Effect of Temperature and Relative Humidity on Growth of Aspergillus and Penicillium spp. and Biocontrol Activity of Pseudomonas protegens AS15 against Aflatoxigenic Aspergillus flavus in Stored Rice Grains

Abstract

In this study, we evaluated the effect of different temperatures (10, 20, 30, and 40 °C) and relative humidities (RHs; 12, 44, 76, and 98%) on populations of predominant grain fungi (Aspergillus candidus, Aspergillus flavus, Aspergillus fumigatus, Penicillium fellutanum, and Penicillium islandicum) and the biocontrol activity of Pseudomonas protegens AS15 against aflatoxigenic A. flavus KCCM 60330 in stored rice. Populations of all the tested fungi in inoculated rice grains were significantly enhanced by both increased temperature and RH. Multiple linear regression analysis revealed that one unit increase of temperature resulted in greater effects than that of RH on fungal populations. When rice grains were treated with P. protegens AS15 prior to inoculation with A. flavus KCCM 60330, fungal populations and aflatoxin production in the inoculated grains were significantly reduced compared with the grains untreated with strain AS15 regardless of temperature and RH (except 12% RH for fungal population). In addition, bacterial populations in grains were significantly enhanced with increasing temperature and RH, regardless of bacterial treatment. Higher bacterial populations were detected in biocontrol strain-treated grains than in untreated control grains. To our knowledge, this is the first report showing consistent biocontrol activity of P. protegens against A. flavus population and aflatoxin production in stored rice grains under various environmental conditions of temperature and RH.

Keywords: Aflatoxin; Aspergillus flavus; biological control; relative humidity; storage fungi; temperature.

Figure 1.

Effect of different temperatures (10, 20, 30, and 40 °C) and relative humidities (RHs; 12, 44, 76, and 98%) at equilibrium on fungal populations 2 weeks after inoculation of rice grains with (A) Aspergillus candidus AC317, (B) Aspergillus flavus AF57, (C) Aspergillus fumigatus AF8, (D) Penicillium fellutanum KU53, or (E) Penicillium islandicum KU101. Surface-sterilized rice grains were inoculated with 10⁶ fungal conidia/g dry weight of rice grains. Fungal populations were evaluated after 4 days of incubation on dichloran 18% glycerol agar amended with 50 mg/ml of chlortetracycline. Different lowercase and uppercase letters on bars (n = 6) are significantly different between different temperatures for a given RH and RHs for a given temperature, respectively, according to Tukey’s HSD test at p < .05. CFU: colony-forming unit.

Figure 2.

Effect of different temperatures (10, 20, 30, and 40 °C) and relative humidities (RHs; 12, 44, 76, and 98%) at equilibrium on (A) fungal population and (B) aflatoxin production in rice grains treated with (+) or without (−) the biocontrol strain, Pseudomonas protegens AS15, followed by inoculation with the aflatoxigenic strain, Aspergillus flavus KCCM 60330. Fungal population and total aflatoxin amount were assessed 2 weeks after inoculation with 10⁶ conidia/g dry weight of rice grains. Different lowercase and uppercase letters on bars with error bars (standard deviations, n = 6) indicate significant differences between different temperatures of rice grains treated with and without P. protegens AS15, respectively, according to Tukey’s HSD test at p < .05. Asterisks (*, **, and ***) on bars at a given temperature indicate significant differences between rice grains treated with or without P. protegens AS15 at p < .05, .01, and .001, respectively. ns, not significant; CFU, colony-forming unit.