THE IN VITRO ANTAGONISTIC EFFECTS OF SOME BACILLUS SPP. ON THE GROWTH AND MYCOTOXIN PRODUCTION OF TOXIGENIC ASPERGILLI

This study aimed to investigate the antagonistic effect of three strains of the genus Bacillus on toxigenic fungi contaminating food commodities, with emphasis on two ochratoxigenic isolates (A. ochraceus, A. westerdijkiae) and two aflatoxigenic isolates (A. flavus and A. parasiticus). In vitro studies were carried out using two different methods for cultivation tested bacilli with fungal isolates – coincubation and dual culture method. The most sensitive isolate was A. ochraceus by both used methods. B. mycoides (5.72 ± 6.4 mm) and B. subtilis (5.08 ± 2.84 mm) were able to inhibit its growth and sporulation during ten days of cultivation (both inhibited the sporulation of A. ochraceus 100%) in coincubation. The most effective in the dual culture method were B. subtilis and B. thuringiensis against A. ochraceus (growth inhibition rate 84.40%; 90.55%) and A. flavus (growth inhibition rate 91.54%; 92.43%). The most effective sporulation inhibitors were B. subtilis and B. thuringiensis, which completely inhibited the sporulation of A. ochraceus and A. parasiticus after ten days of coincubation. Likewise, all tested bacterial strains showed complete inhibition of ochratoxin A synthesis in A. ochraceus and A. westerdijkiae exposure to bacterial volatiles. So, the current study illustrated that strains of the genus Bacillus could significantly inhibit the growth, sporulation, and mycotoxin production of toxigenic aspergilli and showing the great potential as a biocontrol agent of pre- and post-harvest food diseases caused by microscopic filamentous fungi.


Fungal isolates, identification, growth condition and inoculum preparation
The following toxigenic isolates of fungi were used in this study: Aspergillus flavus, A. parasiticus, A. ochraceus and A. westerdijkiae. All the fungal isolates were obtained from green coffee beans samples, marketed in Slovakia. The isolates were identified to the species level according to morphological characteristics based on microscopy used follow diagnostic literature: Samson et al., 2002, Chen et al., 2017 2019 and Yamairi et al., 2019. Aspergillus isolates were tested for aflatoxin B1 and G1, and ochratoxin A, and their production was confirmed by thin layer chromatography (TLC) (Labuda and Tancinová, 2006). To prepare a fungal inoculum the isolates were cultivated on Sabouraud dextrose agar (SDA) (HiMedia, India) for 5 days at 25 °C. Then the inoculum was prepared according to Císarová et al. (2020) to a final concentration 10 6 CFU/mL by adjusting the density to 0.7-1.2 McFarland units, depending on the strain.

In vitro screening antagonistic activity of Bacillus spp. by volatiles
The antifungal activity of volatiles potentially produced by Bacillus species against toxicogenic Aspergillus species (Aspergillus flavus, A. parasiticus, A. ochraceus and A. westerdijkiae) was tested using the coincubation method (Ul Hassan et al., 2019), with some modification (Fig 1). A 100 μl of the prepared bacterial inoculum suspension (10 6 cells/ml) was applied on MPA plates (Meat peptone agar; HiMedia, India) and cultivated without sealing for 24 hours at 30 °C. After 24 hours, the cover of the bacterial plate was replaced by a base SDA plate containing 5 μl of fungal inoculum (10 6 CFU/ml). Subsequently, both parts of the plates were tightly sealed with parafilm to prevent volatiles escape. The plates were cultivated for 10 days at 30 °C in the dark. The plates were cultivated in an inverted position (the plate containing the bacterial inoculum was on top). The control treatment was represented by fungus inoculated plates without bacterial inoculum. The effects of bacterial volatiles on fungal growth and sporulation were evaluated by measured diameters (Ø mm) of the growing fungal colonies at the 2 n d, 4 th , 6 th , 8 th , and 10 th day of cultivation with a digital caliper. All analyzes were performed in 6 replicates.
To evaluate the reversibility effects of bacteria volatiles on the fungal growth, on the 5 th day of cultivation, a small piece of a fungal colony (Ø 10 mm) from the margin was removed with a sterile cork borer and transferred to fresh SDA plates. These plates were incubated for 10 days at 30 °C, and mycelial growth and sporulation were monitored (in the same way as before on 2 n d, 4 th , 6 th , 8 th and 10 th day) and compared with the control group (plates with fungal inoculum never exposed to bacterial volatiles).

In vitro screening antagonistic activity of Bacillus spp. by contact
The strains of genus Bacillus were also evaluated in vitro for their antagonistic properties against toxicogenic Aspergillus species by using dual culture technique according to Rajkumar et al. (2018) with slight modification (Fig. 2). Firstly, the Petri dishes (Ø 60 mm) with PDA (Potato dextrose agar, HiMedia, India) were divided in half by a dividing line and then were inoculated with the bacterial inoculum (10 6 cells/ml) by spreading (comma) approximately 1 cm from the center of the plate dividing line to the left side of plates. After 24 hours of cultivation at 30 °C in the dark, the plates were inoculated with fungal inoculum (1 cm from the center of the plate dividing line). Each fungus was inoculated to the right side of plates with inoculation needle. The antagonistic activity of the Bacillus spp. was monitored in comparison with a control sample (plates with fungal inoculum in the absence of bacteria). The prepared plates were sealed with parafilm and cultivated at 30 °C for 8 days. The mycelial growth of fungi was recorded with a digital caliper on the 2 nd , 4 th , 6 th , and 8 th day of cultivation in mm. The Percentage of Mycelial Growth Inhibition (PGMI) of the tested species was calculated using the following equation: where FC is the growth of the fungal mycelium control (measured in mm from the inoculation point to the edge of the mycelial colony), FB is the growth of the fungal mycelium in the presence of bacteria (measured in mm from the inoculation point to the edge of the mycelial colony towards the antagonist). All analyzes were performed in 6 replicates. The antitoxigenic effect of the genus Bacillus was evaluated only after coincubation method (including the reversibility test). For the detection of mycotoxins, the qualitative TLC (thin layer chromatography) screening method according to Labuda and Tančinová (2006) was used. After 10 days of cultivation (coincubation and reversibility) the ability of the genus Bacillus to suppress the production of aflatoxin B1 (AFB1) in A. flavus, AFB1 and aflatoxin G1 (AFG1) in A. parasiticus and ochratoxin A (OTA) produced by A. westerdijkiae and A. ochraceus was evaluated. The used method is described in Císarová et al. (2015). Visualization of mycotoxins AFB1, AFG1, ochratoxin A and their authentic standards (Sigma Aldrich, Germany) was performed directly under a UV lamp at 366 nm, where aflatoxin B1 and ochratoxin A appeared as a blue spot and AFG1 as a green spot.

Statistical analysis
Obtained results were evaluated by analysis of variance (one-way ANOVA) and Tukey HSD 95% multiple range test (p˂0.05) using the program STATGRAPHICS Centurion XVI (version 16.1.11). The results of the percentage inhibition of spore germination and percentage of the mycelial growth inhibition (PMGI %) were calculated by using the MS Office Excel 2016 program.

Antagonistic activity of the genus Bacillus
Contamination of food with various perishable microorganisms, mainly microscopic filamentous fungi, is a global problem that leads to large-scale economic losses. Nowadays, synthetic preservatives or antifungals are used to prevent their growth in food. However, these substances can affect humans and the environment, so more environmentally friendly alternatives are still being sought In this study, three strains of the genus Bacillus (B. subtilis, B. mycoides, B. thuringiensis) were assayed against four isolates of ochratoxigenic (A. westerdijkiae and A. ochraceus) and aflatoxigenic (A. flavus and A. parasiticus) aspergilli using two different methods. The first coincubation method investigated the antifungal activities of potentially produced volatiles of the genus Bacillus. The second method was used to evaluate the antifungal activity of Bacillus spp. by the method of dual culture, where the tested bacilli were inoculated on one plate together with the fungi and were in contact with each other. Furthermore, there are many reports focused on the antioxygenic potential of the genus Bacillus

Effect of Bacillus spp. on the growth and sporulation of tested fungi by bacterial volatiles
The result of the coincubation method showed that in the presence of the Bacillus spp. and their released volatiles, the growth of tested fungi was inhibited significantly (p<0.05). The strains showed different antagonistic activity on mycelia growth and sporulation of toxigenic aspergilli. A. ochraceus was the most sensitive isolate from the tested ochratoxigenic fungi. B. mycoides (5.72 ± 6.4 mm) and B. subtilis (5.08 ± 2.84 mm) were able to inhibit vegetative growth and sporulation during ten days of cultivation (both inhibited the sporulation of A. ochraceus 100%) of this isolate significantly. After ten days of coincubation with these species, the radial growth of A. ochraceus resulted in smaller colony diameters compared to the control set (13.18 ± 7.07 mm). B. thuringiensis showed the ability to inhibit this species only until day 6 of cultivation ( Fig. 1A) with the inhibition of sporulation 33,33%. The better results for B. thuringiensis were noted by Arfaoui et al. (2019). They found that B. thuringiensis J4F was able to inhibit 78% of A. ochraceus growth. Also, partial inhibition of A. westerdijkiae was found in treatment with B. thuringiensis (20.75 ± 12.44 mm) compared to a control set (23.65 ± 12.65 mm). However, B. thuringiensis did not show any inhibitory effect on A. westerdijkiae sporulation (Table 1). B. subtilis and B. mycoides were the most effective species in inhibiting the growth of this isolate. Antagonism of B. subtilis by volatiles was studied in work of Petchkongkaew et al. (2008). They found that the growth of A. westerdijkiae tested by the coincubation method for seven days was lower, about 34%. Inhibition of growth was associated with prolongation of the lag phase of the microscopic fungus, thereby reducing its ability to compete and utilize the nutrients in the environment. Also, Einloft et al. (2017) observed the inhibitory effect of B. safeness RF69, B. amyloliquefaciens RP103, and B. subtilis RP242 on the growth of A. westerdijkiae as well as their ability to inhibit spore formation. They found that B. subtilis RP242 was the most effective biological control agent of fungal growth and sporulation. The results showed a 95.6% reduction in growth and spore germination after ten days of coincubation.

Figure 1
The antagonistic effect of the tested Bacillus spp. on the growth: A) A. ochraceus (n=6) and B) A. westerdijkiae (n=6) during 10 days of cultivation in the dark (in mm) at 30 °C The inhibition activity of Bacillus spp. on the growth and sporulation of aflatoxigenic aspergilli was much more potent, especially for A. flavus isolate.
Results showed that among all tested toxinogenic fungi of the genus Aspergillus, A. flavus was the most sensitive to the action of bacterial antagonists. The mycelial growth of A. flavus reached the smallest averages throughout all periods of cultivation (10 days), mainly in the presence of B. subtilis (1.60 ± 1.87 mm) and B. thuringiensis (0.85 ± 1.15 mm) ( Fig. 2A). Nayak et al. (2020) tested the antifungal activity of 4 isolates of Bacillus spp. against the growth and sporulation of two strains of A. flavus (A2, A28). Their results showed that Bacillus vallismortis BC5 was the most potent in mycelial growth inhibition of A. flavus (92.3%), followed by B. subtilis BC6 (90%) and B. amyloliquefaciens BC1 and BC2 (88.6% and 89.3%). However, in our study, their efficacy did not correlate with inhibition of sporulation. B. subtilis showed no inhibitory effect on sporulation, and the effect of B. thuringiensis on A. flavus sporulation was minimal (16.67%) ( Table 1) In this study, 100% inhibition of sporulation was observed in treatment with B. mycoides, but this species had only a mediatory inhibition effect on A. flavus growth after ten days of cultivation (18.72 ± 13.98 mm) in comparison with a control set (29.55 ± 10.20 mm). On the other hand, B. subtilis and B. thuringiensis in comparison to the control and A. flavus species had better inhibitory effects on the sporulation of A. parasiticus (100%), but these species inhibited its growth only moderately (Fig. 2B). The best inhibitory effect for A. parasiticus was found in treatment with B. mycoides (6.11 ± 3.78 mm) during the whole cultivation period (10 days). However, B. mycoides was not able to inhibit A. parastiticus sporulation (Table 1) In their study, the species A. clavatus MG103 and A. parasiticus MG51 showed the least susceptibility. Similar to our study, the growth of these species was not affected by Bacillus spp. volatiles production.

Figure 2
The antagonistic effect of the tested Bacillus spp. on the growth: A) A. flavus (n=6) and B) A. parasiticus (n=6) during 10 days of cultivation in the dark (in mm) at 30 °C 100.00 Control of tested fungi ----Legend: -: no effect (without presence of Bacillus strains) The cell wall of fungi is necessary for their homeostasis, and various environmental or antifungal stresses can cause modifications such as suppression of sporulation, insufficient development of hyphae, or pigmentation losses (Garcia-Rubio et al.,  2020). In this study, similar changes were observed. Compared to the control, some fungal isolates whose sporulation was partially or entirely suppressed showed an observed change or loss of mycelial pigmentation (Fig. 3).

Figure 3
Changes observed in mycelial pigmentation: A) normal fungal mycelium (never exposed to bacterial volatiles) (control); B) change of mycelial pigmentation after treatment with volatiles of tested bacilli After five days of coincubation with individual species of bacilli, the cultured plates were opened, and one small pieces of fungal mycelia were removed and subsequently cultivated on fresh SDA plates for another ten days without the presence of tested bacteria. However, upon transfer to the fresh SDA media, in the absence of bacterial volatiles, all the fungal isolates showed normal growth and sporulation, comparable with control sets. Our results showed that the inhibition was poor and that the coincubation of fungi with the volatiles of tested bacilli during the 5-days was insufficient for more significant inhibition of their growth and sporulation. For this reason, the obtained results are not presented in this work.  (2021), similar reversibility in mycelial growth and sporulation was observed in toxigenic fungi included A. flavus CECT, 2687after removal from volatiles of B. megaterium BM344-1. So, these results suggested that the microbial volatiles effect was insufficient and that bacterial volatiles were continuously required to allow effective biocontrol of spoilage fungi.

Effect of Bacillus spp. on the growth of tested fungi by contact
In the dual culture method, only the effect of Bacillus species on the growth of toxigenic fungi was observed. The results of dual culture method showed that antifungal ability of bacilli had a certain inhibitory potential on the growth of all tested fungal isolates. Significant differences (p<0.05) between the antagonistic effect of tested bacilli were found (Table 2). B. mycoides showed the best inhibitory ability against toxigenic aspergilli. 27.83 ± 11.70 ab 36.00 ± 0.00 b 21.34 ± 7.38 a 36.00 ± 0.00 ab Legend: * -data in the columns followed by the same letter are not significantly different in 95.0% Tukey HSD test, SD standard deviation B. mycoides showed the best inhibitory ability against ochratoxigenic aspergilli. The percentage of mycelial growth inhibition (%) calculated after all days of cultivation (8 days) with comparison to control sets was 67.36 % for A. westerdijkiae and 82.96 % for A. ochraceus (Fig. 4). From the tested fungi, A. ochraceus was the most sensitive to the action of all Bacillus species (Fig. 5B). The most potent inhibition against this isolate exhibited B. subtillis (growth inhibition rate 91.54%) followed by B. thuringiensis (growth inhibition rate 90.55 %) and B. mycoides (growth inhibition rate 82.96%). The sensitivity of A. flavus isolate was lower compared to A. ochraceus, but its growth was significantly inhibited by B. thuringiensis (growth inhibition rate 92.43 %) and B. subtilis (growth inhibition rate 91.54%) compared to the control sets (Fig. 5A). Similar results with Bacillus subtillis against S. rolfsii obtained by authors Rajkumar et al. (2018) and Nayak et al. (2020) in their study found that Bacillus spp. were able to inhibit the growth of A. flavus up to more than 90%. A very poor inhibition effect was observed using the dual culture method with B. thuringiensis and A. westerdijkiae. B. thuringiensis inhibited the growth of this isolate only up to the sixth day of cultivation (Ø 8.97 mm) compared to control (Ø 13.71 mm). After the cultivation period (8 days), the diameters of A. westerdijkiae colonies were larger (18.52 ± 0.97 mm) than the colony diameters of control sets (17.28 ± 0.28 mm). So, its growth was moderately stimulated by B. thuringiensis.   Based on the results, can be concluded that the coincubation method was better than dual culture method used. In the case of aflatoxigenic aspergilli, the difference between two used methods in the action of Bacillus spp. were observed. Significant and stronger antagonistic effects of selected species of the genus Bacillus could be seen mainly in the case of A. flavus and A. parasiticus used coincubation method in comparison with the dual culture method. The different effects of bacteria in the case of coincubation method, which were resulted in a higher inhibition of fungal growth, may be due to the use of a more suitable culture medium for bacteria, and fungi, respectively. The more appropriate used medium in coincubation method may have affected the potential production of Bacillus spp. volatile compounds, and since the bacteria did not come into direct contact with fungi, can be assume that the highest growth inhibition was due to their production. Also, Morita et al. (2019) studied the antifungal effect of Bacillus pumillus volatiles by cultivating fungi and bacteria on three different media: Trypt-Soya agar (TSA), Lauria-Betani agar (LBA) and TM Enterprise agar (TMES). They found that the bacterium grown showed the strongest inhibition effect against Penicillium italicum only on TMEA medium. Tyc et al. (2017) report, that the volatile organic compounds produced by Bacillus spp. can easily evaporate and diffuse through pores filled with air and water. Due to these physicochemical properties, volatile substances are ideal candidates for cooperation or antagonism between soil microorganisms that do not live directly next to each other.

Inhibitory effect of Bacillus spp. on mycotoxin production
Bacillus spp. volatiles not only inhibited the vegetative growth but also affected the mycotoxin production of toxigenic fungi. Although some species of the genus Bacillus did not completely inhibit the growth of the tested fungi, but they were able to suppress the production of their mycotoxins, especially in the case of inhibition of ochratoxin a (OTA) production. All three bacterial isolates inhibited OTA production by A. westerdijkiaae and A. ochraceus totally (100%) in comparison with control sets. Successful inhibition of mycotoxin biosynthesis was also observed in the case of reversible action of Bacillus spp., when 5 days exposure of microscopic fungi to bacterial volatiles was sufficient to completely inhibit their toxigenic potential (  A. parasiticus AFB1 4/6 0/6 2/6 6/6 AFG1 4/6 0/6 2/6 Legend: ndmycotoxin not detected (below the limit of detection of the TLC method), OAochratoxin A, AFB1aflatoxin B1, AFG1aflatoxin G1, ** -number of mycotoxins produced isolates, * -number of tested isolates B. thuringiensis demonstrated the best antitoxigenic potential against AFB1 production by A. flavus. Its production was completely inhibited even in the case of tested reversibility. Kong et al. (2014) found that aflatoxin accumulation was totally (more than 98 %) inhibited and cause the down-regulation of the aflatoxins genes by co-cultivation with B. megaterium. The biosynthesis of AFB1 and AFG1 produced by A. parasiticus totally inhibited only B. mycoides in all repetitions. B. subtilis inhibited the production of AFB1 and AFG1 produced by A. parasiticus at least. In the case of coincubation was able to suppress the production these aflatoxins only in half replicates, and in the case of a reversible effect the production of these mycotoxins was observed in up to 4 replicates. Better results with B. subtilis obtained Siahmoshteh et al. (2018). In their study B. subtilis was able to suppress A. parasiticus aflatoxins production up to 100 % and the degradation of aflatoxins has been proved after two days of co-cultivation the fungal strain with bacteria.

CONCLUSION
In summary, the results obtained in this study represent the promising characteristics of Bacillus spp. to be used as biocontrol agents of toxigenic fungi of genus Aspergillus. The antagonistic activity of Bacillus spp. was evaluated using two different methods (coincubation and dual culture technique), where some differences were observed, especially in the case of growth inhibition of aflatoxigenic isolates. Our findings demonstrated that the coincubation method showed increased inhibitory efficiency. Among all fungal isolates, we observed the strongest inhibitory effect in the case of Bacillus spp. on the growth of A. ochraceus in both coincubation and dual culture methods, respectively. This fungal isolate was indicated as the most sensitive compared to the related species (A. westerdijkiae) and the aflatoxigenic species. Antitoxinogenic properties of Bacillus spp. by using coincubation method were very effective, mainly in inhibition of ochratoxin production, but also had the potential to inhibit aflatoxins synthesis. Our research confirmed the antagonistic abilities of Bacillus spp. against microscopic fungi, contaminating many crops and food. Their bioprotective character and their volatile compounds have also been confirmed in many in vivo and in situ assays. Therefore, these results suggest the potential application of Bacillus spp. for the preservation of food commodities, and further studies should be carried out to develop the commercial usage of the selected biocontrol agents of the genus Bacillus.