Biological control of Aspergillus flavus growth and its aflatoxin b1 production by antagonistic yeasts

Abstract

The presence of mycotoxins in food and feed poses a risk to human health and animal productivity and result large economic losses. In the field and during storage period, Aspergillus flavus infect grains. Aflatoxin B1, is classified as group 1 carcinogen, having hepatotoxic, genotoxic, and teratogenic properties, may be produced by generally A. flavus and Aspergillus parasiticus in addition to grain deterioration and yield loss. Aflatoxins have been found in a variety of foods including oilseeds, nuts, dried figs and spices. Dried figs, pistachios, hazelnuts and groundnuts cultivated in Turkey are risky products in terms of aflatoxins. The application of synthetic fungicides is the most common method for controlling decay in most crops. However, because of the fungal resistance and detrimental impacts on human and animal health, as well as the environmental concerns in general, their use is being tried to diminished. Due to these concerns, researchers have tended to investigate more eco-friendly and healthy methods to manage fungal diseases. As a result, detecting and preventing Aspergillus species contamination, as well as lowering the level of aflatoxins in foodstuffs used in many agricultural products. To reduce usage of synthetic fungicide, biological control is an important strategy as a promising alternative with low environmental impact in reducing fungal infection and mycotoxin production in the field and during postharvest period. In addition, among microorganisms, yeast species have been extensively studied as antagonist due to simple nutritional requirements, able to colonize dry surfaces for long periods of time and able to grow rapidly in bioreactors with inexpensive substrates. Furthermore, yeast is simply adapted to microenvironments. The crucial and most important step is to develop biocontrol agent is isolation and screening of yeast isolates. Antagonistic yeasts have been showed several different mechanism to control of different moulds such as competition for space and nutrients, biofilm formation, parasitism, production of antimicrobial volatile organic compounds and production of lytic enzymes. The antagonist mechanism generally explained with cell wall-degrading enzyme synthesis including chitinases, β-1,3-glucanase, protease, cellulase and pectinase. Yeasts with high cell wall degrading enzyme activity, also showed high biocontrol efficacy. Biocontrol of aflatoxin has been generally documented by non-aflatoxigenic Aspergillus species. However, the studies to control Aspergillus flavus by yeasts limited. In various industrial processes, the Metschnikowia yeast has wide range of biotechnological application and generally isolated from fruits and flowers. Several Metschnikowia based biocontrol products have been industrialized to control postharvest diseases including Botrytis or Monilinia spp. However, only a few biocontrol agents are converted into industrial products. Meyerozyma, Moesziomyces and Metschnikowia sp. yeasts antifungal activity have been studied by several researchers. Among antagonistic microorganism mechanism of action, production of antimicrobial volatile organic compounds is one of the least studied. Primarily, volatile organic compounds produced by antagonistic yeasts have great potential used as biocontrol agents of filamentous fungi. In this study, four yeast isolates have been isolated and identified by different plant parts including hawthorn, hoşkıran, bean and grape leaf collected from Turkey different region. Four previously isolated and identified isolates from grapes, blueberry have been also included in this study. The eight antagonistic yeasts have been belong to Moesziomyces sp., Meyerozyma sp. and Metschnikowia sp. Yeasts secrete fungal lytic enzymes which are typically associated with biocontrol mechanism. Lytic enzyme activities of yeasts were examined with screening method. All of the isolates have β-glucosidase and chitinase activity, which are crucial for antifungal mechanism, however absence of the pectinase activity. Among antagonistic yeasts, only Metschnikowia pulcherrima (26-BMD) found as protease negative. Dual culture assay have been conducted to observe antagonistic effect of yeasts against aflatoxin B1 producer Aspergillus flavus. All of the antagonistic yeasts formed inhibition zones in dual culture assay against to Aspergillus flavus due to secretion of diffusible antifungal compounds. After that, yeasts antifungal and antiaflatoxigenic impact on aflatoxin producer Aspergillus flavus by spot inoculation method with different incubation period by in vitro studies. Different yeasts used to investigate to control Aspergillus flavus growth. In addition to that, origin of the yeast affect their biocontrol efficacy. All of the antagonistic yeasts formed inhibition zones in dual culture assay against to Aspergillus flavus due to secretion of diffusible antifungal compounds. While Aspergillus flavus mycelial growth of inhibition 86-97% after three days. All isolated and identified yeasts were effective to control Aspergillus flavus, as well as aflatoxin. Aflatoxin B1 production was reduced from 1773 ng/g (in control samples) to 1.26-10.15 ng/g with the application of antagonistic yeast. Metschnikowia aff. pulcherrima (32-AMM) was found as the most effective yeasts to inhibit mycelial growth of Aspergillus flavus among other yeasts. In addition, yeasts plant origin and incubation period also affect their inhibition potential (p<0.05). All of the yeasts might be used as biocontrol agent against Aspergillus flavus growth. Additionally, all of the yeast volatile organic compounds (VOCs) reduced sporulation however among antagonistic yeasts only Moesziomyces bullatus (DN-FY), Metschnikowia aff. pulcherrima (DN-MP) and Metschnikowia aff. pulcherrima (32-AMM) were reduced Aspergillus flavus mycelial growth with in vitro studies. But only VOCs produced by Metschnikowia aff. fructicola (1-UDM) was also found effective in reduction of Aflatoxin B1 production in in vitro experiments. This activity was associated to different volatile organic compounds. As a result, more investigation into the role of volatile organic compounds in Aspergillus flavus and aflatoxin B1 control is required. Further field experiments would indicate yeasts biocontrol potential on the products prone to contaminated from Aspergillus flavus. By the way, also isolation of volatile organic compounds from yeasts should be used to protect products from contamination without harmless to humans and environmentally friendly.