of action.

Application of antifungal metabolites from Streptomyces philanthi RL-1-178 for maize grain coating formulations and their efficacy as biofungicide during storage.

The major safety risk of maize grain is contamination with mycotoxins. In this study, a maize-coating formulation containing freeze-dried culture filtrate of Streptomyces philanthi RL-1-178 (DCF RL-1-178) was developed and evaluated to prevent the growth of mycotoxins during maize grain storage. In vitro studies using confrontation tests on PDA plates indicated that S. philanthi RL-1-178 inhibited the growth of Aspergillus parasiticus TISTR 3276 (89.0%) and A. flavus PSRDC-4 (95.0%). The maize grain coating formulations containing the DCF RL-1-178 (0, 5, 10, and 15% (v/v)) and the polymer polyvinylpyrrolidone (PVP-K90, 4.0% (w/v)) were tested for their efficacy in In vitro and during 5 months storage. In In vitro assay, maize coating formular containing the optimum concentration (15.0%, v/v) of the DCF RL-1-178 exhibited 54.80% and 54.17% inhibition on the growth of A. parasiticus TISTR 3276 and A. flavus PSRDC-4 respectively. The inhibition was also illustrated by the microstructures of interactions between the coated maize grains with or without the DCF RL-1-178 and the fungal pathogens observed under microscope and SEM. Incorporating the DCF RL-1-178 or fungicidal Metalaxyl® into the polymer PVP-K90 maize grains coating resulted in the complete inhibition of the production of aflatoxin B1 (analysed by HPLC) by the two aflatoxigenic pathogens after 5 months storage at room temperature. However, the shelf-life was shortened to only 3 months during storage at room temperature with 90% relative humidity. Overall, the application of the 10-15% DCF RL-1-178 into the maize grain coating formular provides a new alternative measure to control the mycotoxins during storage for at least 5 months. The In vitro cell cytotoxicity study showed that a concentration of 15% (v/v) or 1000 µg/mL of the DCF RL-1-178 had a strong cytotoxic effect on Vero cells. These findings indicate that DCF RL-1-178 is a potential biofungicide for controlling mycotoxins contamination in maize seed storage for planting, but not maize grain storage for animal feed.

Utilization of palm oil mill effluent as a novel substrate for the production of antifungal compounds by Streptomyces philanthi RM-1-138 and evaluation of its efficacy in suppression of three strains of oil palm pathogen.

AIMS: This study aimed to use palm oil mill effluent (POME) as a renewable resource for the production of antifungal compounds by Streptomyces philanthi RM-1-138 against Ganoderma boninense, Ceratocystis paradoxa and Curvularia oryzae. METHODS AND RESULTS: The efficacy of antifungal compounds RM-1-138 against the three strains of fungal oil palm pathogen was evaluated both in vitro and on oil palm leaf segments. In vitro studies using confrontation tests on glucose yeast-malt extract (GYM) agar plates indicated that the strain RM-1-138 inhibited the growth of all three fungal pathogenic strains. The antifungal compounds produced in the GYM medium exhibited significantly higher inhibition (79%-100%) against the three fungal pathogens than using the diluted POME (50%) medium (80%-83% inhibition). The optimum condition for the production of antifungal compounds from the strain RM-1-138 was as following: POME of 47,966 mg L-1 chemical oxygen demand (COD), the initial pH at 7.0 and supplemented with yeast extract (0.4%). Meanwhile, severe morphological and internal abnormalities in C. oryzae hyphae were observed under a scanning electron microscope and transmission electron microscope. In vivo experiment on oil palm leaf segments indicated that the efficacy of the antifungal compounds RM-1-138 (DSI = 1.3) were not significantly difference in the suppression of Curvularia leaf spot compared with the two commercial chemical fungicides of mancozeb® (DSI = 1.0) and tetraconazole® (DSI = 1.3). CONCLUSIONS: Antifungal compounds produced by S. philanthi RM-1-138 grown in POME have the potential to inhibit fungal pathogens. SIGNIFICANCE AND IMPACT OF THE STUDY: The POME (about 47 mg L-1 COD) with the initial pH of 7.0 and supplementation of 0.4% nitrogen could be used as a culture medium for the growth and production of antifungal compounds of S. philanthi RL-1-138. In addition, the antifungal compound RM-1-138 could suppress the three strains of oil palm fungal pathogen tested on oil palm leaf segment.

Efficacy of the antifungal metabolites of Streptomyces philanthi RL-1-178 on aflatoxin degradation with its application to prevent aflatoxigenic fungi in stored maize grains and identification of the bioactive compound.

Aflatoxin B1 is a potent carcinogen produced by Aspergillus flavus (A. flavus) and Aspergillus. parasiticus (A. parasiticus), mainly during grain storage. The efficacy of the freeze-dried culture filtrate of Streptomyces philanthi (S. philanthi) strain RL-1-178 (DCF) on degradation of aflatoxin B1 (AFB1) were evaluated and its bioactive compounds were identified. The DCF at a concentration of 9.0% (w/v) completely inhibited growth and AFB1 production of A. parasiticus TISTR 3276 and A. flavus PSRDC-4 after 7 days tested in yeast-extract sucrose (YES) medium and on stored maize grains after 28 and 14 days incubation, respectively. This indicated the more tolerance of A. parasiticus over A. flavus. The DCF and bacterial cells of S. philanthi were capable to degrade AFB1 by 85.0% and 100% for 72 h and 8 days, respectively. This confirmed the higher efficacy of the DCF over the cells. After separation of the DCF on thin-layer chromatography (TLC) plate by bioautography bioassay, each active band was identified by liquid chromatography-quadrupole time of flight mass spectrometer (LC-Q-TOF MS/MS). The results revealed two compounds which were identified as azithromycin and an unknown based on mass ions of both ESI+ and ESI- modes. The antifungal metabolites in the culture filtrate of S. philanthi were proved to degrade aflatoxin B1. It could be concluded that the DCF may be applied to prevent the growth of the two aflatoxin-producing fungi as well as the occurrence of aflatoxin in the stored maize grains.

Evolutionary stability of antibiotic protection in a defensive symbiosis.

The increasing resistance of human pathogens severely limits the efficacy of antibiotics in medicine, yet many animals, including solitary beewolf wasps, successfully engage in defensive alliances with antibiotic-producing bacteria for millions of years. Here, we report on the in situ production of 49 derivatives belonging to three antibiotic compound classes (45 piericidin derivatives, 3 streptochlorin derivatives, and nigericin) by the symbionts of 25 beewolf host species and subspecies, spanning 68 million years of evolution. Despite a high degree of qualitative stability in the antibiotic mixture, we found consistent quantitative differences between species and across geographic localities, presumably reflecting adaptations to combat local pathogen communities. Antimicrobial bioassays with the three main components and in silico predictions based on the structure and specificity in polyketide synthase domains of the piericidin biosynthesis gene cluster yield insights into the mechanistic basis and ecoevolutionary implications of producing a complex mixture of antimicrobial compounds in a natural setting.

Efficacy of volatile compounds from Streptomyces philanthi RL-1-178 as a biofumigant for controlling growth and aflatoxin production of the two aflatoxin-producing fungi on stored soybean seeds.

AIMS: This study aimed to apply the volatile organic compounds from Streptomyces philanthi RL-1-178 (VOCs RL-1-178) as a fumigant to protect soybean seeds against the two aflatoxin-producing fungi in stored soybean seeds. METHODS AND RESULTS: The antifungal bioassay tests on potato dextrose agar (PDA) dishes showed that 30 g l-1 wheat seed inoculum of S. philanthi RL-1-178 exhibited total (100%) inhibition on Aspergillus parasiticus TISTR 3276 and Aspergillus flavus PSRDC-4. Identification of the VOCs RL-1-178 using GC-MS revealed 39 compounds with the most abundant substances being geosmin (13·75%) followed by l-linalool (13·55%), 2-mercaptoethanol (9·71%) and heneicosane (5·96%). Comparison on the efficacy of the VOCs RL-1-178 (at 30 g l-1 wheat seed culture) and their four major components (100 µl l-1 each) on the suppression of the two aflatoxin-producing fungi on PDA plates revealed that the VOCs RL-1-178 as well as geosmin, l-linalool and 2-mercaptoethanol completely inhibited (100%) mycelial growth while heneicosane showed only 70·7% inhibition. Use of the VOCs RL-1-178 (30 g l-1 ) as a biofumigant on stored soybean seeds resulted in complete protection (100%) against the infection as well as complete inhibition on production of aflatoxin (B1 , B2 and G2 ) (analysed by HPLC) by the two aflatoxin-producing fungi. CONCLUSIONS: The VOCs RL-1-178 displayed strong inhibitory effects on A. parasiticus TISTR 3276 and A. flavus PSRDC-4 as well as inhibited aflatoxin (B1 , B2 and G2 ) production. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings suggest that the VOCs RL-1-178 can be applied as a biofumigant to control the two aflatoxin-producing fungi on stored seeds products.