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

Tuyển tập Hội nghị Nấm học Toàn quốc lần thứ 4
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*
1

Institute of Food and Biotechnology, Industrial University of Ho Chi Minh City,
*

Email:
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.

1 .. 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.
2 . MATERIAL AND METHODS

2.1 . Microorganisms and culture conditions
One bacterial strain Bacillus licheniformis KN12 and 5 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 3 - 5 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
6 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, 4 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 5 - 7 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 2 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).
3 . 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 5 pathogens: Fusarium oxysporum, F. equiseti,
Neoscytalidium dimidiatum, Aspergillus sp., and A. fumigatus. After 7 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 1. 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 2. 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 3. 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 4. 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 3 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 6 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 5. 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 2 hours). The relatively
remaining antagonistic activity of the supernatant was checked and displayed in Figure 6.
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 6. Effect of temperature (A) and pH (B) on stability of antifungal agents against F. oxysporum
and F. equiseti of B. licheniformis KN12

4 . 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 min 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 research.

REFERENCES
[1]. Fausto, A., Rodrigues, M. L. & Coelho, C. (2019). The still underestimated problem of fungal
diseases worldwide. Frontiers in Microbiology, 10, 214-214.
[2]. Fisher, M. C., Gurr, S. J., Cuomo, C. A., Blehert, D. S., Jin, H., Stukenbrock, E. H.,… &
Cowen, L. E. (2020). Threats posed by the fungal kingdom to humans, wildlife, and
agriculture. mBio, 11(3), [e00449-20]. />[3]. Dean, R., Van Kan, J. A., Pretorius, Z. A., Hammond-Kosack, K. E., Di Pietro, A., Spanu, P.
D.,... & Foster, G. D. (2012). The Top 10 fungal pathogens in molecular plant pathology.
Molecular
Plant
Pathology,
13(4),

414-430.
/>
207


Nguyen Ngoc An et al.

[4]. Ordonez, N., Seidl, M. F., Waalwijk, C., Drenth, A., Kilian, A., Thomma, B. P.,... & Kema,
G. H. (2015). Worse Comes to Worst: Bananas and Panama Disease-When Plant and
Pathogen Clones Meet. PLoS pathogens, 11(11), e1005197.
[5]. Thangavelu, R. & Mustaffa, M. M. (2010). First Report on the Occurrence of a Virulent Strain
of Fusarium Wilt Pathogen (Race-1) Infecting Cavendish (AAA) Group of Bananas in India.
Plant Disease, 94(11), 1379.
[6]. García-Bastidas, F., Ordóđez, N., Konkol, J., Al-Qasim, M., Naser, Z., Abdelwali, M., Salem,
N., Waalwijk, C., Ploetz, R. C. & Kema, G. (2014). First Report of Fusarium oxysporum f. sp.
cubense Tropical Race 4 Associated with Panama Disease of Banana outside Southeast Asia.
Plant Disease, 98(5), 694. />[7]. Maymon, M., Sela, N., Shpatz, U., Galpaz, N. & Freeman, S. (2020). The origin and current
situation of Fusarium oxysporum f. sp. cubense tropical race 4 in Israel and the Middle East.
Scientific Reports, 10(1), 1590.
[8]. Qi, Y. X., Zhang, X., Pu, J. J., Xie, Y. X., Zhang, H. Q. & Huang, S. L. (2008). Race 4
identification of Fusarium oxysporum f. sp. cubense from Cavendish cultivars in Hainan
province, China. Australasian Plant Disease Notes, 3(1), 46-47.
[9]. FAO, Forecasting threats to the food chain affecting food security in countries and regions.
2020: Rome, Italy.
[10]. Koike, S. T.& Gordon, T. R. (2015). Management of Fusarium wilt of strawberry. Crop
Protection, 73, 67-72.
[11]. Choi, I. Y., Kim, J. H., Lee, W. H., Park, J. H. & Shin, H. D. (2015). First Report on Fusarium
Wilt of Zucchini Caused by Fusarium oxysporum, in Korea. Mycobiology, 43(2), 174-8.
[12]. Roberts, P., Dufault, N., Hochmuth, R., Vallad, G. & Paretet, M. (2019). Fusarium wilt
(Fusarium oxysporum f. sp. niveum) of watermelon: PP352, 10/2019. EDIS, 2019 (5), 4.

[13]. Rahman, M. Z., Ahmad, K., Siddiqui, Y., Saad, N., Hun, T. G., Hata, E. M.,… & Kutawa, A.
B. (2021). First report of Fusarium wilt disease on watermelon caused by Fusarium
oxysporum f. sp. niveum (FON) in Malaysia. Plant Dis.
[14]. Nucci, M. & Anaissie, E. (2007). Fusarium infections in immunocompromised patients. Clin
Microbiol Rev, 20(4), 695-704.
[15]. Thomas, B., Audonneau, N. C., Machouart, M. & Debourgogne, A. (2020). Fusarium
infections: Epidemiological aspects over 10 years in a university hospital in France. Journal of
Infection and Public Health, 13(8), 1089-1093.
[16]. Nuangmek, W., Aiduang, W., Suwannarach, N., Kumla, J., Kiatsiriroat, T. & Lumyong, S.
(2019). First report of fruit rot on cantaloupe caused by Fusarium equiseti in Thailand.
Journal of General Plant Pathology, 85(4), 295-300.
[17]. Afroz, T., Jee, S., Choi, H. W., Kim, J. H., Assefa, A. D., Aktaruzzaman, M., Hahn, B. S. &
Lee, H. S. (2020). First Report of Fusarium wilt caused by Fusarium equiseti on Cabbage
(Brassica oleracea var. capitate L.) in Korea. Plant disease, 10.1094/PDIS-06-20-1278-PDN.
Advance online publication. />
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
indigenous biocontrol agents. Plant Disease, 104(9), 2498-2508.
[19]. Wang, W., Wang, B., Sun, X., Qi, X., Zhao, C., Chang, X., Khaskheli, M. I. & Gong, G.
(2021). Symptoms and pathogens diversity of Corn Fusarium sheath rot in Sichuan province,
China. Scientific Reports, 11(1), 2835.
[20]. Cui, L., Yang, C., Yang, L., Jin, M. & Wei, L. (2021). First Report of Fusarium equiseti
causing Fusarium wilt on potato (Solanum tuberosum) in China. Plant Disease,
10.1094/PDIS-02-21-0281-PDN. Advance online publication. />[21]. Khan, M. F., Liu, Y., Bhuyian, M., Lashman, D., Liu, Z. & Zhong, S (2021). First report of
Fusarium equiseti Causing seedling death on sugar beet in Minnesota, USA. Plant Disease,
10.1094/PDIS-10-20-2102-PDN. Advance online publication. />[22]. Xuan Hai, T., Thai Ngoc Minh, N., Ngoc Anh, D., Ngoc Dung, T., Thi Minh Chau, N. &

Tran-Anh, L. (2021). A rare Fusarium equiseti infection in a 53-year-old male with burn
injury: A case report. Current Medical Mycology, 7(1), 59-62.
[23]. Oliver, R. P., & Hewitt, H. G. (2014). Fungicides in crop protection. Wallingford: CABI
Publishing, United Kingdom.
[24]. Ribas E Ribas, A. D., Spolti, P., Del Ponte, E. M., Donato, K. Z., Schrekker, H. & Fuentefria,
A. M. (2016). Is the emergence of fungal resistance to medical triazoles related to their use in
the agroecosystems? A mini review. Brazilian Journal of Microbiology: [publication of the
Brazilian Society for Microbiology], 47(4), 793-799.
[25]. Bastos, R. W., Rossato, L., Goldman, G. H. & Santos, D. A. (2021). Fungicide effects on
human fungal pathogens: Cross-resistance to medical drugs and beyond. PLoS Pathogens,
17(12), e1010073.
[26]. Fira, D., Dimkić, I., Berić, T., Lozo, J. & Stanković, S. (2018). Biological control of plant
pathogens by Bacillus species. Journal of Biotechnology, 285, 44-55.
[27]. Pandit, M. A., Kumar, J., Gulati, S., Bhandari, N., Mehta, P., Katyal, R., Rawat, C. D.,
Mishra, V. & Kaur, J. (2022). Major biological control strategies for plant pathogens.
Pathogens (Basel, Switzerland), 11(2), 273.
[28]. Muras, A., Romero, M., Mayer, C. & Otero, A. (2021). Biotechnological applications of
Bacillus licheniformis. Critical Reviews in Biotechnology, 41(4), 609-627.
[29]. Khan, N., Martínez-Hidalgo, P., Ice, T. A., Maymon, M., Humm, E. A., Nejat, N., Sanders, E.
R., Kaplan, D. & Hirsch, A. M. (2018). Antifungal activity of Bacillus species against
Fusarium and analysis of the potential mechanisms used in biocontrol. Frontiers in
Microbiology, 9, 2363.
[30]. Ul Hassan, Z., Al Thani, R., Alnaimi, H., Migheli, Q. & Jaoua, S. (2019). Investigation and
application of Bacillus licheniformis volatile compounds for the biological control of
toxigenic Aspergillus and Penicillium spp. ACS Omega, 4(17), 17186-17193.

209


Nguyen Ngoc An et al.

[31]. Zhaolina, J., Huiwen, H., Huijuan, Z., Feng, H., Yunhui, T., Zhengwen, Y. & Jingyou, X.
(2015). The biocontrol effects of the Bacillus licheniformis W10 strain and its antifungal
protein against brown rot in peach. Horticultural Plant Journal, 1(3), 131-138.
[32]. Nawaz, H., Nelly Rajaofera, M.J., He, Q., Anam, U., Lin, C. & Miao, W. (2018). Evaluation
of antifungal metabolites activity from bacillus licheniformis OE-04 against Colletotrichum
gossypii. Pesticide Biochemistry and Physiology, 146, 33-42.
[33]. Cui, T. B., Chai, H. Y., & Jiang, L. X. (2012). Isolation and partial characterization of an
antifungal protein produced by Bacillus licheniformis BS-3. Molecules (Basel, Switzerland),
17(6), 7336-7347.
[34]. Nigris, S., Baldan, E., Tondello, A., Zanella, F., Vitulo, N., Favaro, G.,… & Baldan, B.
(2018). Biocontrol traits of Bacillus licheniformis GL174, a culturable endophyte of Vitis
vinifera cv. Glera. BMC Microbiology, 18(1), p.133.
[35]. Asaturova, A. M., Homyak, A. I., Tomashevich, N. S., Pavlova, M. D., Zhevnova, N. A. &
Dubyaga, V. M. (2015). Conditions for the cultivation of new Bacillus bacteria being micro
bioproduct producers. Journal of Pure and Applied Microbiology, 9(4), 1-9.
[36]. Wang, Z., Wang, Y., Zheng, L., Yang, X., Liu, H. & Guo, J. (2014). Isolation and
characterization of an antifungal protein from Bacillus licheniformis HS10. Biochemical and
Biophysical Research Communications, 454(1), 48-52.
[37]. Lebbadi, M., Gálvez, A., Maqueda, M., Martínez-Bueno, M. & Valdivia, E. (1994). Fungicin
M4: a narrow spectrum peptide antibiotic from Bacillus licheniformis M-4. The Journal of
Applied Bacteriology, 77(1), 49-53.

210


Effects of culture conditions on Bacillus licheniformis KN12 antifungal activity against Fusarium…

TĨM TẮT

ẢNH HƯỞNG CỦA CÁC ĐIỀU KIỆN NI CẤY LÊN HOẠT TÍNH
KHÁNG NẤM Fusarium oxysporum VÀ Fusarium equiseti
CỦA Bacillus licheniformis KN12
Nguyễn Ngọc Ẩn1, Trịnh Tiền Kim Ngân1, Nguyễn Thị Diệu Hạnh1, Phạm Tấn Việt1*
Viện Công nghệ Sinh học và Thực phẩm, Đại học Công nghiệp TP. HCM

1

*

Email:

Fusarium spp. là nấm mốc gây bệnh héo rũ trên các loại cây trồng và dẫn đến thiệt hại kinh tế lớn trên
tồn thế giới. Hiện nay, để kiểm sốt các loại nấm gây bệnh này, nhiều chủng vi khuẩn đã được nghiên cứu
và ứng dụng rộng rãi trên nhiều cây trồng nhằm tìm ra các hoạt chất sinh học thay thế hóa chất tổng hợp an
tồn và hiệu quả. Trong nghiên cứu này, các điều kiện ni cấy thích hợp đã được xác định để chủng Bacillus
licheniformis KN12 tổng hợp được các hợp chất kháng nấm Fusarium oxysporum và Fusarium equiseti. Hoạt
tính kháng nấm cao nhất của dịch ni cấy được ghi nhận khi nuôi chủng vi khuẩn này trên môi trường
Luria-Bertani với pH ban đầu 7,0, ở 37 °C trong điều kiện lắc 150 vịng/phút trong 15 giờ. Hoạt tính ức chế
F. oxysporum và F. equiseti của dịch nuôi cấy vẫn được duy trì khi được xử lý nhiệt lên đến 90 °C trong 15
phút. Ngồi ra, hoạt tính kháng của dịch nuôi cấy đối với nấm F. oxysporum vẫn được duy trì trong khoảng
pH 5,0-10,0 trong khi hoạt tính này đối với F. equiseti vẫn được ghi nhận trong khoảng pH 3,0-11,0. Những
kết quả này cho thấy B. licheniformis KN12 là một chủng vi khuẩn tiềm năng có thể được ứng dụng trong
tương lai nhằm kiểm soát bệnh héo rũ bởi Fusarium trong nơng nghiệp bền vững.
Từ khóa: Bacillus licheniformis, Fusarium oxysporum, Fusarium equiseti, hoạt tính kháng mốc, điều
kiện nuôi cấy.

211

212