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.

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.

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.

Evaluation of Strains of Metarhizium anisopliae and Beauveria bassiana against Spodoptera litura on the Basis of Their Virulence, Germination Rate, Conidia Production, Radial Growth and Enzyme Activity.

Ten strains of the entomopathogenic fungi Metarhizium anisopliae and Beauveria bassiana were evaluated to find the most effective strain for optimization studies. The first criterion tested for strain selection was the mortality (> 50%) of Spodoptera litura larvae after inoculation of the fungus for 4 days. Results on several bioassays revealed that B. bassiana BNBCRC showed the most virulence on mortality S. litura larvae (80% mortality). B. bassiana BNBCRC also showed the highest germination rate (72.22%). However, its conidia yield (7.2 × 10(8) conidia/mL) was lower than those of B. bassiana B 14841 (8.3 × 10(8) conidia/mL) and M. anisopliae M6 (8.2 × 10(8) conidia/mL). The highest accumulative radial growth was obtained from the strain B14841 (37.10 mm/day) while the strain BNBCRC showed moderate radial growth (24.40 mm/day). M. anisopliae M6 possessed the highest protease activity (145.00 mU/mL) while M. anisopliae M8 possessed the highest chitinase activity (20.00 mU/mL) during 96~144 hr cultivation. Amongst these criteria, selection based on virulence and germination rate lead to the selection of B. bassiana BNBCRC. B. bassiana B14841 would be selected if based on growth rate while M. anisopliae M6 and M8 possessed the highest enzyme activities.