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Article

Antagonistic Activity and Potential Mechanisms of Endophytic Bacillus subtilis YL13 in Biocontrol of Camellia oleifera Anthracnose

by
Yandong Xia
,
Junang Liu
,
Zhikai Wang
,
Yuan He
,
Qian Tan
,
Zhuang Du
,
Anqi Niu
,
Manman Liu
,
Zhong Li
,
Mengke Sang
and
Guoying Zhou
*
Key Laboratory of National Forestry and Grassland Administration on Control of Artificial Forest Diseases and Pests in South China, College of Life Science and Technology, Central South University of Forestry and Technology, Changsha 410004, China
*
Author to whom correspondence should be addressed.
Forests 2023, 14(5), 886; https://doi.org/10.3390/f14050886
Submission received: 4 March 2023 / Revised: 14 April 2023 / Accepted: 21 April 2023 / Published: 26 April 2023
(This article belongs to the Special Issue Management of Forest Pests and Diseases II)
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Abstract

:
Anthracnose, caused by the fungus Collectotrichum fructicola (C. fructicola), is a major disease affecting the quality and yield of Camellia oleifera (C. oleifera); it reduces C. oleifera yield by 40%–80%. Bacillus subtilis (B. subtilis) YL13 is an antagonistic endophytic bacteria strain isolated from healthy C. oleifera leaves. This study was aimed at investigating the potential of YL13 for the biocontrol of C. oleifera anthracnose and the possible mechanisms involved. In in vitro assays, YL13 demonstrated remarkable antagonistic activity of C. fructicola. Its cell-free filtrates displayed antagonistic activity, which suggested that the metabolites of YL13 might play important roles. In vivo tests showed that the disease index of YL13-treated plants was obviously reduced under greenhouse conditions. YL13 secretes a variety of bioactive metabolites, including protease, cellulase, and siderophore, which might participate in the resistance to C. fructicola. In addition, C. oleifera treated with the fermentation broth of YL13 demonstrated different defense responses, e.g., accumulation of hydrogen peroxide (H2O2) and activation of the defense-related enzyme peroxidase (POD), which might contribute directly or indirectly to overcome external stresses. The significant biocontrol effect and host defense-induction activity of YL13 suggested that this B. subtilis strain as well as its metabolites have the potential to be exploited as microbial control agents for the efficient management of C. oleifera anthracnose.

1. Introduction

Camellia oleifera (C. oleifera) is a unique woody edible oil tree species in China. Camellia oil from the C. oleifera seeds contains a high amount of unsaturated fatty acids, and it is widely used as cooking oil because of its high nutritional value and healthful properties [1,2]. With the rapid increase in C. oleifera planting areas, the occurrence of C. oleifera diseases is becoming more and more serious. In particular, C. oleifera anthracnose is the most common disease in C. oleifera planting areas. Its main harm is to cause flower and fruit drops, which will reduce the yield. Generally, it can cause the loss of C. oleifera seeds to be about 20%, and the high loss can reach more than 40%. The oil content of diseased seeds is only 50% of that of healthy seeds, or even lower [3].
The pathogens causing C. oleifera anthracnose are various, involving several species of the genus Anthrax. It is difficult to distinguish the species of the microbes only based on the morphological characteristics of colonies, conidia, and appressoria. At present, it is mainly identified by morphological features and multi-gene sequence information. According to previous reports, there are mainly eight kinds of anthrax pathogens in C. oleifera, including C. fructicola, C. camellia, C. siamense, C. horii, C. karstii, C. boninense, and C. cliviae, and the dominant pathogen is C. fructicola [4,5,6,7].
Nowadays, the main method for controlling C. oleifera diseases is chemical control. The use of chemical pesticides can quickly reduce losses. However, the pathogens of C. oleifera anthracnose have strong adaptability and are prone to developing drug resistance [8]. In addition, the use of large doses of chemical pesticides will not only seriously pollute the environment but also increase the pesticide residues in C. oleifera seeds, reduce the quality of camellia oil (tea seed oil), and affect the healthy development of the camellia oil industry [9]. With the enhancement of people’s awareness of environmental protection, global researchers are also increasingly concerned about biological control. The market demand for biological control agents is growing gradually, and the search for non-toxic and efficient means to control C. oleifera diseases has also received widespread attention [10]. Endophytes colonize plants and establish a co-evolutionary relationship with their hosts. It can be said that plant endophytes are an important component of plant micro-ecosystems, and they might work better for controlling pathogen attacks than microorganisms that act outside the plant without causing visible diseases of host plants [11]. Endophytes can promote host plant growth, participate in the host defense response, and enhance the host’s ability to resist disease and stress; therefore, they have received extensive attention from researchers. Screening and utilization of antagonistic endophytes has become a new research direction in the efficient management of C. oleifera anthracnose [12].
Bacillus has been commonly identified as biocontrol endophytic bacteria in different plants [13], such as B. amyloliquefaciens and B. subtilis, which can form spores and resist various adverse environments [14]. The direct or indirect mechanisms of Bacillus in controlling plant diseases mainly include the synthesis of various metabolic substances, such as cell wall degrading enzymes, antagonists, iron carriers, hormones, etc., to inhibit the growth of pathogens, compete with pathogens for nutrients, induce plant system resistance, and promote plant growth [15]. For example, the three Bacillus strains isolated by Zhang inhibited the growth of pathogens due to their ability to secrete antagonistic enzymes [16]. Siderophores production gives endophytes an advantage in the competition with pathogens for iron and enhances Fe absorption in plants, promoting plant growth [17]. Moreover, B. velezensis ZW10 showed a significant biocontrol effect in Chen’s study. The antimicrobial compounds of ZW10 inhibited the growth of pathogen hyphae and elicited host defense responses, e.g., accumulation of hydrogen peroxide, activation of defense-related enzymes, and up-regulated expression of defense-related pathway genes [18]. Xu isolated and identified a B. subtilis strain with antagonistic activity against different pathogens, while the biocontrol effect and mechanisms remained to be revealed [19]. Up to now, there are relatively limited reports on the control of C. oleifera anthracnose by B. subtilis in greenhouses or fields, and the mechanisms have not yet been studied.
The bacteria B. subtilis YL13 was isolated from healthy C. oleifera leaves. Previous studies have shown that B. subtilis YL13 displayed an antagonistic effect against the different pathogens of C. oleifera diseases, such as C. gloeosporioides, C. siamense, and C. camelliae [20]. YL13 exhibited a certain inhibitory effect on C. oleifera anthracnose in detached leaf assays. However, the mechanisms involved are still unclear. In this study, we tested and analyzed the characteristics of YL13 against C. oleifera anthracnose and primarily revealed its interaction mechanism with the host plants.

2. Materials and Methods

2.1. Plant Material, Strain and Pathogen

Two-year-old cuttings of C. oleifera cultivar “Huashuo” were purchased from Hunan Tianhua C. Oleifera Technology Co., Ltd., Youxian County of Zhuzhou, Hunan, China (26°52′5″ N, 113°19′25″ E). The phytopathogenic fungus C. fructicola was deposited in the China General Microbiological Culture Collection Centre (CGMCC 3.17371). The endophytic bacteria YL13 were isolated and maintained by the Key Laboratory of National Forestry in South China (Changsha, China). C. fructicola was grown on the potato dextrose agar medium (PDA) at 28 °C. PDA medium contains peeled potatoes (200 g), dextrose (20 g), and agar (15 g), all in 1 L of ddH2O. YL13 was grown in the Luria-Bertani broth medium (LB) at 30 °C. LB medium contains yeast extract (5 g), tryptone (10 g), and sodium chloride (10 g), all in 1 L of ddH2O.

2.2. Antagonistic Activity of YL13 on the Pathogen of C. oleifera Anthracnose In Vitro

Plate-culture assays were conducted to test the antagonistic ability of the fermentation broth and the cell-free filtrate of the strain YL13 against the pathogenic C. fructicola [21]. The spores of C. fructicola on PDA were scraped and dipped in sterile distilled water. The concentration of the fungal spore suspension was adjusted to 106 spores mL−1 using a hemocytometer. PDA with the C. fructicola spore suspension (19:1, v/v) was poured into the plates after they were quickly mixed well in a tube. Oxford cups were placed on the plates in an equilateral triangular arrangement.
As for preparing the cell-free filtrate of the strain YL13, the supernatant was harvested by centrifugation (6000× g, 10 min) and filtered by a membrane filter (0.22 μm). The filtrate for each day (per 24 h) was pipetted into the Oxford cup in the center of the PDA plate, mixed with the C. fructicola spore suspension (106 spores mL−1, 1:19, v/v), 100 μL per cup, then put into the incubator at 28 °C and observed. Each assay was repeated twice.
The hyphal structure of C. fructicola was observed by scanning electron microscopy (SEM) to detect the effect of the antagonistic strain on the pathogen. A mycelial plug of the fungus C. fructicola (6 mm diameter) was inoculated at the center of the PDA plate, and the bacteria YL13 was inoculated at the three vertices of the triangle distribution. The control group was only inoculated with a mycelial plug. All plates were incubated at 28 °C for 7 days. The hyphal of C. fructicola towards the antagonist on dual-culture assay plates was collected. The hyphal of C. fructicola not exposed to the antagonistic bacteria YL13 on PDA plates was collected as the control. Samples were fixed overnight in 2% glutaraldehyde (4 °C) and washed with phosphate buffered saline (0.1 M, pH 7.4), and then dehydrated through the ethanol solutions (sequentially, 10%, 30%, 50%, 70%, 90%). For ultrastructure observation by SEM, the freeze-drying and gold-coating of the samples were performed as previously described [22].

2.3. Biocontrol Activity of YL13 against C. oleifera Anthracnose In Vivo

The YL13 strain was inoculated in LB liquid medium and shaken at 180 rpm and 30 °C for 72 h. The YL13 fermentation broth was adjusted into three concentrations with sterile water (1.0 × 104 CFU mL−1; 1.0 × 106 CFU mL−1; 1.0 × 108 CFU mL−1) by an ultraviolet spectrophotometer. C. oleifera mature leaves were sprayed with about 25 mL fermentation broth or LB medium as the control. 24 h later, the leaves were sprayed with the C. fructicola spore suspension at concentrations of 106 spores mL−1. The disease index was estimated and calculated 30 days after the inoculation of the pathogen. Each assay was carried out three times, and each treatment had six replicates. Assays were conducted in a greenhouse with a 10 h light 28 °C and 14 h dark 25 °C cycle, light 800 μmol (m2 s) −1, and humidity 70%. The disease level was estimated and scored from zero to four; the disease index as well as biocontrol efficacy were determined based on the previous studies [23,24]:
Disease index (%) = [∑ (number of the diseased plants × the assessed disease index)/(total number of samples × the highest disease index)] × 100%;
Biocontrol efficacy (%) = [(the disease index of the control—the disease index of the treatment)/disease index of the control] × 100%.

2.4. Detection of the Metabolites Produced by YL13

The production of various lytic enzymes and bioactive metabolites is a prominent mechanism of many biocontrol bacteria [25]. The ability of the bacteria YL13 to secrete different functional compounds, like β-1,3-glucanase, protease, cellulase, and siderophore, was determined [26,27,28]. Each assay was carried out three times. Three replications were conducted for each group.

2.5. Measurement of H2O2 Levels in C. oleifera Leaves

The foliar spraying treatment with YL13 fermentation broth (1.0 × 108 CFU mL−1) on C. oleifera plants was performed as described above. Spraying with LB medium was set as the control. For the measurement of H2O2 levels, the leaf samples were picked at different time points after treatment (2, 4, 8, 12, 24, 48, 72, and 168 h). Samples (0.1 g) were ground in precooled acetone (1 mL) in a mortar. Then the homogenate was centrifuged at 12,000 rpm (for 10 min at 4 °C), and the supernatants were prepared for detection. H2O2 levels were assayed using the reagent kits (Suzhou Grace Biotechnolgy Co., Ltd., Suzhou, China) according to the instruction manual. Each assay was carried out three times. Three replications were conducted for each group.

2.6. Measurement of POD Activity in C. oleifera Leaves

The peroxidase (POD) levels of YL13 fermentation broth (1.0 × 108 CFU mL−1) inoculated leaves were tested after 0, 6, 12, 24, 48, 72, and 168 h. Treatments with LB medium were set as the control. The leaf samples (0.5 g) were homogenized with the sodium phosphate buffer (4.5 mL, 0.1 mol L−1, pH 7.0) in an ice bath. Then the homogenate was centrifuged at 3500 rpm (for 10 min at 4 °C), and the supernatants were retained for the measurement. The POD activity was detected according to the protocol of the detection kits (Jiancheng Bioengineering Research Institute, Nanjing, China). Each assay was carried out three times. Three replications were conducted for each group.

2.7. Statistical Analyses

The data were statistically analyzed with SPSS software (Version 19.0, Armonk, NY, USA). Statistical significance was detected according to the Fisher’s least-significant difference test (p < 0.05), and the independent-samples test was performed to determine the significance of the difference: *p < 0.05, ** p < 0.01, *** p < 0.001.

3. Results

3.1. Antagonistic Activities of YL13 In Vitro

The fermentation broth of strain YL13 exhibited a critical inhibition effect on the growth of C. fructicola according to the dual-culture assays (Figure 1A). The antagonistic effect of YL13 increased with an increase in culture time. The YL13 fermentation broth, which was cultured for 72 h, displayed the most remarkable antagonistic activity. YL13 cell-free filtrate obviously revealed antagonistic activity on C. fructicola. The diameters of the inhibition zone on the PDA plate with YL13 treatment filtrate previously cultured for 72 h were significantly larger than those on the plate with YL13 treatment filtrate just cultured for 24 h (Figure 1B). Consistently, the observation of C. fructicola hyphae by SEM confirmed this. Compared with the control that was not exposed to the antagonistic bacteria YL13, C. fructicola towards YL13 showed obvious changes in the hyphae; for instance, the hyphae were wrinkled and irregularly twined into clumps (Figure 1C).

3.2. Biocontrol Efficacy of YL13 on C. oleifera Anthracnose in Greenhouse

In this study, pot experiments demonstrated that YL13 could significantly suppress anthracnose in C. oleifera plants under greenhouse conditions (Table 1). LB medium was set as the control. The disease incidence of the control uninoculated with YL13 was 68.3%, significantly higher than that of the other treatments. Similarly, thirty days after leave inoculation with YL13 fermentation broth, the biocontrol efficacy of treatments 1, 2, and 3 were 41.3%, 56.7%, and 67.9%, respectively. The control effect was significantly negatively correlated with the disease index. With the increase in concentration of the YL13 fermentation broth, the control effect increased obviously.

3.3. The Metabolic Substances Synthetized by YL13

The metabolic substances synthesized by YL13 were qualitatively tested and shown in Table 2. YL13 can grow well on the casein medium and cellulose medium, and a clear circle surrounding the colonies revealed that YL13 can not only produce protease (Figure 2A) but also produce cellulase (Figure 2B). However, it was unable to detect the release of β-1,3-glucanase by YL13. YL13 also produced iron chelator siderophore as a result of a clear halo around the colonies (Figure 2C), which might inhibit pathogen growth by nutrient competition of ions [29].

3.4. Effect of YL13 on H2O2 Accumulation in C. oleifera Leaves

The elevated accumulation of hydrogen peroxide (H2O2) should be the earliest and most essential defense response in plants [30]. The H2O2 levels in C. oleifera leaves are shown in Figure 3. At 0–4 h, a quick accumulation of H2O2 was observed, which then started to decline. Subsequently, the H2O2 level increased and reached its peak at 12 h. In other words, at 0–72 h, the H2O2 burst was particularly greater in the YL13-inoculated leaves, with two remarkable peaks, while no significant effect on the POD activities was detected in the control group (LB medium was set as the control). The present results revealed that treatment with strain YL13 could lead to H2O2 accumulation in C. oleifera leaves.

3.5. Effect of YL13 on POD Levels in C. oleifera Leaves

POD is important for balancing H2O2 levels in plants. The POD levels in C. oleifera leaves are shown in Figure 4. At 6 and 12 h, a notable increase in POD activity was observed in the YL13-inoculated leaves, with a peak value at 12 h. Then it gradually decreased. However, there was no significant increase in POD activity in the control group. These data suggested that the defense-related enzyme POD activity was significantly increased due to the treatment with the strain YL13.

4. Discussion

Compared with rhizosphere and soil microorganisms, endophytes colonized in plants have stable living spaces and are not vulnerable to environmental conditions [31]. In the long-term process of co-evolution, endophytes and plants have established a mutually beneficial relationship [32]. Plants can provide nutrition for endophytic microbes, and the microbes help plants resist the invasion of pathogens [33]. Therefore, endophytes have shown a good development prospect in the exploitation and application of microbial agents resistant to plant diseases, greatly promoting the green evolution of agriculture. Studies have shown that the main mechanisms of endophytes in plant protection include: (1) producing metabolic substances to directly inhibit pathogen growth, such as antibacterial compounds [34]; (2) nutrition and niche competition with pathogens [35]; (3) secreting a series of hydrogen enzymes, such as the β-1,3-glucanases, chitinases, and cellulases [35]; (4) initiating defense responses of host plants (ISR and SAR); (5) promoting the growth of host plants and indirectly improving the disease resistance of plants [36,37].
At present, the Bacillus group used for biological control of plant disease mainly includes B. subtilis, B. amyloliquefaciens, B. pumilus, and so on. Many studies have shown that B. subtilis has strong antagonistic activity against a variety of plant pathogens [38]. Rajkumar et al. found out that the biocontrol effect of strains determined by the pot test was more reliable than that determined by the detached leaf assay in vitro [39]. The pot experiment carried out by Liu et al. demonstrated that the control efficacy of the strain B. subtilis RSS-1 against soybean phytophthora root, which is caused by the pathogen Phytophthora sojae, reached 65.5% [40]. The application and mechanism investigation of B. subtilis in the prevention and control of C. oleifera anthracnose has already attracted researchers’ attention. In this study, we used the fermentation broth of the strain YL13 to conduct pot experiments in greenhouses, further verifying the biocontrol effect of this strain or its bioactive metabolites. YL13 played a constructive role in controlling C. oleifera anthracnose in vitro as well as in vivo. The B. subtilis strain YL13 and its metabolites have the potential to be exploited as microbial control agents against C. oleifera anthracnose.
It has been proven that some Bacillus spp. can produce a variety of enzymes, e.g., β-1,3-glucanase, cellulase, and protease, which play a crucial role in the degradation of the cell walls of fungi [41,42]. The B. cereus YN917 strain isolated by Zhou et al. exhibited disease prevention properties due to the production of several metabolic substances, like β-1,3-glucanase, protease, as well as siderophores [43]. Zheng et al. investigated in their research that the control efficacy of the strain B. velezensis D61-A against rice sheath blight caused by Rhizoctonia solani was 61.5% in greenhouses, and one of the action mechanisms was that this bacteria could secrete siderophores and lytic enzymes like cellulase and protease [44]. Similarly, in this study, the stain YL13 could produce relevant active enzymes, including cellulase and protease, to affect the morphology of the pathogen hyphae. This was in agreement with the SEM results. The microstructure observation results proved that the hyphae of the pathogen C. fructicola towards the antagonistic bacteria YL13 were deformed and irregularly twined into clumps when compared to the control that was not exposed to YL13. In addition, siderophore production is also one of the mechanisms occupied by YL13 to inhibit pathogen growth.
When plants are invaded by pathogens or microorganisms, different defense responses can be stimulated [45]. Systemic acquired resistance (SAR) and induced systemic resistance (ISR) are two key models for plants to respond to the pathogen attack. ISR is usually mediated by the jasmonic acid (JA) and ethylene signaling (ET) pathways, while SAR is generally mediated by the salicylic acid signaling (SA) pathway [46]. Endophytes induce plant systemic resistance via regulating stomatal closure and callose deposition, reactive oxygen species (ROS) burst, enhancing defense enzyme activity, and increasing expression levels of defense-related genes in hosts [47]. The endophytic antagonistic bacteria Azospirillum sp. B510 isolated from rice by Kusajima et al. triggered systematic resistance in the host and significantly enhanced the resistance of rice to rice blast disease. A higher level of POD and phenylalanine ammonia lyase (PAL) activities was tested within rice samples treated with strain B51, and expression levels of the genes associated with the JA/ET signal transduction pathway were upregulated [48]. In the study of Wang et al., treatment with B. cereus AR156 on loquat fruit increased defense-related enzyme activities, such as PAL and POD, and enhanced H2O2 accumulation, resulting in inducing the defense responses of the host and reducing the disease incidence of anthracnose rot [49]. In recent years, most of the antagonistic bacteria isolated from healthy C. oleifera are Bacillus, such as B. amylolyticus and B.subtilis. Bacillus can not only form spores to prevent plant diseases but also induce host systemic resistance [46]. However, reports about the management of C. oleifera anthracnose by B. subtilis are relatively limited. As for the present research, spraying treatment with YL13 fermentation broth noticeably increased the H2O2 levels in C. oleifera leaves. Hydrogen peroxide accumulation is one of the most direct responses of plants to external stresses. However, excessive hydrogen peroxide can damage plant cells, even leading to cell death [50]. Defense-related enzymes, such as POD, play critical roles in balancing hydrogen peroxide levels in cells [51]. Similarly, the POD activities were particularly enhanced with YL13 treatment in this study. The results illustrated the action of YL13 as a defense inducer in its host plants. That is to say, YL13 can induce essential defense reactions by activating H2O2 accumulation and the activities of relevant enzymes.
The issues proposed for further research include: (1) analyzing the signal transduction pathway initiated by YL13 treatment using a transcriptome sequencing approach; (2) isolating and identifying more functional metabolites of YL13, especially in inducing the systemic resistance of hosts; (3) meanwhile, the field trials are also being carried out and the test period is set to be longer.

5. Conclusions

In vitro assays, both the fermentation broth and the cell-free culture filtrate of the endophytic bacteria YL13 demonstrated antagonistic activity against the strain C. fructicola, which is the dominant pathogen of C. oleifera anthracnose. The cell-free filtrates displayed antagonistic activity, indicating that the metabolic substances of YL13 might play important roles. In the greenhouse trial, YL13 showed a biological control effect on C. oleifera anthracnose. YL13 synthesizes a variety of metabolic substances, including protease, cellulase, and iron carriers, which can directly inhibit pathogen growth or indirectly enhance plant disease resistance. Additionally, C. oleifera treated with YL13 fermentation broth showed a variety of defense responses, including the accumulation of reactive oxygen species and the enhancement of defense related enzyme activity. The significant antagonistic activity of YL13 and the ability to stimulate host defense responses showed that this strain could be regarded as an effective biological control agent (BCA) in the control of C. oleifera anthracnose.

Author Contributions

Conceptualization, Y.X. and G.Z.; methodology, A.N. and M.L.; formal analysis, Z.W., Y.H. and M.S.; writing—original draft preparation, Y.X. and Z.L.; writing—review and editing, Q.T. and Z.D.; supervision, G.Z.; project administration, G.Z. and J.L.; funding acquisition, J.L. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the National Natural Science Foundation of China (Grant No. 32271900) and the Postgraduate Science and Technology Innovation Fund of Central South Forestry University (No. CX20220715).

Data Availability Statement

All relevant data are within this paper.

Conflicts of Interest

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

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Forests 14 00886 g001 550
Figure 1. The antagonistic effect of YL13 on C. fructicola in vitro. (A) The antagonistic activity on C. fructicola by YL13 fermentation broth; (B) The antagonistic activity on C. fructicola by YL13 cell-free filtrate; (C) SEM observation of C. fructicola hyphal structures. CK, control, not exposed to YL13; YL13, exposed to YL13; h, hours; bars: 50 μm, 10 μm; red box indicates the hyphae were broken, wrinkled, and irregularly twined into clumps.
Figure 1. The antagonistic effect of YL13 on C. fructicola in vitro. (A) The antagonistic activity on C. fructicola by YL13 fermentation broth; (B) The antagonistic activity on C. fructicola by YL13 cell-free filtrate; (C) SEM observation of C. fructicola hyphal structures. CK, control, not exposed to YL13; YL13, exposed to YL13; h, hours; bars: 50 μm, 10 μm; red box indicates the hyphae were broken, wrinkled, and irregularly twined into clumps.
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Figure 2. The ability of YL13 to produce protease (A), cellulase (B), and siderophore (C).
Figure 2. The ability of YL13 to produce protease (A), cellulase (B), and siderophore (C).
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Figure 3. YL13-induced H2O2 accumulation in C. oleifera leaves. CK, control, spraying with LB medium; YL13, spraying with YL13 fermentation broth (1.0 × 108 CFU mL−1). Bars represent the standard errors of three replicates. * p < 0.05, ** p < 0.01, *** p < 0.001.
Figure 3. YL13-induced H2O2 accumulation in C. oleifera leaves. CK, control, spraying with LB medium; YL13, spraying with YL13 fermentation broth (1.0 × 108 CFU mL−1). Bars represent the standard errors of three replicates. * p < 0.05, ** p < 0.01, *** p < 0.001.
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Figure 4. Peroxidase (POD) activity in YL13-inoculated C. oleifera leaves. CK, control, spraying with LB medium; YL13, spraying with YL13 fermentation broth (1.0 × 108 CFU mL−1). Bars represent the standard errors of three replicates. * p < 0.05, ** p < 0.01, *** p < 0.001.
Figure 4. Peroxidase (POD) activity in YL13-inoculated C. oleifera leaves. CK, control, spraying with LB medium; YL13, spraying with YL13 fermentation broth (1.0 × 108 CFU mL−1). Bars represent the standard errors of three replicates. * p < 0.05, ** p < 0.01, *** p < 0.001.
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Table
Table 1. Biocontrol effects of YL13 on C. oleifera anthracnose under greenhouse.
Table 1. Biocontrol effects of YL13 on C. oleifera anthracnose under greenhouse.
TreatmentsDisease Index (%)Biocontrol Efficacy (%)
CK68.3 ± 2.5 a--
Treatment 140.2 ± 2.2 b41.3 ± 2.3 a
Treatment 229.6 ± 3.9 c56.7 ± 2.8 b
Treatment 323.1 ± 4.0 d67.9 ± 3.5 c
CK: spraying with LB medium; Treatment 1: spraying with YL13 fermentation broth, 1.0 × 104 CFU mL−1; Treatment 2: spraying with YL13 fermentation broth, 1.0 × 106 CFU mL−1; Treatment 3: spraying with YL13 fermentation broth, 1.0 × 108 CFU mL−1. Values with different letters within the same column are significant differences based on Fisher’s least-significant difference test (p < 0.05). Numbers followed by “±” are standard errors. The “--” represents a lack of detection.
Table
Table 2. The metabolic substances synthesized by YL13.
Table 2. The metabolic substances synthesized by YL13.
Metabolic SubstancesYL13
β-1,3-glucanase−
Protease+
Cellulase+
Siderophore+
The “+” represents a positive result, and the “−” represents a negative result.
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Xia, Y.; Liu, J.; Wang, Z.; He, Y.; Tan, Q.; Du, Z.; Niu, A.; Liu, M.; Li, Z.; Sang, M.; et al. Antagonistic Activity and Potential Mechanisms of Endophytic Bacillus subtilis YL13 in Biocontrol of Camellia oleifera Anthracnose. Forests 2023, 14, 886. https://doi.org/10.3390/f14050886

AMA Style

Xia Y, Liu J, Wang Z, He Y, Tan Q, Du Z, Niu A, Liu M, Li Z, Sang M, et al. Antagonistic Activity and Potential Mechanisms of Endophytic Bacillus subtilis YL13 in Biocontrol of Camellia oleifera Anthracnose. Forests. 2023; 14(5):886. https://doi.org/10.3390/f14050886

Chicago/Turabian Style

Xia, Yandong, Junang Liu, Zhikai Wang, Yuan He, Qian Tan, Zhuang Du, Anqi Niu, Manman Liu, Zhong Li, Mengke Sang, and et al. 2023. "Antagonistic Activity and Potential Mechanisms of Endophytic Bacillus subtilis YL13 in Biocontrol of Camellia oleifera Anthracnose" Forests 14, no. 5: 886. https://doi.org/10.3390/f14050886

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