Characterization and Inhibitory Effects of an Antifungal
Protein from the Bacillus cereus Strain YN917
Hu Zhou1,2,3, Huajun Zhu1,2,3,
Ling Hu1, Xiyue Yu1, Zuohua Ren1,2,3
and Erming Liu1,2,3*
1College of Plant Protection,
Hunan Agricultural University, Changsha, Hunan Province, P. R. China
2Hunan Provincial Key Laboratory for Biology and Control
of Plant Diseases and Plant Pests, Changsha, Hunan Province, P. R. China
3Southern Regional Collaborative Innovation Center for
Grain and Oil Crops in China, Changsha, Hunan Province, P. R. China
*For
correspondence: ermingliu@163.com
Received 19 October 2020;
Accepted 19 February 2021; Published 16 April 2021
In this research, a type of antifungal protein derived from the Bacillus
cereus strain YN917, was precipitated by ammonium sulfate after
fermentation. We investigated the antifungal effects and antifungal mechanism
of the protein as well as its stability under different stress conditions such
as the variation of temperature, pH value, exposure to ultraviolet radiation
and proteases. Furthermore, the in vitro needle inoculation method was
carried out to explore the antagonistic effects of 40% isoprothiolane Wettable
powder (WP), 20%
tricyclazole WP and the YN917 antifungal protein against the rice blast
pathogen, Magnaporthe oryzae. Results showed that 20% tricyclazole WP and 40% isoprothiolane
EC treatments had significant antagonistic effects on rice blast, and that the
YN917 protein also had a dual inhibitory effect on mycelium growth and
conidiospore germination in M. oryzae, which can cause this mycelium to
swell and twist. The YN917 protein had a broad inhibitory spectrum with
different effects on 15 plant- associated pathogenic fungi and oomycetes, such
as Botrytis cinerea, Bipolaris
maydis and M. oryzae. In
addition, the physical and chemical properties of this antifungal protein were
relatively stable and have potential for application in the biocontrol of
fungal pathogens in plants. Overall, the results revealed that this protein has
a wide tolerance to different temperatures, pH values, exposures to ultraviolet
radiation and proteases, which is required for it to function as a bio-control
agent against rice blast under a wide range of environmental conditions. © 2021
Friends Science Publishers
Keywords: Antifungal protein; Bacillus cereus; Biocontrol;
Hyphal growth; Magnaporthe oryzae
Rice blast is a fungal disease that seriously affects rice yield and
quality and is caused by the pathogen known as Magnaporthe oryzae. The
annual worldwide loss of rice yield accounts for about 10–30% of the total
yield and has caused extreme losses to the agricultural industry sector (He et al. 2019). Extensive research on methods
to prevent and control rice blast has been undertaken worldwide. At present,
rice blast is mainly controlled by resistant varieties and by both chemical and
biological control resources (Kusajima et al. 2018; Kong et al.
2018; Kimura and Fukuchi 2018; Javaid et
al. 2019). Among these, the application of chemical fungicides is
effective, but it presents challenging downsides, such as the presence of
fungicide residues in rice grains, increased production costs and a harmful
impact on human health and the environment (Liu et al. 2017; Wu et al. 2018). Moreover, plant pathogens can
become resistant to fungicides, and cause biological imbalances and
environmental contamination (Stadnik and Borzecki 2009). In order to maintain
the common goal of sustainable agriculture development, the production and
utilization of bio-fungicides to control rice blast have seen a rapid growth in
recent years. A variety of microorganisms such as bacteria, fungi, oomycetes
and their related metabolites can be developed as biological control agents to
replace chemical fungicides (Al-Reza et al. 2010; Hammami et al. 2011; Ali et al. 2020; Shoaib et al.
2020; Sharf et al. 2021). At present,
many bio-control microorganisms exist, such as Bacillus spp., Pseudomonadaceae spp., Agrobacterium
radiobact, Actinomycesbovis spp.,
Coniothyrium, Micromonospora spp.,
Achromobacter spp., Serratia
marcescens, Bipolaris spp.
(Johansson et al. 2014), which can be
used to control rice blast.
Ongena and Jacques (2008)
reported that Bacillus spp. could be one of the major sources of
potential microbial fungicides due to their valuable characteristics. They
can prevent and control several plant diseases, including rice blast. A
variety of Bacillus strains are currently commercialized as
bio-fungicides (Bodhankar et al. 2017; Etesami et al. 2019); for
example, the American AgraQuest Company developed a bactericide preparation
SerenadeTM with B. subtilis strain QST 713 and Souata AS with B. pumilus
strain QST 2808 (Dorighello et al. 2015). Bayer, a German crop science
company developed Kodiak® Concentrate with a B. subtilis
strain (Dowd et al. 1998). China also has developed microbial
preparations, Yunnan Xingyao Biological Products Factory, Yunnan Agricultural
University and China Agricultural University cooperative developed the microbial
fungicide Baikang with B. subtilis strain B908 (Ryder et al.
2005) and Nanjing Agricultural University made a live bactericide preparation
named Maifengning with B. subtilis strain B3 (Zhang et
al. 1994). Bacillus spp. is the
main bio-control agent used against rice blast. By June 2019, there were 19
products derived from Bacillus spp. registered in China (including B.
subtilis, B. cereus, B. amyloliquefaciens), 15 of which (80% of the total)
were derived from B. subtilis.
In the early stage, a bio-control strain of B. cereus,
named YN917, was successfully isolated from a healthy rice leaf sample of the
susceptible rice cultivar ‘Xiangzaoxian No. 24’, which had a significant
inhibitory effect on M. oryzae. Therefore, considering the priority of
searching for natural alternatives to chemical fungicides, the research aim was
to separate a type of antifungal protein from the B. cereus YN917
strain, and investigate its antifungal effects; specifically, we used ammonium
sulfate for the precipitation of the YN917 antifungal protein. The stability,
inhibition spectrum, characteristics and antifungal mechanism of this protein
were investigated to offer a theoretical basis for the biological control of
rice blast and development of natural bio-fungicides.
Experimental material
The B. cereus strain
YN917 (16S rRNA GenBank: MT990515.1) used in this study was isolated
from a healthy rice leaf sample of the susceptible rice cultivar ‘Xiangzaoxian
No 24’ and stored in the Laboratory of Plant Pathogenic Microorganisms and Rice
Diseases, Hunan Agricultural University, Changsha, China. Unless otherwise specified, the strain was grown on
Luria-Bertani medium
(10.0 g L-1 tryptone, 5.0 g L-1 yeast extract, 10.0 g L-1
NaCl and pH 7.0) at 28°C. Stock cultures were maintained in 50% (v/v)
glycerol at -80°C to provide a stable inoculum during the
study period.
The tested fungus was rice blast (M.
oryzae) and other phytopathogenic fungi such as Botrytis cinerea, Bipolaris
maydis, Ustilaginoidea virens, Fusarium graminearum, Phytophthora
parasitica var. nicotianae, Alternaria solani, Rhizoctonia
solan, Verticillium dahliae, A. alternata, Diaporthe
citri, F. oxysporum,
Colletotrichum gloeosporioides and Sclerotinia
sclerotiorum.
Indica rice ‘Yuzhenxiang’ and ‘Xiangwanxian
No.12’ (Gold Nongfeng Seed Industry Technology Co., Ltd., China), and hybrid rice
‘YLiangyou 1998’ (Hunan hope Seed Technology Co., Ltd., China) were used as the test rice varieties. The tested fungicides were ‘40% isoprothiolane EC’ (500 times dilution), (Jiangsu Jiangnan agrochemical Co., Ltd., China), and ‘20% tricyclazole WP’ (750 times dilution), (Jiangsu Changqing agrochemical Co., Ltd., China).
The antifungal proteins of strain YN917 were extracted using the
ammonium sulfate fractional salting out method. After fermentation, the culture
broth of the B. cereus strain YN917 was centrifuged at 10000 r min−1
for 15 min at 4°C to remove bacterial cells, and then the cell-free supernatant
was adjusted to 20, 30, 40, 50, 60, 70, 80 and 90%, in this order, with solid
ammonium sulfate under ice bath conditions. It was then stored at 4°C for 48 h.
The precipitate was obtained by centrifugation at 10000 rmin−1
for 15 min at 4°C and it was completely dissolved in phosphate buffered saline
(PBS; pH 7.4). Subsequently, the precipitation was dialyzed against the same
buffer for 2 d, ammonium sulfate was removed, and the antifungal activity test
was then assayed. The crude extract was filtered through a 0.22 μm bacterial filter and stored at
−20°C until use. Each sample was examined to determine the strongest
antifungal activity, which had the optimum ammonium sulfate saturation required
to prepare the antifungal protein. The experiment was repeated three times.
The M. oryzae samples were placed
in the center of a potato dextrose agar medium firstly, which was then cut
through with a cork borer to form a hole of 6 mm in diameter at the edge, 25 mm
away from the plate center. The treated protein was prepared and 40 µL
of respective treatments were added to each hole. Distilled water was used as
the negative control. Each treatment was replicated three times. The dishes
were incubated at 28°C until mycelial growth was reached in more than 3/4 of
the culture dish, the colony diameter was measured and the inhibition rate of
the antifungal YN917 protein on the growth of pathogens was calculated.
The dual culture method was used in this study to determine the
inhibition of the YN917 antifungal protein on 15 plant pathogenic fungi and
oomycetes: M. oryzae, two B. cinereal strains,
B. maydis, U. virens, F. graminearum,
P. parasitica var. nicotianae,
A. solani, R. solan, V. dahliae, A. alternata,
D. citri, F. oxysporum,
C. gloeosporioides, S. sclerotiorum.
The experiment was carried out as described in Arpita et al. (2019).
High temperature tolerance: To study the thermostability of
the YN917 antifungal protein, equal sample volumes were placed at −20°C,
4°C, 20°C, 30°C, 40°C, 50°C, 60°C, 70°C, 80°C, 90°C, 100°C and 121°C, for 30
min and then they were quickly cooled down to room temperature on ice.
pH tolerance: The pH values of equal sample
volumes (1 mL) were adjusted to 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0
and 12.0, by HCl (1 mol L−1) or NaOH (1 mol L−1).
The solution was kept at 4°C for 24 h. Afterwards, the samples were readjusted
to pH 7.0.
Protease tolerance: Equal sample volumes were
treated with 1 mg mL−1 protease K, papain, pepsin, trypsin and
alkaline protease, at 37°C for 1 h, and then kept in a water bath at 100°C for
5 min to inactivate the protease.
Ultraviolet radiation tolerance: Equal sample volumes were placed under 28W ultraviolet
radiation at 10 cm, and were irradiated for 0.25 h, 0.5 h, 0.75 h, 1 h, 2 h, 4
h, 6 h and 12 h. A sample of untreated antifungal protein of the same
concentration was used as control. The experiment was conducted twice, and
three independent replicates were conducted for each treatment.
The activity of the antifungal protein against M.
oryzae was assessed based on the
experiments described above.
To assess the effect of the YN917
antifungal protein on the mycelial morphology of M. oryzae, 1 mg
mL−1 of protein was added to a 250 mL Erlenmeyer flask filled with 50 mL of fresh
PDB medium containing M. oryzae. The flask was kept at 28°C and shaken
at 180 r min−1; it was then observed by microscope (× 40 magnification) after 1 d, 2 d, 3
d, 4 d and 5 d. All assays were conducted
in triplicate, and an intact mycelium showing normal growth with an equal
volume of dd-H2O addition served as negative control.
M. oryzae spores were mixed with the YN917 antifungal protein in a ratio of 1:1 (v/v), sterile distilled water was used as negative control. The treatments were placed at 28°C, and spore germination was observed by microscope (× 40 magnification) after 2 h, 4 h, 6
h, 8 h, 10 h and 12 h. One hundred single conidia per dish were examined, the
experiment was duplicated, and three independent replicates were conducted for
each treatment.
Control of rice blast M. oryzae was evaluated by in
vitro inoculation performed on rice leaves (Shi et al. 2015). In brief, same-sized healthy leaves were selected for testing at the 5-leaf stage. For the preventive treatment, the surface of the
leaves was sprayed with 100 µg mL−1 of YN917 antifungal
protein first, and after 24 h a M. oryzae spore
suspension was
inoculated. For the curative treatment, leaf surfaces were sprayed with 100 µg
mL−1 of antifungal protein 24 h after being inoculated with
the spore suspension. Negative controls were treated the same way using
sterile distilled water, and the positive controls were treated with a 500
times diluent of 40% isoprothiolane EC, and a 750 times diluent of 20%
tricyclazole WP, in both the preventive and curative assays. All experiments
were carried out in triplicate. The samples were placed at conditions of 85–100% relative humidity and temperature of 28°C, with
a 12 h photoperiod for 7 d. They were moisturized regularly with water to
maintain hydration, and the antagonistic effect was analyzed.
The data were analyzed in S.P.S.S. 20.0 and were
subjected to Duncan’s analysis of
variance; the means were separated by Duncan’s multiple range tests at P ≤ 0.05.
Optimum saturation of ammonium sulfate precipitation
Different proteins have different saturation
characteristics when they are precipitated by (NH4)2SO4.
Therefore, the specific optimum (NH4)2SO4
precipitation saturation of the YN917 protein
was investigated in this study. The
results (Fig. 1) showed that the protein precipitated (NH4)2SO4
at 20–30% saturation had no inhibition on rice blast fungus, M. oryzae. When precipitated by
(NH4)2SO4 at 40% saturation, a slight
inhibition on M.
oryzae was observed. When precipitated by (NH4)2SO4
at 60% saturation, the protein increased considerably, and the inhibition was significantly
enhanced. The protein precipitated by (NH4)2SO4
at 80% saturation showed a stronger antifungal activity than at other
saturation levels, while the inhibition effect declined when the saturation was
more than 80%. The results indicate that the optimum saturation
of the precipitated antifungal crude protein from strain YN917 was 80%.
The antagonistic test using common plant pathogens and oomycetes showed that the antifungal protein had a broad-spectrum antagonistic activity against the pathogens chosen
for this research (Table 1).
The maximum inhibition in the radial growth caused by the antifungal
protein was 51.67% ± 1.73% for grape gray-mold fungus B. cinerea, 46.10% ± 3.22% for strawberry gray-mold fungus B. cinerea, 42.86% ± 1.35% for southern
corn-leaf blight fungus B. maydis and 39.97% ± 1.23% for rice blast fungus M. oryzae. When compared with the controls, each of the treatments
showed a significant difference (P ≤ 0.05) suggesting this protein has a potential to be successfully used in disease prevention.
Table 1: Antagonistic effects of B. cereus YN917 antifungal crude protein
against fungal pathogens
Item |
Host |
Pathogen |
Inhibition
(%) |
1 |
Grape |
51.67 ± 1.73 |
|
2 |
Strawberry |
B. cinerea |
46.10 ± 3.22 |
3 |
Maize |
Bipolaris maydis |
42.86 ± 1.35 |
Fig. 1: Inhibitory results with
different ammonium sulfate saturation Fig. 2: Stability of YN917 antifungal protein at different temperatures Fig. 3: Stability of YN917 antifungal protein at different pH values 4 |
Rice |
Magnaporthe oryzae |
39.97 ± 1.23 |
5 |
Rice |
Ustilaginoidea virens |
39.25 ± 0.43 |
6 |
Wheat |
Fusarium graminearum |
38.82 ± 2.04 |
7 |
Tobacco |
Phytophtora parasitica var. nicotianae
|
38.02 ± 2.19 |
8 |
Potato |
Alternaria solani |
32.63 ± 1.15 |
9 |
Rice |
Rhizoctonia solan |
31.94 ± 0.09 |
10 |
Cotton |
Verticillium dahliae |
30.26 ± 1.12 |
11 |
Tobacco |
A. alternata |
29.14 ± 3.94 |
12 |
Orange |
Diaporthe Citri |
28.70 ± 1.23 |
13 |
Spicy |
F. oxysporum |
24.58 ± 0.84 |
14 |
Camphor |
Colletotrichum gloeosporioides |
22.97 ± 0.51 |
15 |
Rape |
Sclerotinia sclerotiorum |
22.48 ± 1.43 |
Mean ± standard deviation
Physicochemical properties
Temperature tolerance: To examine thermo-stability, the antifungal crude
protein was placed at different temperatures for 1 h and antifungal activity
was determined. The results (Fig. 2) showed that the inhibition rate on M.
oryzae changed significantly after being exposed to different temperatures
(P ≤ 0.05). The antifungal activity was stable after exposure to
temperatures between −20°C
and 40°C.
When the temperature was higher than 40°C, the inhibition rate and associated antifungal
activity was significantly decreased. After exposure to 70–121°C, the inhibition
rate continued to decrease significantly. This indicated that the crude
antifungal protein has thermostability, and it can function adequately in the
natural environment, showing a significant potential for its development and
application.
pH tolerance: To assess pH stability, the antifungal protein was incubated at different pH values for 24 h and antifungal activity was determined. The results (Fig.
3) showed that the inhibition rate on M. oryzae changed significantly
after different pH treatments (P ≤ 0.05). The inhibition rate of the YN917 antifungal protein was
stable at pH values between 5.0 and 8.0, and the inhibitory activity sharply
decreased at pH values < 5.0 or pH values > 8, which indicated that the
protein is sensitive to overly acidic or overly alkaline conditions, but it
maintains a sufficient antifungal activity against M. oryzae when pH is
between 5.0 and 8.0.
Protease tolerance: Compared with the
control, the inhibition rate of the antifungal protein did not change during
protease treatments. However, when comparing protease treatments, the protein
was more sensitive to protease K, alkaline protease, trypsin, pepsin and
papain, and its antifungal activity was weakened after these treatments (Fig.
4). It was particularly sensitive to trypsin and pepsin, which indicated that
the antifungal protein can be digested by proteases in the human digestive
system and it will not adversely affect human health. Therefore, this protein
can be safely used in the biological control of crops.
Ultraviolet radiation tolerance: To
assess UV stability, the antifungal protein was irradiated by ultraviolet
radiation for 0.25–12 h and antifungal activity was determined.
The activity remained stable (Fig. 5), which indicated that the protein is not
sensitive to ultraviolet radiation and has good anti-ultraviolet properties.
Fig. 4: Stability of YN917 antifungal protein in the presence of proteases
Fig. 5: Stability of YN917 antifungal protein exposed to ultraviolet radiation
Effect of the YN917 protein on M. oryzae spores
Spore production
and germination of M.
oryzae were determined in the
presence of YN917. The results obtained showed the protein preparation had a significant potent inhibitory
effect on the fungal spore production of M.
oryzae at varied times. Distilled water, used as negative control, did not
inhibit spore production (Fig. 6 and
Table 2). Moreover, the spores treated with the protein exhibited a swollen
shape, while non-treated spores maintained their normal morphology. This
suggests that the antifungal protein has a strong inhibitory effect on the
spore germination of M. oryzae, affecting also the permeability
of the conidiospore membranes.
Effect of the YN917 protein on M. oryzae mycelium
To assess the
strength of the protein’s antifungal effect, the mycelium of M. oryzae
was microscopically observed near the zone where inhibition occurred, (Fig. 7)
which revealed signs of extensive lysis of the spores; in particular abnormal
distortion, slender shapes, multiple swelling and spore disintegration were
observed. In contrast, the untreated negative control presented a normal
mycelium and smooth surfaces. This suggests that the YN917 antifungal protein is effective in inhibiting
spore formation and germination.
In order to
evaluate the antifungal activity of B. cereus YN917 against M. oryzae, in vitro experiments were
carried out on infected rice leaves of the rice cultivars ‘Yuzhenxiang’,
‘Y-liangyou1998’ and ‘Xiangwanxian No.12’.’ The efficacy of biocontrol in the separate leaf assays is shown
in Table 3. In both the preventive and curative treatments, the YN917 antifungal
protein showed a significant bio-control efficacy against M. oryzae.
In the prevention treatment, leaves treated with the protein had a lower incidence rate of
disease (18.4% in Yuzhenxiang, 19.63% in Yliangyou 1998 and 14.26% in
Xiangwanxian No 12) and presented smaller lesions than leaves without any
treatment (97.26% in Yuzhenxiang, 97.66% in Yliangyou 1998 and 98.33% in
Xiangwanxian No 12). In the curative treatment, the
disease indexes of the protein were 22.53, 25.31 and 24.51%, respectively. The
results were similar in both the preventive and curative samples,
indicating that the YN917 antifungal protein
may be effective as a potential bio-control agent against rice blast.
Table 2: Effect of YN917 antifungal protein on M.
oryzae spore germination
Treatment |
2 h |
4 h |
6 h |
8 h |
10 h |
12 h |
CK |
7.69 ± 1.49 |
34.48
± 3.46 |
55.26
± 2.61 |
74.45
± 2.32 |
88.19
± 2.55 |
88.72
± 3.25 |
YN917 protein |
1.96 ± 0.64 |
4.00 ± 1.03 |
13.16
± 1.83 |
12.30 ± 2.31 |
24.16
± 3.07 |
22.72
± 2.16 |
Table 3: Effect of YN917 antifungal protein on
rice blast control in vitro
Treatments |
Yuzhenxiang |
Y-liangyou1998 |
Xiangwanxian No.12 |
|||
–24 h |
+ 24 h |
–24 h |
+24 h |
–24 h |
+24 h |
|
CK |
97.26
± 2.48 a |
92.41
± 3.66 a |
97.66 ± 1.66 a |
94.75
± 1.67 a |
98.33 ± 2.36 a |
98.30
± 0.42 a |
25% Tricyclazole WP |
7.36 ± 0.55 c |
54.46 ± 2.29 b |
10.67 ± 0.94 c |
52.96
± 2.28 b |
9.00 ± 0.82 d |
56.11
± 2.83 b |
20.15
± 1.52 b |
12.22 ± 1.38 d |
22.96 ± 2.77 b |
11.04
± 0.82 d |
30.99 ± 1.67 b |
16.40
± 1.85 d |
|
100 ug/mL protein |
18.40
± 1.36 b |
22.53 ± 4.16 c |
19.63± 2.29 b |
25.31 ± 1.43 c |
14.26 ± 2.33 c |
24.51
± 2.00 c |
Mean ± standard deviation. Values sharing same letters do not
significantly differ (P > 0.05)
Fig. 6: Effect of the YN917 antifungal protein on M.
oryzae spores
(A)
Normal spores (CK); (B), (C) abnormal spores
of M. oryzae
appear
bulbous and swollen
Fig. 7: Effect of the YN917 antifungal protein on M.
oryzae mycelium
A
negative control, without YN917 protein treatment showing normal mycelium
morphology and spore germination; B mycelium treated with YN917 protein showing
extensive lysis of the mycelia and destructed spores
In
relation to the present study specifically, the results have confirmed that the
YN917 protein has a wide temperature adaptation range, and can maintain a high
antifungal activity after being exposed to 80°C for 30 min. This protein is considerably adapted to withstand
high temperatures, providing an advantage when used in cultivation crops
(specifically in the summertime). In addition, it is not sensitive to
ultraviolet radiation, but acidic pH values in in vitro experiments seemed to significantly restrict its activity.
The suitable soil pH value ensuring antifungal
activity was between 5.0 and 8.0, which does correspond to the natural
growth ring of common crops. Moreover, the protein is sensitive to trypsin and
pepsin treatment, which indicates that it can be digested by protease in the
human digestive system and is therefore harmless to human health. In summary,
this study has shown that from an ecological point of view, the YN917 protein
has a wide tolerance to various stressors such as temperature, pH, ultraviolet radiation,
and protease, which suggests that it could be applied to a wide range of
environmental conditions.
Many
antifungal proteins derived from Bacillus spp. have a strong inhibition
activity (Gotor-Vila et al. 2017). For example, the antifungal protein derived from B. subtilis ZL2-70 was shown to strongly
inhibit V. dahliae, Fusariumum graminearum,
C. gloeosporioides, and 19 other plant pathogens;
the Bacisubin protein isolated from B. subtilis B-916 can inhibit the
growth of various pathogens such as M. oryzae, B. cinerea, and A.
brassicae (Luo et al. 2009). The
antifungal protein YN917 derived from the B. cereus YN917 strain
presents a broad antifungal spectrum, which can effectively inhibit the
mycelial growth of 15 pathogenic fungi and oomycetes, such as M. oryzae,
B. cinerea and B. maydis. The experiments carried out in this
particular study showed that this antifungal protein has a strong inhibitory
effect on the growth and germination of M. oryzae spores. Furthermore,
several antifungal proteins have been shown to inhibit fungal mycelium and the germination of spores
(Leelasuphakul et al. 2008; Matar et al. 2009). For example, a
novel protein produced by B. subtilis B29 (Li et al. 2009), can
inhibit the conidial spore germination of F. oxysporum, suppress
germ-tube elongation, and induce distortion, tumescence and rupture of a
portion of the germinated spores. The Bacisubin protein, produced by B.
subtilis B-916 (Liu et al. 2007) can cause the R. solani mycelia to enlarge and rupture, and the crude
antifungal protein RY3 produced by B. amyloliquefacens RY3 can
inhibit the germination of P. digitatum spores (Chen et al.
2013). Finally, the protein produced by B. cereus HS24, can
significantly inhibit conidium germination and mycelial growth in M. oryzae (Huang
et al. 2019).
Bacillus spp. in nature can produce different secondary
metabolites, which are related to
microorganisms in different ecological environments. These secondary
metabolites include antifungal and antiseptic substances, biofertilizers, as well as various active enzymes. Therefore,
the determination of the antifungal components produced by the YN917 protein
will be the objective of our future research.
In
the present work, results indicate that there are natural antifungal substances
in the YN917 strain of B. cereus. The
YN917 protein showed a broad-spectrum antagonistic activity against 15 plant
pathogens. Its physical and chemical properties were relatively stable and had
a dual inhibitory effect on mycelium growth and conidiospore germination in M.
oryzae. In vitro experiments revealed that its control effect against rice
blast was considerable. Therefore, protein extracts from the YN917 strain can
be considered as a promising and novel bio-control resource to be used in
agricultural production.
This work was funded by the National Key R&D Program of China
(2016YFD0300700), central government financed projects, China (2014ZX0800102B)
and the Public welfare industry (agriculture) special scientific research
projects, China (201203014).
Author Contributions
Conceptualization, HZ; Methodology, HZ; Validation, HZ, HZ, LH, XY;
Formal Analysis, HZ and HZ; Data Curation, HZ; Writing-Original HZ;
Writing-Review and Editing, HZ and EL; Supervision, ZR; Funding Acquisition, ZR
and EL
Conflict of Interest
The authors declare that they have no conflict of interest.
Data Availability
All data will be available
upon reasonable request to the corresponding author.
Ethics Approval
Not applicable.
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