Introduction

In South Korea, Protaetia brevitarsis (Sacarbaeidae) larvae are produced both as an alternative source of protein and as a functional food and animal feed. However, during the mass production of P. brevitarsis larvae, inappropriate control of temperature and humidity in breeding facilities can result in larval death, as a result of infection by various fungi, including species of Metarhizium and Beauveria. Accordingly, various studies have investigated the use of beneficial microorganisms to prevent such infections. One study demonstrated that the growth of Beauveria bassiana is inhibited by the yeast Saccharomyces cerevisiae (Santoro et al., 2008). B. bassiana is an anamorphic entomopathogenic fungus, often found in soil, that causes white muscardine disease and is widely used as a mycoinsecticide for pest control (Meyling and Eilenberg, 2007), owing to its ability to produce toxins, such as mycotoxin and beauvericin (Genthner et al., 1994). The infection of insect larvae by B. bassiana occurs via the mouth and cuticle and is followed by fungal proliferation, hardening of the host body, and dispersal of spores from the host epidermis. In general, beneficial microorganisms are microbial biocontrol agents that have the ability to inhibit fungal infections by secreting organic acids and antimicrobial compounds (Shetty et al., 2006, Larsen et al., 2015). For example, yeast is an active antagonist of filamentous spoilage fungi and can absorb aflatoxins (B1, B2, G1, and G2), which are a type of mycotoxin (Armando et al., 2012). Specifically, S. cerevisiae can control to product aflatoxin B1 occurrence in vitro via temperature, pH, and oxygen (Armando et al., 2012, Chulze et al., 2015), and both Pichia anomala and S. cerevisiae have been reported to inhibit the growth of fungal wheat pathogens that produce ochratoxin A and aflatoxin (Petersson et al., 1998, Petruzzi et al., 2015). In addition, S. cerevisiae is generally recognized as safe and is commonly used in baking and brewing (Petersson et al., 1998, Campagnollo et al., 2015). Therefore, the species could potentially improve the safety of P. brevitarsis production by inhibiting the growth of toxin-producing fungal pathogens.

Materials and Methods

P. brevitarsis and B. bassiana culture conditions

Healthy P. brevitarsis were bred in a 35 cm × 25 cm enclosure with wet sawdust as a food source, a temperature of 24 ± 2 ℃, relative humidity of 60 ± 5%, and photoperiod of 16:8 (h light:h dark). Meanwhile, B. bassiana (KACC40039) was obtained from the Korean Agricultural Culture Collection (KACC, Rural Development Administration, Wanju, Korea) and cultured on potato dextrose agar (PDA; BD, Franklin Lakes, NJ, USA) at 24 ℃ for 14 d.

Antifungal activity of Effective Microorganisms

Bacillus subtilis (KACC17047), Lactobacillus plantarum (KACC10552), and Saccharomyces cerevisiae (KACC30068) were obtained from the KACC and cultured in liquid MRS, R2A, and YEPD media, respectively, at 30 ℃. Subsequently, 100 μL S. cerevisiae culture (1 × 107 cfu/mL) was inoculated onto 20 mL PDA in sterile Petri dishes, and B. bassiana was seeded with a sterile steel borer by punching fungus into the center of the plate with a diameter of 5 mm and incubated at 24℃. Antifungal activity was measured after 30 d. Each condition (Negative and Positive Controls and EM treated) was performed in triplicate. For a negative control, triple distilled water spread over the entire PDA medium, which was followed by B. bassiana seeding. For a positive control, 1.2% sodium hypochlorite was diluted by 10-fold with triple distilled water and spread over the entire surface PDA medium, which was followed by B. bassiana seeding. Meanwhile, L. plantarum and S. cerevisiae were cultured at 37 ℃ for 8 h, and 100 μL of the supernatants (1 × 107 cfu/mL) of the cultured media were spread in PDA medium, which was followed by seeding with B. bassiana. At 30 d after seeding with B. bassiana, the radius of the fungal colonies were measured, and we calculated the percent growth inhibition according to the agar well diffusion assay method described by Grover and Moore (1962), using the following equation: MGI (Inhibition of mycelial growth) = (DC-DT)/DC × 100%, DC = diameter of control, DT = diameter of test (Padency et al., 1982). The statistical analysis was conducted using SPSS (Statistical Package for the Social Sciences, USA). The B. bassiana mycelia growth was analyzed by ANOVA (analysis of variance).

Effects of S. cerevisiae injection on the mortality of third instar P. brevitarsis

Third instar P. brevitarsis larvae were fasted for 72 h, and then each larva was injected with 50 μL of either S. cerevisiae (1 × 107 cfu/50 μL); triple-distilled water, as a negative control; or YPD broth, as an another positive control, using a BD ultrafine insulin syringe (31 gauge, 6 mm). Ten larvae were included in each group, and each treatment was performed in triplicate. After injection, specimens were fed with fermented sawdust, and mortality was assessed at 7 d after injection.

Effect of S. cerevisiae consumption on the mortality of percutaneously infected second instar P. brevitarsis larvae

Second instar P. brevitarsis larvae were individually fed 20 g S. cerevisiae-inoculated sawdust (100 mL S. cerevisiae [1 × 107 cfu/mL], 300 g autoclaved sawdust) in Petri dishes for 14 d. Next, a 1:3 weight mixture of yeast and fermented sawdust was fed to the P. brevitarsis larvae for 14 d. In addition, B. bassiana was diluted to 1 × 109 cfu/10 mL triple-distilled water and 50 μL Tween20/Tween80, and the resulting solution was inoculated onto second instar P. brevitarsis larvae that had fasted for 24 h. The larvae were inoculated twice, with a 2-h interval, and the larvae were also inoculated a third time at 24 h after the second inoculation. The experiment was performed in triplicate, and 10 larvae were used for each repetition. Following inoculation with B. bassiana, the humidity inside the breeding cage was increased to promote fungal growth, and the mortality of the P. brevitarsis larvae was measured every 5 d.

Effect of S. cerevisiae consumption on B. bassiana infection rate

Ten larvae of Second instar P. brevitarsis were reared in an airtight cage for 20 d and fed a mixture of 100 mL S. cerevisiae (1 × 105 cfu/mL) and 300 g fermented sawdust. After that, larvae that had previously been killed by B. bassiana were ground with a mortar and pestle, and 0.3 g of the powder was mixed with 30 g of fermented sawdust. The mixture was then fed 10 s instar P. brevitarsis larvae and one larva that treated the B. bassiana were reared in petri dish (10cm diameter) respectively. The mortality of the larvae was measured after 10 d. The experiment was performed in triplicate.

Results and Discussion

Although P. brevitarsis larvae are infected by B. bassiana less frequently than by Metarhizium anisopliae, B. bassiana still an important pathogen; however, previous studies have shown that it can be prevented using beneficial microorganisms. In the present study, we found that S. cerevisiae was the most effective at inhibiting B. bassiana growth in vitro (Fig 1) and that the microorganism reduced the mortality of B. bassiana-fed P. brevitarsis larvae. S. cerevisiae is almost harmless to humans and is used in the fermentation of various food products, including bread, wine, and beer, as well as in the production of edible insects, owing to its antifungal activity, especially against B. bassiana. The mean radius of B. bassiana colonies in the control group (Table 1), which did not receive any beneficial microorganisms, was 5 cm, whereas that of the S. cerevisiae treatment group was 0.25 ± 0.1 cm, which represented ~97% inhibition. When each treatment group was compared, the S. cerevisiae, B. subtilis, and L. plantarum treatment groups had similar inhibitory effects with p >0.05. However, S. cerevisiae exhibited the strongest inhibition of B. bassiana, which indicated its superior antifungal effect (One-way ANOVA, p < 0.001; Post Hoc: Tukey HSD).

Fig. 1.In vitro inhibition of Beauveria bassiana by beneficial microorganisms.

Table 1.TDW: Triple distilled water; Different alphabet above number of radius of B. bassiana means statistically significant differences (One-way ANOVA, p < 0.001; Post Hoc: Tukey HSD).

For the in vivo evaluation of S. cerevisiae, we found that, after 5 d, 20% (3 of 15) of the control larvae had died, whereas none of the S. cerevisiae-treated larvae had died, and after 10 and 15 d, four of the larvae had died in both the control and S. cerevisiae treatment groups (Fig. 3). Therefore, a similar mortality was observed between the control and S. cerevisiae treatment groups when a high B. bassiana concentration was used. When the larvae were infected percutaneously, with 1 × 109 cfu/mL B. bassiana, the mortality of the S. cerevisiae treatment group was similar to that of control after 15 d, but the larvae’s survived time after treatment was extended. Also, almost no mortality when P. brevitarsis larvae were infected with S. cerevisiae via the hemolymph (Fig. 2.).The mean survived time of the S. cerevisiae treatment group larvae was significantly different than that of the control group (T-test, p < 0.5). Almost no mortality when P. brevitarsis larvae were infected with S. cerevisiae via the hemolymph. In addition, we also found that the consumption of S. cerevisiae decreased the mortality of infected larvae by ~12.2% (Fig. 3), although the difference was not significant (T-test, p > 0.05). Therefore, the S. cerevisiae treatment group exhibited that the difference of mortality rate was not significant with TDW and YPD groups.

Fig. 2.Mortality of Protaetia brevitarsis larvae injected with Saccharomyces cerevisiae. Con, negative control; YPD broth, positive control; S. cerevisiae, 1 × 107 cfu/50 μL.

Fig. 3.Effect of Saccharomyces cerevisiae on the mortality of Protaetia brevitarsis larvae after percutaneous infection with B. bassiana.

In conclusion, the present study demonstrated that S. cerevisiae, L. plantarum, and B. subtilis inhibited the in vitro growth of B. bassiana; that S. cerevisiae had no harmful effect when injected into the hemolymph of P. brevitarsis larvae; and that a S. cerevisiae-inoculated food source delayed B. bassiana infection and reduced P. brevitarsis mortality in vivo. No previous studies have reported the pathogenic potential of S. cerevisiae in P. brevitarsis and S. cerevisiae can be easily obtained as an Effective Microorganisms (EM). Since S. cerevisiae has no harmful effects on humans, it is considered to be effective in the prevention and control of white muscardine caused by B. bassiana in P. brevitarsis larvae.

Conflicts of Interest

The authors have no conflict of interest to declare.