Biocontrol Activity of Volatile-Producing Bacillus megaterium and Pseudomonas protegens Against Aspergillus and Penicillium spp. Predominant in Stored Rice Grains: Study II

In our previous studies, Bacillus megaterium KU143, Microbacterium testaceum KU313, and Pseudomonas protegens AS15 have been shown to be antagonistic to Aspergillus flavus in stored rice grains. In this study, the biocontrol activities of these strains were evaluated against Aspergillus candidus, Aspergillus fumigatus, Penicillium fellutanum, and Penicillium islandicum, which are predominant in stored rice grains. In vitro and in vivo antifungal activities of the bacterial strains were evaluated against the fungi on media and rice grains, respectively. The antifungal activities of the volatiles produced by the strains against fungal development and population were also tested using I-plates. In in vitro tests, the strains produced secondary metabolites capable of reducing conidial germination, germ-tube elongation, and mycelial growth of all the tested fungi. In in vivo tests, the strains significantly inhibited the fungal growth in rice grains. Additionally, in I-plate tests, strains KU143 and AS15 produced volatiles that significantly inhibited not only mycelial growth, sporulation, and conidial germination of the fungi on media but also fungal populations on rice grains. GC-MS analysis of the volatiles by strains KU143 and AS15 identified 12 and 17 compounds, respectively. Among these, the antifungal compound, 5-methyl-2-phenyl-1H-indole, was produced by strain KU143 and the antimicrobial compounds, 2-butyl 1-octanal, dimethyl disulfide, 2-isopropyl-5-methyl-1-heptanol, and 4-trifluoroacetoxyhexadecane, were produced by strain AS15. These results suggest that the tested strains producing extracellular metabolites and/or volatiles may have a broad spectrum of antifungal activities against the grain fungi. In particular, B. megaterium KU143 and P. protegens AS15 may be potential biocontrol agents against Aspergillus and Penicillium spp. during rice grain storage.

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

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.

Biocontrol Activity of Volatile-Producing Bacillus megaterium and Pseudomonas protegens against Aspergillus flavus and Aflatoxin Production on Stored Rice Grains

In our previous study, three bacterial strains, Bacillus megaterium KU143, Microbacterium testaceum KU313, and Pseudomonas protegens AS15, were selected as effective biocontrol agents against Aspergillus flavus on stored rice grains. In this study, we evaluated the inhibitory effects of the volatiles produced by the strains on A. flavus growth and aflatoxin production on stored rice grains. The three strains significantly reduced mycelial growth of A. flavus in dual-culture assays compared with the negative control strain, Sphingomonas aquatilis KU408, and an untreated control. Of these tested strains, volatiles produced by B. megaterium KU143 and P. protegens AS15 markedly inhibited mycelial growth, sporulation, and conidial germination of A. flavus on agar medium and suppressed the fungal populations in rice grains. Moreover, volatiles produced by these two strains significantly reduced aflatoxin production in the rice grains by A. flavus. To our knowledge, this is the first report of the suppression of A. flavus aflatoxin production in rice grains using B. megaterium and P. protegens volatiles.

Influence of Temperature and Water Activity on Deleterious Fungi and Mycotoxin Production during Grain Storage

Cereal grains are the most important food source for humans. As the global population continues to grow exponentially, the need for the enhanced yield and minimal loss of agricultural crops, mainly cereal grains, is increasing. In general, harvested grains are stored for specific time periods to guarantee their continuous supply throughout the year. During storage, economic losses due to reduction in quality and quantity of grains can become very significant. Grain loss is usually the result of its deterioration due to fungal contamination that can occur from preharvest to postharvest stages. The deleterious fungi can be classified based on predominance at different stages of crop growth and harvest that are affected by environmental factors such as water activity (a(w)) and eco-physiological requirements. These fungi include species such as those belonging to the genera Aspergillus and Penicillium that can produce mycotoxins harmful to animals and humans. The grain type and condition, environment, and biological factors can also influence the occurrence and predominance of mycotoxigenic fungi in stored grains. The main environmental factors influencing grain fungi and mycotoxins are temperature and a(w). This review discusses the effects of temperature and a(w) on fungal growth and mycotoxin production in stored grains. The focus is on the occurrence and optimum and minimum growth requirements for grain fungi and mycotoxin production. The environmental influence on aflatoxin production and hypothesized mechanisms of its molecular suppression in response to environmental changes are also discussed. In addition, the use of controlled or modified atmosphere as an environmentally safe alternative to harmful agricultural chemicals is discussed and recommended future research issues are highlighted.

Microbe-Mediated Control of Mycotoxigenic Grain Fungi in Stored Rice with Focus on Aflatoxin Biodegradation and Biosynthesis Inhibition

Rice contaminated with fungal species during storage is not only of poor quality and low economic value, but may also have harmful effects on human and animal health. The predominant fungal species isolated from rice grains during storage belong to the genera Aspergillus and Penicillium. Some of these fungal species produce mycotoxins; they are responsible for adverse health effects in humans and animals, particularly Aspergillus flavus, which produces the extremely carcinogenic aflatoxins. Not surprisingly, there have been numerous attempts to devise safety procedure for the control of such harmful fungi and production of mycotoxins, including aflatoxins. This review provides information about fungal and mycotoxin contamination of stored rice grains, and microbe-based (biological) strategies to control grain fungi and mycotoxins. The latter will include information regarding attempts undertaken for mycotoxin (especially aflatoxin) bio-detoxification and microbial interference with the aflatoxin-biosynthetic pathway in the toxin-producing fungi.