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Article

Antifungal Activity of Bacillus velezensis X3-2 Against Plant Pathogens and Biocontrol Effect on Potato Late Blight

by
Peixia Wei
1,†,
Mengying Gao
1,†,
Shuang Zhou
2,
Guohui Liu
3,
Pan Wang
1,
Chunguang Liu
1,
Fengshan Yang
1 and
Haiyan Fu
1,*
1
Engineering Research Center of Agricultural Microbiology Technology, Ministry of Education & Heilongjiang Provincial Key Laboratory of Ecological Restoration and Resource Utilization for Cold Region & Key Laboratory of Molecular Biology, College of Heilongjiang Province & School of Life Sciences, Heilongjiang University, Harbin 150080, China
2
Institute of Rural Revitalization Science and Technology, Heilongjiang Academy of Agricultural Scienses, Harbin 150027, China
3
Heilongjiang Agricultural Environment and Cultivated Land Protection Station, Harbin 150031, China
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Agriculture 2024, 14(12), 2224; https://doi.org/10.3390/agriculture14122224
Submission received: 12 November 2024 / Revised: 27 November 2024 / Accepted: 3 December 2024 / Published: 5 December 2024
(This article belongs to the Section Crop Protection, Diseases, Pests and Weeds)
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Abstract

Late blight of potato is caused by the pathogen Phytophthora infestans, which has been considered to be the most destructive disease affecting potato crops worldwide. In recent years, the use of antagonistic microorganisms to control potato late blight has become a green and environmentally friendly means of disease control, greatly reducing the use of chemical pesticides. To obtain antagonistic bacteria with a high biocontrol effect against potato late blight, a total of 16 antagonistic bacterial strains with an inhibition rate of more than 50% against P. infestans were screened from potato rhizosphere soil by double-culture method, among which the bacterial isolate (X3-2) had the strongest inhibitory activity against P. infestans, with an inhibition rate of 81.97 ± 4.81%, respectively, and a broad-spectrum inhibitory activity. The bacterial isolate (X3-2) was identified as Bacillus velezensis based on its 16S rDNA gene sequence and morphological as well as biochemical properties. The results of our in vitro experiments demonstrated that X3-2 was a potent inducer of resistance in potato tubers and leaflets against late blight. In greenhouse experiments, it was confirmed that the biological preparation X3-2 exhibits an anti-oomycete effect, demonstrating a significant control efficacy on potato late blight. Further analyses showed that the antagonistic substances of X3-2 were distributed both intracellularly and extracellularly. In addition, screening for plant-growth-promoting (PGP) traits showed that X3-2 has the ability to produce siderophores and secrete indole acetic acid (IAA). The findings from this research suggest that B. velezensis X3-2 exhibits promise as a biocontrol agent for managing late blight. In the future, the composition and mechanism of the action of its antimicrobial substances can be studied in depth, and field trials can be carried out to assess its actual prevention and control effects.

1. Introduction

Potato (Solanum tuberosum L.) is a versatile crop that can be grown in a wide range of habitats, and its tubers are particularly rich in starch [1]. Nevertheless, potato yield and quality are threatened by a number of devastating diseases, among which the pathogen Phytophthora infestans, the causative agent of late blight, is a highly prevalent epidemic plant disease that can lead to the drying out of stems and foliage and the rotting of tubers, posing a huge risk to the safe production of potatoes [2]. Potato late blight has been a significant burden on the agricultural industry, with conservative estimates suggesting that it causes more than USD 6 billion in terms of management and economic losses each year, severely hampering potato production and industrialization [3]. To protect potatoes from the infection of P. infestans, people have adopted various integrated control strategies, such as using fungicides, using biological control agents, and using resistant varieties [4,5,6,7]. Currently, the prevention and control of late blight still rely primarily on synthetic fungicides [8]. While pesticides are fast and effective in controlling this disease, their excessive use endangers the safety of agricultural products and human health, leads to the increased resistance of pathogenic fungi, and causes environmental pollution and ecological imbalance [9,10]. Hence, it is imperative that we develop and explore more efficient, ecologically relevant, and environmentally friendly alternatives to protect safe potato production. Currently, the importance of sustainability has become a prominent issue that is widely acknowledged by the public. As a result, research on alternative control strategies, particularly in the realm of biological control, has seen a significant increase in attention and interest.
Beneficial microorganisms, as important components of natural ecosystems, play a critically important part in promoting plant productivity and health [11,12,13]. In an effort to control late blight, several antagonistic microorganisms, including Trichoderma harzianum HNA14, Bacillus megaterium WL-3, Bacillus velezensis SDTB038, Rhodopseudomonas palustris GJ-22, Myxococcus xanthus B25-I-1, and Pseudomonas fluorescens LBUM636, have been studied and reported as potential biological control agents (BCAs) [7,14,15,16,17,18]. In recent times, the efficacy of Bacillus species, particularly B. velezensis strains, in combating plant diseases has garnered significant attention. Notably, apart from the well-studied B. velezensis SDTB038, an escalating number of B. velezensis strains have exhibited robust anti-oomycete properties against P. infestans, such as B. velezensis VB7, B. velezensis AFB2-2, B. velezensis SQR9, and other strains [19,20,21]. Antagonistic microorganisms play a vital part in inhibiting the mycelial growth of P. infestans. These antagonistic microorganisms could secrete single compounds or mixtures of molecules with varying modes of action that effectively combat the spread of P. infestans [22]. Despite the availability of numerous strains of Bacillus velezensis, research focused on the genetic enhancement and functional optimization of superior strains is still insufficient. This gap in knowledge restricts the full exploitation of their biocontrol potential and impedes the development of environmentally sustainable and effective pest control strategies.
Plants and microorganisms have a complex relationship that can greatly benefit both parties. These interactions can improve plant fitness through various mechanisms that promote growth, stress relief, and defense against pathogens [23,24]. In the realm of beneficial microorganisms in the rhizosphere, Bacillus spp. Stand out as one of the most extensively studied. The isolation of the Bacillus velezensis group from many plants, rhizospheres, and soils has shown that they have powerful survival mechanisms in nature. Evidence has accumulated to show that bacteria from the B. velezensis group can release several metabolites involved in metal chelation, insecticidal and nematocidal activities, biofilm formation, the induction of plant tolerance to stress, and the initiation of microbial cell signaling [25]. Studies of the B. velezensis group isolates on plants suggest that the use of these traits in commercial formulations for green and regenerative agriculture will be expanded. However, there are only a few reports of using formulated bioagents to inhibit P. infestans.
In the study, a strain of B. velezensis was meticulously examined for its robust inhibitory properties against P. infestans within a controlled laboratory environment. The primary aims of the investigation were as follows: (1) to assess the ability of strain X3-2 to inhibit the mycelial growth of P. infestans and its effectiveness in combatting potato late blight through a pot experiment; (2) to identify strain X3-2 by means of 16S rDNA and to detail its morphological and biochemical features; (3) to characterize the antagonistic substances of X3-2 by using the dual culture assay; and (4) to demonstrate that strain X3-2 could stimulate resistance in potato tubers and leaves against P. infestans. Overall, this study furnishes significant insights into a late-model biological agent for managing potato late blight.

2. Materials and Methods

2.1. Culture Media, Sample Source, and Test Strains

To facilitate optimal growth and propagation of various strains, P. infestans was cultured in a rye agar medium composed of 70 g yeast extract, 20 g sucrose, 15 g agar, and 1000 mL of sterile water. LB (Luria−Bertani) and PDA (potato dextrose agar) plates (20 g potato, 20 g dextrose, 15 g agar, and 1000 mL sterile water) were utilized to culture bacteria and fungi. Potato late blight can grow well on rye agar medium, which provides nutrients that can satisfy the filamentous fungi’s need for carbon and nitrogen sources; LB medium is a commonly used bacterial medium; and PDA medium is commonly used to cultivate a variety of fungi. The rhizosphere soils were obtained from potato (Kexin 37) plants grown in Hulan District, Harbin City, Heilongjiang, China (46°00′ N, 126°38′ E), and considered dark soil. Rye agar medium was prepared according to the method that was outlined by Medina and Platt [26]. The method of preparation is as follows: Firstly, 70 g of rye seeds was washed twice to remove the residue and transferred to soak in 0.5 L of sterilized water overnight. The next day, the wort was collected after autoclaving for 20 min. The solution was filtered and fixed to 1 L. Subsequently, both 15 g of agar and 20 g of sucrose were incorporated into the solution and autoclaved for 20 min. Finally, the mixture was poured into a plate for further use. Chemical reagents for physiological biochemistry were bought from Biotech (Shanghai, China).
The P. infestans CJ-13 strain used in this study was purchased from Hebei Academy of Agricultural and Forestry, which was used for potting experiments of potatoes infected with late blight. Geotrichum candidum H was isolated from sweet potato sour rot in September 2021 (GenBank: PP911431.1). Fusarium oxysporium f. sp. cucumberinum (ACCC37438), provided by the Agricultural Culture Collection of China (ACCC), is a strain capable of causing cucumber wilt disease. Rhizoctonia solani AG-3 can cause potato black scurf. Fusarium solani F3 can cause potato dry rot. These strains are used to test isolates for broad-spectrum antifungal activity and stored in our laboratory (Heilongjiang Provincial Key Laboratory of Ecological Restoration and Resource Utilization for Cold Region, Heilongjiang University). The aforementioned five pathogens have been tested and proven to cause typical symptoms of disease on plants.

2.2. Isolation of Rhizosphere Bacteria

We used a quincunx five-point sampling method to collect rhizosphere soil samples from potato plants, and the subsamples from different points were pooled into a single sample. The isolation of bacteria was performed according to Feng et al. [27]. Briefly, 1 g of soil sample from rhizosphere of potato plants was weighed into a triangular flask containing small steel bead and sterile distilled water (10 mL) and shaken for 20 min. The solution was then diluted to a concentration of 10−4 to 10−6 with sterilized water. Next, 100 µL of the suspension was aspirated with a pipette gun and applied to LB agar plates (10 g tryptone, 5 g yeast extract, 10 g NaCl, 15 g agar, and 1000 mL sterile water), respectively. Then, LB culture plates were incubated inverted at 37 °C, respectively. The growth of isolates in LB Petri dishes was checked daily. After incubation, individual colonies were purified by plate streaking and sealed and stored in a 4 °C refrigerator.

2.3. Screening of P. infestans Antagonist Bacteria

The ability of the isolates against P. infestans was measured using a dual culture assay. A P. infestans mycelial disk (8 mm in diameter) was inoculated in the center of the rye agar plate (9 cm in diameter). The isolates were then inoculated onto both sides of the P. infestans disk, while plates only with P. infestans disk were set as a control. The colony inhibition rate of P. infestans was measured after culturing for 7 d at 20 °C in the dark. The tests were performed in triplicate. Then, the inhibition rates (I%) were calculated as follows [28]. Finally, the strains that were able to suppress P. infestans infection of potato were screened, purified, and stored for subsequent tests.
I % = control   colony   diameter −   colony   diameter   of   treated control   colony   diameter − initial   colony   diameter   ( 8   mm ) × 100 %

2.4. The Effect of X3-2 on the Growth of Other Plant Pathogens In Vitro

The antifungal activity of X3-2 was tested in confrontation assays against G. candidum, F. oxysporium, R. solani, and F. solani. These indicator fungi were individually made into cakes and inoculated in the center of fresh PDA plates. Subsequently, strain X3-2 was spotted with sterile toothpicks at 20 mm from the indicated strains. Controls were PDA media inoculated with pathogenic fungi only. The tests were performed in triplicate. The inhibition zone was observed after 7 d of incubation at 20 °C.

2.5. Taxonomic Identification of Strain X3-2

Genomic DNA of X3-2 was subjected to PCR amplification using primers 27F (5′-AGTTTGATCMTGGCTCAG-3′) and 1492R (5′-GGTTACCTTGTTACGACTT-3′) [29], and gene fragments obtained after PCR were transported to Sangon Biotech (Shanghai, China) for sequencing. Subsequently, the 16S rDNA sequences of the strain X3-2 was compared with homologous sequences in National Center for Biotechnology Information (NCBI) (https://blast.ncbi.nlm.nih.gov/Blast.cgi). At the same time, a phylogenetic tree of the 16S rDNA sequence of strain X3-2 was constructed and visualized by one table (tvBOT) [30]. Additional physiological and biochemical (anaerobic test, starch hydrolysis, contact enzyme test, glucose oxidation, etc.) bacterial identification methods were used to confirm the taxonomic affiliation of X3-2. Morphological characteristics (edge, viscosity, color, surface texture, etc.) of the single colony in the medium were recorded by microscopic observation [31,32].

2.6. Strain X3-2 Biocontrol Assays on Potato Tissues In Vitro

Healthy potatoes were selected for cutting, and their surfaces were sterilized according to Jiang et al. [33]. The surface of potato tubers of uniform size was disinfected with 1% NaClO solution, cleaned three times with distilled water, and then placed in an ultra-clean bench to dry naturally. Mycelia of P. infestans was incubated at 20 °C for 7 d and then collected with sterile ice water and shaken well to obtain a solution of sporangia. In addition, in order to release the zoospores from the sporangia, the sporangial suspension was left in the dark at 4 °C for 3 h before the start of the experiment [34], and the concentration of zoospores was adjusted to 1 × 106 CFU/mL using sterile water in hemocytometer (XB-K-25) counting. In vitro experiments were conducted with potato leaves and tubers (1.5 cm × 1.5 cm × 0.5 cm). Biocontrol experiments were carried out under the following conditions: B. velezensis X3-2 suspension (20 µL) was applied to the surface of tuber cuttings and the surface of leaves for 48 h ahead of time under dark conditions at 20 °C. Subsequently, 20 µL zoospore suspension of P. infestans was evenly applied to the abaxial surface of leaves, and an 8 mm diameter mycelium disk of P. infestans was placed on the tubers. An equal volume of liquid LB and bacterial suspension only without infection were used as controls. The tests were performed in triplicate. The disease index was counted, and the relative protection rate was calculated according to the classification criteria of the disease class [17,35,36].

2.7. Talc-Based Formulation of X3-2 Bioagents

The talc-based formulation of X3-2 bioagent was performed according to Islam et al. [37], and some modifications were made to it. Talc powder was used as carrier, CaCO3 and carboxymethyl cellulose were used as stabilizer and protectant. Firstly, 50 g of talc powder, 0.75 g of CaCO3, and 0.5 g of carboxymethyl cellulose were mixed and sterilized at 120 °C for 20 min. Secondly, to prepare the X3-2 bioagents, the strain X3-2 was inoculated into LB broth and shaken at 30 °C for 24 h to obtain the bacterial fermentation broth. Next, the X3-2 bacterial fermentation broth was centrifuged in a centrifuge (L530R) at 4500 rpm for 5 min, and then obtained bacterial cells were resuspended in fresh liquid LB medium (20 mL) with 0.5 mL of sterile glycerol. These cultures (2 × 109 CFU/mL) were stirred into a beaker containing 50 g of powdered talc. Finally, the formulated bacterial antagonists were air-dried in a blast-drying oven (DH-101-2) and air-dried at 30 °C overnight, then ground into powder, and stored in self-sealing bags.

2.8. Effect of Formulated X3-2 Bioagents on Potato Late Blight in a Pot Experiment

Uniformly sized and apparently disease-free potato tubers were washed three times with sterilized water, and then the suberized tubers were submerged with the formulated X3-2 bioagents (2% w/v) for 30 min. Both treated and nontreated tubers were planted in the same pot filled with nutrient soils (one tuber per pot). The prepared bioagents (100 mL/pot) were sprayed at 40 DAPs (days after planting) on the surface of potato plants in the treated pots. The zoospores were obtained like described in Section 2.5. Then the zoospores (1 × 106 CFU/mL) of P. infestans were inoculated into the root system of potato plants after three days of treatment with X3-2, 20 mL per pot. The experiments were performed in triplicate, and each treatment within an experiment contained 12 pots. In the control group, potato tubers were treated with equal volumes of sterile water instead of X3-2 bioagents. All potted potatoes were grown in a growth chamber at 25 °C in light–dark (16 h:8 h). Disease severity in potato plants of different treatments was counted by estimating the percentage of infected area of individual plants 5, 10, and 15 d after inoculation with late blight according to Fu et al. [4]. Disease incidence in the different treatment groups was expressed as a percentage of infected plants to the total number of plants. Disease index was recorded according to the 0–9 classification standard (Table S1) and calculated as follows.
D i s e a s e   i n d e x = ∑ ( representative   value   at   each   level   ×   number   of   plants   in   the   corresponding   disease   level ) highest   representative   value   ×   total   number   of   plants × 100

2.9. Antagonistic Effects of X3-2 Against P. infestans

Cell-free filtrates (CFSs) of X3-2 were used to evaluate their antagonistic effects on the growth of P. infestans as described by Jeong et al. [38]. Living cells from strain X3-2 were incubated for 24 h at 37 °C and grown on LB agar medium. The bacterial cells were then transferred to LB broth at 5% of inoculum and incubated at 37 °C for 24 h. The concentration of culture was diluted to 1 × 107 CFU/mL. The fermentation culture was centrifuged at 4000 rpm for 20 min to obtain the extracellular supernatant, which was further filtered through a membrane (0.22 mm) to obtain CFS. P. infestans mycelial discs were inoculated in the center of rye agar medium, and CFS (10%, 20%, and 30%) was added to the medium to test the effect of CFS on P. infestans mycelial growth. Rye agar medium supplemented with 10% LB was added as a control. A mycelium disk of P. infestans was applied to rye agar medium and cultured for 5 days. The tests were performed in triplicate. Finally, colony diameters of P. infestans were counted by the crossover method, and colony inhibition rates of P. infestans were computed as described previously.
To determine the distribution of antagonistic substances of X3-2 against P. infestans, bacterial suspensions, cell suspensions, and bacterial crushing fluid of X3-2 were obtained, and we incubated them separately against P. infestans. To obtain a cell suspension, the bacteria were resuspended in 0.90% sterile normal saline, and we adjusted the final concentration to 1 × 107 CFU/mL with distilled water. Next, bacterial pellets were washed three times with sterile water and then resuspended in sterile normal saline and broken up in an ultrasonic cell crusher isolation chamber after an ice bath. Subsequently, the lysed cells were centrifuged again, and the X3-2 supernatant was filtered through membranes (0.22 µm) to acquire the bacterial crushing fluid. According to the punch method, each punch (8 mm) was separately added to 100 µL of bacterial suspension, cell suspension, cell-free filtrates, and bacterial crushing fluid. In the control group, the same volume of LB liquid medium was added. The tests were performed in triplicate. The plates were placed in the dark at 20 °C for 5 d, and colony inhibition rates of P. infestans were calculated.

2.10. Determination of Extracellular Enzymes and Traits of Plant Growth Promoting (PGP) of X3-2

The production of extracellular enzymes such as glucanase, cellulase, protease, and amylase was assessed by inoculating overnight-grown strain X3-2 in the respective media, followed by incubation at 30 °C for 4 d to observe whether transparent circles were produced around the colonies [39,40,41,42,43]. The tests were performed in triplicate. After incubation, cellulase activity was decontaminated with 1 M NaCl solution for 10 min to measure the clear area around the colony. The amylase activity was checked using a 1% iodine solution, and the clear transparent zones around the colony were observed, indicating a positive test result. The ability of strain X3-2 to secrete siderophores was assayed in the CAS solid medium. Indole-3-acetic acid (IAA) production of the X3-2 was examined by the Salkowski reagent method as described by Jia et al. [44].

2.11. Data Analysis

All treatments were performed with three replicates. The data were subjected to estimate using one-way analysis of variance (ANOVA) with SPSS version 25.0 software (IBM, New York, NY, USA). Dunnett’ s multiple comparisons test and Student’ s t-test were used to determine the significance of data. All experimental data were expressed as average ± standard deviation (SD).

3. Results

3.1. Isolation and Screening of P. infestans Antagonist Bacteria

The experiment isolated and purified 238 different bacterial isolates from 21 rhizosphere soil samples. After three rounds of screening, a total of 16 antagonistic strains with inhibition rates greater than 50% against P. infestans were obtained (Figure S1). In particular, the X3-2 strain showed a strong inhibitory effect (Figure 1a,b), and its colony inhibition rate for P. infestans were calculated as 81.97 ± 4.81% (Figure 1c). In this study, we only selected strain X3-2 for follow-up studies to preliminarily explore its antifungal activity against plant pathogens and biocontrol effect on potato late blight.

3.2. Identification of Strain X3-2

The morphology of colonies of strain X3-2 on LB plates was characterized as white, opaque, and sticky, with a rough surface and a wrinkled edge of the colony (Figure 2a,b). Further physiological and biochemical tests showed that X3-2 is an aerobic Gram-positive bacterium capable of producing oxidase, gelatinase, and catalase and reducing nitrate (Table S2). Additionally, the amplified 16S rDNA product of strain X3-2 provided a sequence of 1487 bp. The sequence of strain X3-2 16S rDNA was submitted to the GenBank database, and the accession number was PP858053.1. Moreover, a phylogenetic tree was constructed based on the 16S rDNA sequence, which showed that strain X3-2 and Bacillus velezensis (orange blocks) clustered in the same branch of phylogeny (Figure 2c). Based on the combined morphological and molecular characteristics, strains X3-2 were identified as B. velezensis.

3.3. The Effect of X3-2 Against Other Plant Pathogens In Vitro

Strain X3-2 was tested for its inhibitory effect on four major plant pathogens (Figure 3a). The results indicated that strain X3-2 exhibited a significant inhibitory effect towards four other plant pathogens, with an average inhibition rate of 65.59 ± 11.39% after 7 d of inoculation. Additionally, G. candidum and R. solani demonstrated the highest inhibition rates compared to the untreated control at 67.17 ± 0.21 and 79.63 ± 0.34%, respectively (Figure 3b, Table S3).

3.4. Effect of X3-2 on Inducing Potato Tissue Resistance to Late Blight

Whether X3-2 enhances potato plant resistance to late blight was assessed by inoculating potato tubers and leaflets pretreated with X3-2 bacterial suspension with P. infestans. Bacterial suspension inhibitory activity demonstrated a significant biocontrol effect on late blight. Furthermore, the bacterial suspension was not pathogenic to the tissues of the potato in vitro, and there were no obvious lesions the tubers and leaflets exposed to P. infestans. The mean disease index (DI) of potato tubers and leaflets in the treatment group was reduced by 80.95 ± 3.43, 62.31 ± 6.56%, respectively, compared to the control (Table 1). The results showed that strain X3-2 could reduce the infestation of P. infestans on the potato by enhancing the resistance of the potato.

3.5. Effect of Formulated X3-2 Bioagents on Potato Plants in a Pot Experiment

The results of the pot experiment showed that the formulated X3-2 bioagents had a well-controlled effect on late blight of potato (Figure 4). Potted potato plants pretreated with X3-2 bioagents showed a significantly lower disease index and disease incidence compared to the control. The potato plants infected only with P. infestans suffered from infection and grew more slowly; additionally, the leaves began yellowing, and most of the leaves fell off (Figure 4a). The incidence of late blight was reduced by 64.28 ± 12.38%, 50.01 ± 8.65%, and 46.66 ± 5.77% after 5, 10, and 15 d of inoculation, respectively, as compared to the control (Figure 4b). Accordingly, the disease index of potato late blight was reduced by 56.82 ± 7.87%, 42.22 ± 3.85%, and 21.43 ± 3.10%, respectively, compared to the control (Figure 4c). Additionally, the potato plants treated with X3-2 bioagents exhibited better growth and were more resistant to late blight than the untreated plants. These results demonstrated that strain X3-2 can slow down the development of late blight symptoms.

3.6. Evaluation of the Antagonistic Effects of X3-2 Against P. infestans

Strain X3-2 with different concentrations of CFS showed a strong inhibitory effect on P. infestans. With an increasing CFS concentration, the inhibitory effect was significantly enhanced, showing a dose-dependent effect (Figure 5a,b). The inhibition rate was 39.80 ± 1.66%–86.73 ± 1.03% when the CFS concentration was 10–30% (Figure 5c). The different components of strain X3-2 suppressed the mycelial growth of P. infestans by more than 60%. Furthermore, both the bacterial suspensions and cell-free filtrates of X3-2 exhibited strong inhibitory effects (Figure 5d,e). The above results showed that antagonistic substances of strain X3-2 against P. infestans were distributed both intracellularly and extracellularly. Extracellular substances are easier to purify and apply, while intracellular substances need to be extracted and purified by different methods before they can be used as biocontrol agents.

3.7. Detection of Extracellular Enzymes and Traits of Plant Growth Promoting (PGP) of X3-2

After 3 d of culture, there were transparent circles that appeared on the protease detection plate (Figure 6a), cellulase detection plate (Figure 6b), amylase detection plate (Figure 6c), and glucanase detection plate (Figure 6d), indicating that X3-2 has the ability to produce varying levels of protease, cellulase, amylase, and glucanase (Table S4). The siderophore detection plate after 3 days of culturing exhibited orange colony hydrolysis circles, indicating that X3-2 produced siderophores (Figure 6e). The supernatant of strain X3-2 turned pink, indicating that X3-2 secreted IAA (Figure 6f). These phenomena suggest that X3-2 has the potential to degrade pathogenic fungi cell walls and promote plant growth.

4. Discussion

Antagonistic microorganisms serve as valuable assets in the management of plant prevalent epidemic disease by enhancing plant resistance and suppressing pathogen growth [45,46,47]. The isolation and screening of microorganisms with antagonistic characterizations against pathogens is a critical first step in identifying potential natural control agents. This initial phase lays the foundation for exploring the capabilities of these microorganisms in combatting plant diseases effectively [48]. The identification of beneficial microorganisms, such as Bacillus subtilis WL-2, Streptomyces sp. FXP04, Bacillus velezensis AFB2-2, Bacillus pumilus W-7, Trichoderma harzianum HNA14 and Bacillus Amyloliquefaciens MB40 for the biological control of this devastating disease is a topic that is gaining increasing attention in scientific research [4,18,20,34,49,50]. In the present study, a total of 238 strains were isolated from healthy potato rhizosphere soils, 16 of which significantly showed inhibitory activity on the mycelial growth of P. infestans. Our findings suggest that rhizosphere soils are key to the isolation of a large number of microorganisms antagonizing pathogens.
Heilongjiang Province is the largest potato planting base in China, with fertile and vast black soil, providing unique geographical conditions for potato cultivation. Late blight of potato is one of the major diseases of potato, and the warm days, cool nights, and high humidity of Heilongjiang provide the necessary conditions for its occurrence and prevalence, resulting in potatoes being highly susceptible to late blight and the poor disease resistance of maincrop potato varieties, which seriously restricts the development of potato industrialization in China. In recent years, there has been increasing interest in the role of bacteria as biocontrol agents due to their typical advantages of diversity, environmental friendliness, and non-pathogenicity. In this paper, the anti-oomycete mechanism of strain X3-2 was preliminarily discussed. Strain X3-2 has been identified as B. velezensis through the examination of its morphological characteristics and 16S rDNA. B. velezensis is renowned for its antagonistic effects against various phytopathogens, making it a prominent biocontrol bacterium [51,52,53,54]. Nevertheless, although there are many strains with antagonistic functions against potato late blight at home and abroad, there are fewer strains with optimized utilization, and most of the studies on their biological control mechanisms are focused on the extracellular metabolites. Additionally, there are few studies on the multifaceted evaluation of antagonistic strains. In our work, the strain X3-2 with a strong inhibitory effect on P. infestans was isolated from potato rhizosphere soil samples from Harbin, Heilongjiang Province, China. In vitro, X3-2 obviously inhibited the growth of P. infestan mycelium by 81.97 ± 4.81% (Figure 1). Previously, B. velezensis 6-5 was showed to significantly suppress the growth of P. infestans mycelium [55]. These reports were similar to our results. However, further studies on the effect of strain X3-2 on the release of P. infestans zoospores are needed in the future. Previous studies have shown that most potential biocontrol strains have a broad antifungal spectrum [28,50]. Similarly, a broad-spectrum bacterium known as X3-2 was chosen for its remarkable inhibitory effects against four distinct pathogenic fungi on PDA, all with inhibition rates above 50%. The results showed that X3-2 has good application prospects in the control of fungal diseases.
Biological control is a green prevention and control technology that has become a research hotspot because of its biosecurity and sustainability [56]. It mainly consists of two methods: the use of beneficial microorganisms to antagonize pathogens or the use of active metabolites to enhance resistance in potato plants. Several studies have indicated that microorganisms may improve host resistance to diseases. Caulier et al. [57] showed that B. subtilis 30B-B6 significantly reduced the occurrence of late blight. B. velezensis KOF112 was shown to induce a defense response in grapevines dependent on the salicylic and jasmonic acid defense pathways [58]. Wu et al. [15] found that the bioactive metabolite produced by myxococcus xanthus B25-I-1 effectively reduced the defense enzyme activity in P. infestans mycelium. In vitro experiments, pretreated potato tissues with X3-2, significantly reduced the severity of the disease, indicating that X3-2 is effective in inducing resistance in potato tissues to late blight. These findings are consistent with this paper. However, the actual effectiveness of the X3-2 strain in the field needs to be further tested. Bacillus species can secrete various biologically active substances that may effectively suppress the growth of P. infestans in vitro while at the same time exerting beneficial effects on potatoes through different mechanisms. B. velezensis FZB42 has been reported to secrete various antimicrobial secondary metabolites such as difficidin, bacillaene, and macrolactin [59]. In this paper, we found that the antagonists of X3-2 against P. infestans were distributed both intracellularly and extracellularly, with those secreted extracellularly having a strong inhibitory effect. Consequently, these antagonistic substances should be further purified and characterized in the future for large-scale production and application in agriculture.
Bacterial extracellular hydrolases such as glucanase, proteases, and cellulase are involved in the biological control of pathogens by lysing cell walls [60,61]. For example, Chaetomium globosum Cg-6 produced large amounts of proteases and cellulases when cultured with P. infestans and plays a key part in the lysis of cell walls, which can contribute to colonization of host tissues by bioprophylactic bacteria, which is one of the mechanisms known to antagonize microbial ginseng for biocontrol [62]. Furthermore, extracellular hydrolases such as β1, 3-glucanases and chitinases produced by Bacillus help to protect potato plants against P. infestans [63]. In summary, proteases and cellulases have been shown to disrupt fungal cell walls, inhibit spore formation, or interfere with nutrient uptake by pathogens, contributing to antagonistic strains against pathogenic fungi. In the extracellular enzyme assay, we found that X3-2 could secrete proteases and cellulases, and the secretion of these hydrolysis enzymes may be one of the mechanisms of biocontrol by X3-2.

5. Conclusions

In the present study, the talc-based formulation of B. velezensis (2% w/v) has shown promising results in combating late blight and enhancing potato growth. Nevertheless, in order to ensure the continued efficacy of beneficial bacteria in protecting plants from diseases and promoting plant growth, it is crucial to consider the colonization of beneficial bacteria in the plant, which can contribute to the sustainable and effective application of such agents in agricultural practices. In the future, the colonization of X3-2 potato plants at different growth stages or under different environmental conditions can be further investigated by labeling X3-2 with green fluorescent protein or combining with other molecular and imaging techniques. In addition, the distribution of antagonistic substances of strain X3-2 inside and outside the cell lays a good foundation for the control of potato late blight by strain X3-2. In conclusion, strain X3-2 could serve as an environmentally friendly alternative for the control of potato late blight.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/agriculture14122224/s1. Figure S1: Bacterial isolates with inhibitory effects on the growth of P. infestans in vitro; Figure S2: Effect of X3-2 on enhancing the resistance of potato tissues to late blight; Table S1: Classification criteria for potato late blight disease; Table S2: Physiological and biochemical characteristics of X3-2; Table S3: The effect of X3-2 on the growth of other plant pathogens in vitro; Table S4: The ability of X3-2 to produce extracellular enzymes.

Author Contributions

Conceptualization, P.W. (Peixia Wei) and F.Y.; writing—original draft, P.W. (Peixia Wei) and M.G.; formal analysis, H.F.; methodology, M.G.; investigation, S.Z.; validation. G.L.; software, P.W. (Pan Wang); data curation, C.L.; resources, H.F.; supervision, H.F.; writing—review and editing, H.F. and F.Y.; project administration, F.Y.; funding acquisition, H.F. and F.Y. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Collaborative Innovation Project of “Double First Class” Disciplines in Heilongjiang Province (LJGXCG2023-036), Heilongjiang Province Agriculture Research System-Ecological Agriculture ([2023] 1197), and the National Key Research and Development Program of China (2018YFD0200800).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data are contained within the article and Supplementary Materials.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Effect of strain X3-2 on the growth of Phytophthora infestans. (a) A single culture of P. infestans. (b) The mycelial morphology of P. infestans under X3-2 confrontation. (c) The colony diameter of P. infestans under X3-2 confrontation. All treatments were performed with three replicates and values were expressed as average ± SD. **** p < 0.0001.
Figure 1. Effect of strain X3-2 on the growth of Phytophthora infestans. (a) A single culture of P. infestans. (b) The mycelial morphology of P. infestans under X3-2 confrontation. (c) The colony diameter of P. infestans under X3-2 confrontation. All treatments were performed with three replicates and values were expressed as average ± SD. **** p < 0.0001.
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Figure 2. Taxonomic identification of strain X3-2. (a) Morphological characteristics of X3-2 colonies on LB medium. (b) Morphological characteristics of X3-2 colonies on LB medium under the stereoscope. (c) The phylogenetic tree of strain X3-2.
Figure 2. Taxonomic identification of strain X3-2. (a) Morphological characteristics of X3-2 colonies on LB medium. (b) Morphological characteristics of X3-2 colonies on LB medium under the stereoscope. (c) The phylogenetic tree of strain X3-2.
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Figure 3. The effect of X3-2 on the growth of other plant pathogens in vitro. (a) Colony morphology under antagonistic conditions on PDA plates of X3-2 and plant pathogens. (b) Colony diameter of other plant pathogens by X3-2. All treatments were performed with three replicates, and values were expressed as average ± SD. **** p < 0.0001.
Figure 3. The effect of X3-2 on the growth of other plant pathogens in vitro. (a) Colony morphology under antagonistic conditions on PDA plates of X3-2 and plant pathogens. (b) Colony diameter of other plant pathogens by X3-2. All treatments were performed with three replicates, and values were expressed as average ± SD. **** p < 0.0001.
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Figure 4. Biological control of potato late blight by the formulated X3-2 bioagents. (a) Photograph of late blight symptoms in treated and control potato plants 10 d after inoculation with P. infestans. (b) Disease incidence in treated and control potato plants after inoculation with P. infestans. (c) Disease index of treated and control potato plants after inoculation with P. infestans. All treatments were performed with three replicates, and values were expressed as average ± SD. * p < 0.05, ** p < 0.01.
Figure 4. Biological control of potato late blight by the formulated X3-2 bioagents. (a) Photograph of late blight symptoms in treated and control potato plants 10 d after inoculation with P. infestans. (b) Disease incidence in treated and control potato plants after inoculation with P. infestans. (c) Disease index of treated and control potato plants after inoculation with P. infestans. All treatments were performed with three replicates, and values were expressed as average ± SD. * p < 0.05, ** p < 0.01.
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Figure 5. Effect on mycelial growth of P. infestans by different concentrations of X3-2 cell-free filtrates (CFS) and distribution of antagonistic components against P. infestans. (a) Effect of different concentrations of X3-2 CFS on the mycelial growth of P. infestans; P. infestans mycelial discs inoculated in the center of rye agar medium served as blank control and named as CK; Rye agar medium supplemented with 10% LB served as positive control and named LB; CFS (10%, 20%, and 30%) was added to the medium to test the effect of CFS on P. infestans mycelial growth. (b) Colony diameter of P. infestans treated with different concentrations of CFS. (c) The inhibition rate of P. infestans by different concentrations of CFS. (d) Colony morphology of P. infestans under different X3-2 component confrontations. (e) Colony diameter of P. infestans under different compositional treatments of X3-2. Each treatment group was performed three times, and values were expressed as average ± SD. **** p < 0.0001.
Figure 5. Effect on mycelial growth of P. infestans by different concentrations of X3-2 cell-free filtrates (CFS) and distribution of antagonistic components against P. infestans. (a) Effect of different concentrations of X3-2 CFS on the mycelial growth of P. infestans; P. infestans mycelial discs inoculated in the center of rye agar medium served as blank control and named as CK; Rye agar medium supplemented with 10% LB served as positive control and named LB; CFS (10%, 20%, and 30%) was added to the medium to test the effect of CFS on P. infestans mycelial growth. (b) Colony diameter of P. infestans treated with different concentrations of CFS. (c) The inhibition rate of P. infestans by different concentrations of CFS. (d) Colony morphology of P. infestans under different X3-2 component confrontations. (e) Colony diameter of P. infestans under different compositional treatments of X3-2. Each treatment group was performed three times, and values were expressed as average ± SD. **** p < 0.0001.
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Figure 6. Detection of extracellular enzymes and traits of plant growth promoting (PGP) of X3-2. (a–d) The appearance of transparent circle around the bacterial colony on the plate indicates extracellular enzyme production by X3-2. (e) The presence of an orange halo around colony on the plate indicates the production of siderophore by X3-2. (f) The pink-colored centrifuge tube contained IAA-producing bacteria and a negative control (LB with 0.5 mg/mL tryptophan). Arrows of different lengths represent the size of the halo.
Figure 6. Detection of extracellular enzymes and traits of plant growth promoting (PGP) of X3-2. (a–d) The appearance of transparent circle around the bacterial colony on the plate indicates extracellular enzyme production by X3-2. (e) The presence of an orange halo around colony on the plate indicates the production of siderophore by X3-2. (f) The pink-colored centrifuge tube contained IAA-producing bacteria and a negative control (LB with 0.5 mg/mL tryptophan). Arrows of different lengths represent the size of the halo.
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Table 1. Biocontrol effect of strain X3-2 on tissues of potato in vitro.
Table 1. Biocontrol effect of strain X3-2 on tissues of potato in vitro.
TreatmentsDisease Index (DI)
TubersLeaflets
Sterile water 0.00 ± 0.00 a0.00 ± 0.00 a
Bacterial suspension only0.00 ± 0.00 a0.00 ± 0.00 a
Bacterial suspension + P. infestans18.52 ± 3.23 b14.81 ± 2.67 b
P. infestans49.14 ± 2.26 c77.78 ± 5.34 c
Each treatment group was performed with three replicates, and values were expressed as average ± SD. Lowercase letters a–c in the same column indicate those that have been subjected to Duncan’s multiple comparison post-hoc test for differences at p < 0.05.
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MDPI and ACS Style

Wei, P.; Gao, M.; Zhou, S.; Liu, G.; Wang, P.; Liu, C.; Yang, F.; Fu, H. Antifungal Activity of Bacillus velezensis X3-2 Against Plant Pathogens and Biocontrol Effect on Potato Late Blight. Agriculture 2024, 14, 2224. https://doi.org/10.3390/agriculture14122224

AMA Style

Wei P, Gao M, Zhou S, Liu G, Wang P, Liu C, Yang F, Fu H. Antifungal Activity of Bacillus velezensis X3-2 Against Plant Pathogens and Biocontrol Effect on Potato Late Blight. Agriculture. 2024; 14(12):2224. https://doi.org/10.3390/agriculture14122224

Chicago/Turabian Style

Wei, Peixia, Mengying Gao, Shuang Zhou, Guohui Liu, Pan Wang, Chunguang Liu, Fengshan Yang, and Haiyan Fu. 2024. "Antifungal Activity of Bacillus velezensis X3-2 Against Plant Pathogens and Biocontrol Effect on Potato Late Blight" Agriculture 14, no. 12: 2224. https://doi.org/10.3390/agriculture14122224

APA Style

Wei, P., Gao, M., Zhou, S., Liu, G., Wang, P., Liu, C., Yang, F., & Fu, H. (2024). Antifungal Activity of Bacillus velezensis X3-2 Against Plant Pathogens and Biocontrol Effect on Potato Late Blight. Agriculture, 14(12), 2224. https://doi.org/10.3390/agriculture14122224

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