Effects of culture conditions on Bacillus licheniformis KN12 antifungal activity against Fusarium oxysporum and Fusarium equiseti

In this study, the suitable culture conditions for synthesizing antifungal compounds produced by Bacillus licheniformis KN12 strain antagonizing Fusarium oxysporum and Fusarium equiseti were identified.

Tuyển tập Hội nghị Nấm học Toàn quốc lần thứ doi: 10.15625/vap.2022.0149 EFFECTS OF CULTURE CONDITIONS ON Bacillus licheniformis KN12 ANTIFUNGAL ACTIVITY AGAINST Fusarium oxysporum AND Fusarium equiseti Nguyen Ngoc An1, Trinh Tien Kim Ngan1, Nguyen Thi Dieu Hanh1, Pham Tan Viet1* Institute of Food and Biotechnology, Industrial University of Ho Chi Minh City, * Email: phamtanviet@iuh.edu.vn ABSTRACT Fusarium spp are emerging fungal pathogens that cause Fusarium wilt in various domestic plants, leading to large economic losses worldwide In order to find a safe and effective way in substitution of synthersized chemicals, many bacterial strains have been widely studied and applied on a variety of plant to control these fungal pathogens In this study, the suitable culture conditions for synthesizing antifungal compounds produced by Bacillus licheniformis KN12 strain antagonizing Fusarium oxysporum and Fusarium equiseti were identified The culture obtained after cultivation of this strain on Luria-Bertani broth with initial pH 7.0 at 37 °C, and 150 rpm for 15 hours was recorded to have the highest antifungal activity The F oxysporum and F equiseti inhibitory activity of the culture supernatant was relatively heat-resistant (up to 90 °C) In addition, the inhibitory activity of the culture supernatant against F oxysporum maintained at pH 5.0 - 10.0 while against F equiseti was retained in wide pH spectrum 3.0 - 11.0 These results suggested that B licheniformis KN12 could be a potential strain for futher studies and applications in controlling Fusarium diseases in sustainable agriculture Keywords: Bacillus licheniformis, Fusarium oxysporum, Fusarium equiseti, antifungal activity, culture condition INTRODUCTION Although great achievements have been achieved in recent years, worldwide agriculture has always faced many difficulties because of adverse weather, pests, and especially pathogenic microorganisms In particular, fungal pathogens are responsible for several serious diseases on many different not only in plants, but also in mushrooms that cause big economic losses [1, 2] Among these fungi, Fusarium spp are considered the fourth and fifth fungal pathogens of scientific and economic importance, respectively [3] Banana wilt caused by Fusarium oxysporum f sp cubense was first reported in 1876 in Australia [4] and then the pathogen has disseminated to many other countries including India [5], Israel and the Middle East [6, 7], China and Southeast Asia [8, 9] Moreover, F oxysporum has become subject of high concerns as it continuously destroy seveal plant crops worldwide including strawberry wilt [10], Zucchini wilt in Korea [11], watermelon wilt in America and Malaysia [12, 13] It is worth noting that F oxysporum is also an emerging pathogen that causes fusariosis in human [14, 15] In addition, another emerging pathogen Fusarium equiseti has been identified as the causal agent of cantaloupe fruit rot in Thailand [16], cabbage wilt in Korea [17], date palm wilt in Qatar [18], corn sheath rot and potato wilt in China [19, 20], sugar beet seedling death in America [21], not to mention that this species has recently been reported to infect patients with burn injury [22] 199 Nguyen Ngoc An et al In order to protect crops, chemical pesticides have been used very commonly [23] However, the abuse of this chemical has significantly threatened the environment and human health [22, 24, 25] Another better perspective to control fungal pathogens is the implement of biocontrol using antagonistic Gram positive spore-forming Bacillus [26, 27] Among them, Bacillus licheniformis is gaining recognition for a variety of highly potential applications in bioremediation, human and animal health promotion, and biopesticide production [28] Therefore, the studies to find new wild strains of Bacillus antagonist to these fungal pathogens are urgent, and this will be the basic for developing bioproducts that help minimize the damages in agriculture in an eco-friendly manner MATERIAL AND METHODS 2.1 Microorganisms and culture conditions One bacterial strain Bacillus licheniformis KN12 and pathogenic fungal strains including Neoscytalidium dimiatum, Fusarium equiseti, F oxysporum, Aspergillus fumigatus, Aspergillus sp were obtained from the collection of Microbiotechnological Laboratory of the Institute of Biotechnology and Food Technology, Industrial University of Ho Chi Minh City Bacillus licheniformis KN12 was cultured under Luria-Bertani broth (LB, Himedia-India) in shaking (150 rpm) condition at 37 C and all fungi were cultured on PGA (Potato Glucose Agar) plate at room temperature for - days prior to use in antagonistic tests 2.2 Agar well diffusion method for antifungal activity determination Antagonistic test was done using agar well diffusion method Mold strains were spotted in the middle of PGA plates and incubated at room temperature After 24 hours, wells with a diameter of mm were aseptically punched with a sterile cork borer at ≈ 1.0 cm from the edge of the plates Culture of B licheniformis KN12 (24-hour growth) was centrifuged (13,000 rpm, 15 minutes, C) and the supernatant was collected Twenty microliters of the supernatant were then added into the wells A negative control well was done by using 20 μL of sterile culture medium Antifungal activity was recorded after - days of incubation at room temperature Anti-Fusarium activity of the culture supernatant in each defined growth condition was evaluated based on the fungal colony inhibition percentage ((colony maximum diameter length - colony inhibited diameter length)/ colony maximum diameter length) 2.3 Impact of nitrogen sources, carbon sources pH, temperature, and incubation time on antifungal metabolite production Bacillus licheniformis KN12 grown in LB broth under shaking (150 rpm) condition at 37 C for 24 hours was used as starter (1.0 % v/v) to test different culture conditions using the agar well diffusion method Bacillus licheniformis KN12 was grown in the basal medium containing (gL-1) 0.5 MgSO4.7H2O, 3.0 NaH2PO4.2H2O, 3.0 Na2HPO4.12H2O, 5.0, pH 7.0 supplemented with different nitrogen sources (NH4Cl, NaNO3, NH4NO3, urea, peptone, yeast extract) and then different carbon sources (D-glucose, sucrose, maltodextrin, potato soluble starch, lactose) at 1.0 % w/v concentration, under shaking (150 rpm) condition at 37 C for 24 hours to find the suitable nitrogen and carbon source Subsequently, the strain was grown in the medium adjusted to different pH (4.0, 5.0, 6.0, 7.0, 8.0, 9.0 ± 0.1) at 37 C for 24 hours and then different temperatures (25 C, 28 C, 200 Effects of culture conditions on Bacillus licheniformis KN12 antifungal activity against Fusarium… 33 C, 37 C, 45 C ± 0.1 C), monitored after 3, 6, 12, 18, 24 hours of cultivation to determine the suitable culture conditions A negative control well was done by using 20 μL of the respective sterile culture medium 2.4 Effect of high temperature and pH on anti-Fusarium activity The agar well diffusion method was used to study the effect of temperature and pH on the stability of antifungal activity of Bacillus licheniformis KN12 culture supernatant in the determined suitable growth condition (control condition) Effect of temperature was done by incubating 1.0 mL of the culture supernatant at different point from 55 °C to 95 °C for 15 minutes and subjected to the wells Effect of pH was done by adjusting 1.0 mL of the culture supernatant to different pH levels of 1.0 to 12.0 using hydrochloric acid/potassium chloride buffer (pH 1.0 - 2.0), citric acid/sodium citrate buffer (pH 3.0 - 5.0), phosphate buffer (pH 6.0 - 7.0), Tris amino methane/hydrochloric acid buffer (pH 8.0 - 9.0), sodium bicarbonate/sodium hydroxide buffer (pH 10.0), sodium phosphate dibasic/sodium hydroxide buffer (pH 11.0 - 12.0), incubate for hours at room temperature and then adjusted to equal final volume before 20 μL of which was subjected to the wells Negative control wells were done by using 20 μL of the sterile culture medium adjusted to the respective pH value Relative anti-Fusarium activity was evaluated on the basic of the fungal colony inhibition percentage remained in each tested condition compared to the control 2.5 Data visualization and analysis All the experiment was done in triplicate The data were visualized using Microsoft Excel 2013 and ANOVA tests (α = 0.05) were done using Statgraphics Centurion 18 software (Statgraphics Technologies) RESULTS AND DISCUSSION 3.1 Antagonistic activity of B licheniformis KN12 against phytopathogenic fungi Phytopathogenic fungi cause significant damage to agriculture worldwide Excessive use of chemical fungicides in agriculture has affected human health, polluted the environment and formed resistance to pathogens Therefore, the use of naturally occurring antagonistic compounds is an effective alternative Microbial antagonists are widely used to control phytopathogens and have been shown to minimize adverse effects Bacillus licheniformis is considered to have the ability to produce many secondary metabloties with antifungal activity against several fungal pathogens such as Fusarium spp., Colletotrichum gossypii, Aspergilllus, Penicillium, Magnaporthe oryzae, and Rhizoctonia solani [29, 30, 31, 32] In this study, a 24-hour culture supernatant of B licheniformis KN12 was used in order to verify its effect against pathogens: Fusarium oxysporum, F equiseti, Neoscytalidium dimidiatum, Aspergillus sp., and A fumigatus After days of incubation, the culture supernatant showed to moderately inhibit the growth of F oxysporum, F equiseti, N dimidiatum while to insignificantly inhibit the growth of the two Aspergillus strains (Figure 1) Subsequently, F oxysporum and F equiseti antagonistic effect of the supernatant in several culture conditions was studied 201 Nguyen Ngoc An et al Figure Phytopathogenic fungal antagonistic efffect of B licheniformis KN12 (-) Control 3.2 Effect of nitrogen source on the antagonistic activity against F oxysporum and F equiseti B licheniformis KN12 was grown in basal medium supplemented with one of the different sources of NH4NO3, NH4Cl, NaNO3, urea, yeast extract, peptone and the antagonistic activity of the supernatant was checked after 24 hours of incubation at 37 C in order to determine the effect of nitrogen sources (Figure 2) The results showed that B licheniformis KN12 grown in all tested sources of nitrogen was able to display inhibition effect against both Fusarium strains Culture of B licheniformis KN12 showed the highest inhibition effect on F oxysporum when supplemented with NaNO3 or Yeast extract (37.3 % and 39.6 %, respectively) and the lowest when supplemented with NH 4NO3 and NH4Cl (9.4 % and 11.5 % respectively) (Figure 2A, 2B) The highest inhibition effect on F equiseti obtained when using NaNO3, Yeast extract, or pepton (29.2 - 31.1 %) with no statistical difference (ANOVA, P < 0.05), and the lowest in the case of NH4Cl (Figure 2A, 2C) Nitrogen sources containing NH4+ seemed to be unsuitable for anti-Fusarium activity On the contrary, natural nitrogen sources such as yeast extract and peptone which contain rich source of amino acids and peptides could be used directly by the bacterial cell, which could result in higher fungal inhibition Moreover, yeast extract is also rich in vitamin and contains a small amount of minerals that could be useful for the production of antifungal substances In the studies by Nigris et al (2018) and Hassanet et al (2019), peptone and yeast extract were also nitrogen sources of choice for different B licheniformis strains for antifungal activity [30, 34] Therefore, yeast extract was chosen for culturing B licheniformis KN12 202 Effects of culture conditions on Bacillus licheniformis KN12 antifungal activity against Fusarium… Figure Effect of nitrogen sources on the biosynthesis of antifungal agents against F oxysporum (A, B) and F equiseti (A, C) of B licheniformis KN12 (-) Control YE: yeast extract 3.3 Effect of carbon source on the antagonistic activity against F oxysporum and F equiseti B licheniformis KN12 was grown in basal medium supplemented with yeast extract and one of the different sources of glucose, sucrose, lactose, maltodextrin, starch, and the antagonistic activity of the supernatant was checked after 24 hours of incubation at 37 C in order to determine the effect of carbon sources (Figure 3) The results showed that B licheniformis KN12 grown in all tested sources of carbon was able to display good inhibition effect against both Fusarium strains Culture of B licheniformis KN12 showed statistically highest inhibition effect on F oxysporum when supplemented with glucose or lactose (39.2 %) and only lactose in the case against F equiseti (37,5 %) (ANOVA, P < 0.05) Starch, sucrose, and maltodextrin gave the same lowest anti F oxysporum activity (32.4 - 36.5 %) while glucose, sucrose, and maltodextrin gave the same lowest anti F equiseti activity (28.9 35.4 %) with no statistically significance (ANOVA, P < 0.05) Polysaccharide which is starch was unsuitable for the antifungal activity against F oxysporum which was contrary to the case of F equiseti This could be partly explained by the difference in production of antifungal secondary metabolites of B licheniformis KN12 against these two phytopathogens Interestingly, B licheniformis KN12 grown in LB medium displayed the antagonistic effect against both 203 Nguyen Ngoc An et al Fusarium strains statistically the same as the newly defined media (Figure 3A, B, C) This medium was also used in the study of Khan et al (2018) for inhibition of Fusarium spp by several strains of B simplex and B subtilis [29] Therefore, Luria-Bertani broth was chosen for culturing B licheniformis KN12 Figure Effect of carbon source in the culture medium on the biosynthesis of antifungal agents against F oxysporum (A, B) and F equiseti (A, C) of B licheniformis KN12 (-) Control LB: Luria-Bertani broth 3.4 Effect of initial pH of medium on the antagonistic activity against F oxysporum and F equiseti For determining the effect of initial pH, B licheniformis KN12 was grown LB medium adjusted to different pH values (4.0, 5.0, 6.0, 7.0, 8.0, and 9.0) and the antagonistic activity of the supernatant was checked after 24 hours of incubation at 37 C (Figure 4) The results showed that B licheniformis KN12 grown in most of the tested initial pH was able to display good inhibition effect against both Fusarium strains Culture of B licheniformis KN12 in pH 4.0 - 7.0 showed the same inhibition effect on F oxysporum (35.3 - 38.0 %) (Figure 4A, B) and on F equiseti (34.3 - 38.9 %) (Figure 4A, C) A dramatically reduced effect on F oxysporum was observed when the strain was grown in LB with an initial pH of 8.0 - 9.0 but not on F equiseti The value of pH for LB medium is commonly adjusted around 7.0 for growing bacteria in general and Bacillus strains with antifungal activity [29, 35] Therefore, the default pH 7.0 value was chosen for B licheniformis KN12 cultivation 204 Effects of culture conditions on Bacillus licheniformis KN12 antifungal activity against Fusarium… Figure Effect of carbon source in the culture medium on the biosynthesis of antifungal agents against F oxysporum (A, B) and F equiseti (A, C) of B licheniformis KN12 (-) Control 3.5 Effect of culture temperature on the antagonistic activity against F oxysporum and F equiseti In order to determine the effect of culturing temperature and time, B licheniformis KN12 was grown LB medium, pH 7.0 at different temperature (25 °C, 28 °C, 33 °C, 37 °C and 45 °C) and the antagonistic activity of the supernatant was checked every hours during a 24-hour period (Figure 5) The results showed that the growth temperature and time had a high influence on the inhibition effect of B licheniformis KN12 against both Fusarium strains Culture of B licheniformis KN12 displayed an increase in the inhibition effect over time and reached maximum after 15 hours at 37 °C and 45 °C in the case of F equiseti (43.1 % and 42.6 % respectively) and after to 15 hours at 37 °C (40.2 - 43.1 %) in the case of F oxysporum (ANOVA, P < 0.05) The inhibition effect gradually decreased from 18 hours to 24 hours of cultivation In the study of Wang et al (2014), B licheniformis HS10 was grown at 28 C for 48 hours for analysis of antifungal effect [36] Another growth condition for B licheniformis M-4 defined by Lebbadi et al (1994) was 28 C for up to 70 hours [37] At 37 C, B licheniformis KN12 has been shown to require 205 Nguyen Ngoc An et al shorter time with only 15 hours of incubation Therefore, B licheniformis KN12 was grown at 37 C for 15 hours as suitable condition for anti-Fusarium activity Figure Effect of temperature on the biosynthesis of antifungal agents against F oxysporum (A, C) and F equiseti (B, D) of B licheniformis KN12 (-) Control 3.6 Effect of temperature and pH on the stability of the antagonistic activity against F oxysporum and F equiseti To further explore the stability of the antagonistic activity in different practical application conditions as well as storage conditions, the effect of temperature and pH on the antagonistic activity against F oxysporum and F equiseti were investigated Culture supernatant of B licheniformis KN12 grown in LB medium, pH 7.0, at 37 °C for 15 hours was incubated at different temperatures (60 - 95 °C, in 15 minutes) and pH (1.0 - 13.0, in hours) The relatively remaining antagonistic activity of the supernatant was checked and displayed in Figure The culture supernatant of B licheniformis KN12 was shown to have a good temperature stability, which gradually reduced from 60 °C to 90 °C with up to 40 - 60 % of the activity remained (Figure 6A) Interestingly, the culture supernatant expressed slightly higher activity on F oxysporum in pH 5.0 - 10.0 (100.0 - 114.3 %), and even much higher activity on F equiseti in pH 4.0 - 10.0 (94.3 - 125.7 %) (Figure 6B) Moreover, the inhibition effect of the supernatant on F equiseti was relatively high even at the very low pH 3.0 (91.4 %) and very high pH 11.0 (71.4 %) (Figure 6B) The temperature stability of the B licheniformis KN12 supernatant against Fusarium was slightly lower but more stable in a wider pH range than that of B licheniformis HS10 (100 C 206 Effects of culture conditions on Bacillus licheniformis KN12 antifungal activity against Fusarium… 30 minutes, pH 6.0 - 10.0) [36] Besides, temperature stability of the B licheniformis KN12 supernatant was somewhat comparable with Fungicin M4 of B licheniformis M-4 in the study of M Lebbadi et al (1994), with 30 % activity remained after 30 minutes of incubation at 80 °C and stable at pH 2.5 - 9.0 [37] Figure Effect of temperature (A) and pH (B) on stability of antifungal agents against F oxysporum and F equiseti of B licheniformis KN12 CONCLUSION Plant diseases caused by the Fusarium spp are always a threat to agriculture and to farmers' income In this study, B licheniformis KN12 biosynthesized compounds against F oxysporum and F equiseti The appropriate conditions for culturing this strain for the highest antifungal activity included LB medium, at 37 °C for 15 h, and 150 rpm This activity remained after being treated for 15 at 60 - 90 °C The antagonistic activity of this culture against F equiseti and F oxysporum unchanged in the pH range of 3.0 - 11.0 and 6.0 - 10.0, respectively These results reinforce the value of Bacillus genus The strain B licheniformis KN12 can be used to produce biological products for the control, prevention, and treatment of Fusarium diseases on plants ACKNOWLEDGMENT: This study was carried out at the Microbiological Technology laboratory, Institute of Food and Biotechnology, Industrial University of Ho Chi Minh City We would like to express our sincere thanks to the Industrial University of Ho Chi Minh City and the Institute of Food and Biotechnology created favorable conditions for us to carry out this 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Figure Effect of nitrogen sources on the biosynthesis of antifungal. .. 208 Effects of culture conditions on Bacillus licheniformis KN12 antifungal activity against Fusarium? ?? [18] Nishad, R & Ahmed, T A (2020) Survey and identification of date palm pathogens and. .. agents against F oxysporum (A, B) and F equiseti (A, C) of B licheniformis KN12 (-) Control YE: yeast extract 3.3 Effect of carbon source on the antagonistic activity against F oxysporum and F equiseti