Biological Control Effect of Antagonistic Bacteria on Potato Black Scurf Disease Caused by Rhizoctonia solani

Abstract

Potato black scurf, caused by Rhizoctonia solani, is a severe soil-borne disease that affects the quality and production of potatoes worldwide. In our study, we used Paenibacillus polymyxa YF and Bacillus amyloliquefaciens HT to determine the antagonistic ability of R. solani, with a particular focus on the antagonistic action of P. polymyxa YF to R. solani and its biocontrol effect on potato black scurf. In fermentation, filtrate assay, 50% filtrate of P. polymyxa YF and B. amyloliquefaciens HT inhibited the growth of R. solani by 85.55% and 82.86%, respectively. Microscopic observations showed notable morphological changes with mycelial collapse, atrophy, and deformation following treatment with the antagonistic filtrates. Moreover, cell membrane permeability results showed increased conductivity in bacteria-treated samples compared to the control. P. polymyxa YF exhibited stable colonization on potato plants and secreted various extracellular enzymes (protease, amylase, and cellulase), along with the synthetic substances with growth-promoting effects, such as siderophores and Indole-3-acetic acid (IAA). Whether it is in the excised tissue inoculation or potted experiment, the negative control showed the highest rank of disease symptoms. In the pot experiment, after YF treatment, physiological parameters showed remarkable changes in plant height, root length, stem thickness, and dry and fresh weight. Compared to blank control, the activities of the four resistant enzymes increased significantly in the P. polymyxa YF treatment group. The upregulation in the P. polymyxa YF group was 4.04, 0.54, 0.46, and 3.10 times, respectively. PCR analysis identified genes in both bacterial strains coding for antimicrobial lipopeptides, including fenB, ituC, and srfAA, which are associated with fengycin, iturin, and surfactin synthesis. These findings demonstrated that P. polymyxa YF has a prominent antagonistic effect on R. solani, suggesting its potential as an effective biological control agent for controlling potato black scurf.

1. Introduction

Potato black scurf, caused by Rhizoctonia solani, is a destructive soil-borne disease that significantly threatens potato cultivation worldwide [1]. R. solani, known for extensive damage to various agricultural and horticultural crops, has become a significant concern in potato-cultivating countries, including China [2]. For instance, in Dingxi City, Gansu province, the incidence rate in severely affected fields has reached 75%, with a general incidence rate of 10–20% in ordinary fields. Furthermore, the incidence of diseases in plots continuously cultivated for 7 years is as high as 96.71% [3,4].

R. solani, belonging to the genus Rhizoctonia [5], is characterized by colorless, branched mycelium forming right or near-right angles. Studies have shown that R. solani is one species that has many strains. Most countries have proved that the main strain causing potato black scurf is AG-3. R. solani AG-3 TB causes tobacco target spot disease, that can seriously damage the quality and output of tobacco. R. solani AG-3 mainly infects the underground parts of plants and causes ulcers of roots, buds, stems, and stolons through mycelia and sclerotia that survive on soil and seed tubers [6,7,8]. When R. solani encounters a potato plant, it begins to attach to the plant and secrete cell-wall-degrading enzymes, and the hyphae begin to infect plant cells, eventually causing the entire cell structure to fall apart and the cells to die [9]. The disease transmission and damage mainly depend on diseased seed potatoes, which facilitate the disease in low-temperature and high-humidity soil [10]. Currently, the challenge in controlling R. solani is attributable to the stability of its sclerotium, which can survive in the soil for a long time.

Current methods for managing potato black scurf include chemical fumigation [11], breeding resistant varieties [12], crop rotation [13,14,15], and so on. However, the chemical control of R. solani is not very effective as most chemicals cause hazardous effects on non-targeted organisms and the environment [16], and disease-resistant varieties may have problems such as low product yield, poor processing quality, and low output [17]. Crop rotation is not suitable for the large-scale cultivation of potato agriculture [17]. Therefore, it is necessary to explore efficient and environmentally friendly methods for managing potato black scurf, such as biological control. Currently, microorganisms have attracted people’s attention as biological control agents for plant diseases and have thus become a research focus [18,19].

A large number of studies have proved that biocontrol bacteria, such as Bacillus velezensis, Brevibacillus parabrevis, Pseudomonas fluorescens, Bacillus subtilis, Bacillus amyloliquefaciens, Trichoderma harzianum, Trichoderma reesei, and Serratia plymuthica B4, can inhibit the infection of pathogens and can also promote plant growth [20,21,22,23,24,25,26,27]. The Bacillus amyloliquefaciens SB14 significantly controlled damping-off disease caused by R. solani AG-4 by 58% and that caused by R. solani AG-2-2 by 52.5; Bacillus subtilis V26 induced significant suppression of root canker and black scurf tuber colonization in incidence disease of 63% and 81%, respectively [28,29]. Furthermore, there are substances that promote plant growth, such as iron carriers and IAA [30,31]. However, there are few reports on using B. polymyxa YF as a biological control agent for potato black scurf. This study investigates the inhibitory effects of Paenibacillus polymyxa YF and Bacillus amyloliquefaciens HT on R. solani, through plate, pot, and in vitro inoculation experiments. Additionally, it seeks to elucidate the potential disease resistance and growth-promoting effects of these antagonistic bacteria. These findings lay a theoretical foundation for developing P. polymyxa YF as a feasible biological control agent with the potential to control potato black scurf and promote potato growth.

2. Materials and Methods

2.1. Pathogens Strain and Antagonistic Bacteria

The fungal pathogen Rhizoctonia solani (GenBank Accession OR824714) strain was provided by the Gansu Academy of Agricultural Sciences (Lanzhou, China). R. solani was maintained under in vitro conditions on potato dextrose agar (PDA) slants with regular sub-culturing after every 15–20 d [32].

P. polymyxa YF (GenBank ID: 16S rRNA, MW205750; gyrA, OP888972; RpoB, OP888973) and B. amyloliquefaciens HT (GenBank ID: 16SrRNA, MW776428; gyrA, OP888971; RpoB, OP888976) were used in this study. They were isolated from sheep manure compost or the rhizosphere soil of healthy vegetables, and stored on Lysog-enic Agar (10 g/L (Basebio, Hangzhou, China), yeast extract 5 g/L (Macklin, Shanghai, China), NaCl 10 g/L (Guangnuo, Shanghai, China), and agar (BioFroxx, Guangzhou, China) 20 g/L) at 4 °C. P. polymyxa YF were conserved at the China General Microbiological Culture Collection Center (CGMCC No.21517) [33]. The strains of microorganisms used in the study have been preserved in the Microbiology Laboratory of Lanzhou Jiaotong University.

2.2. Antifungal Activity Assay of P. polymyxa YF and B. amyloliquefaciens HT against R. solani

The antagonistic ability of biocontrol bacteria was measured in vitro on PDA plates in a confrontation assay [34]. The 6 mm diameter cakes from R. solani cultured at 28 °C for 7 d were inoculated in the center of all plates. Then, the bacteria isolates were transferred in three symmetric spots 2 cm from the R. solani. In the control, sterile distilled water was used instead of the antagonistic bacteria. Plates were incubated for 7 d at 28 °C in the dark, and the growth of the fungus towards the bacterial and control treatments was measured on day 7 [35]. The percentage of mycelium growth inhibition was calculated by using the following formula: inhibition rate (%) = [(R − r)/R] × 100. Here, R is the radius of fungal growth from the center of the plate towards the control treatment and r is the radius of fungal growth towards the bacterial treatment.

The poisoned food technique investigated the antifungal activity of extracellular metabolites from P. polymyxa YF and B. amyloliquefaciens HT [36]. Two bottles of LB broth (100 mL) were inoculated with 200 μL P. polymyxa YF and B. amyloliquefaciens HT (OD 600 = 1), respectively, and incubated simultaneously at 28 °C for 48 h. The culture was removed by centrifugation for 10 min at a speed of 3640 g, and the supernatant was filtered aseptically by a disk membrane with a diameter of 0.22 µm. The obtained cell-free culture filtrate (CF) was mixed with the molten PDA medium (45 ± 5 °C) so that the final medium volume ratio was 10%, 20%, and 50% (v/v). The media amended with different concentrations of CF were poured into Petri plates and inoculated with R. solani plug in the center. PDA medium amended with LB broth in the same concentrations was used as the control treatment. Inoculated plates were incubated for 7 d (the average time for R. solani to cover the plate surface in the control treatment). Plates were photographed, and pathogenic hyphae were measured. Inhibition rate (%) = [(D1 − D2)⁄D1] × 100; here, ‘D1’ is the colony diameter of R. solani in the control and ‘D2’ is the colony diameter of R. solani in the treatment plates.

2.3. Determination of the Plant-Growth-Promotion Properties of P. polymyxa YF and B. amyloliquefaciens HT

2.3.1. Test of Siderophore Production

Siderophore production was analyzed by Modified CAS Agar Medium Kit (Hopebio, Qingdao, China, CAS: chrome azurol sulphonate 10.87 g, 1 L distilled water) according to the methods outlined previously [37]. P. polymyxa YF and B. amyloliquefaciens HT were inoculated onto Lysogenic Agar plates and incubated at 30 °C for 24 h. The strain was inoculated on CAS medium and the plate was incubated at 30 °C for 3 days. The clear and transparent circle around the colony is believed to indicate the production of iron carriers.

2.3.2. Detection of Extracellular Enzymes

P. polymyxa YF and B. amyloliquefaciens HT can produce extracellular enzymes such as protease, cellulase, and amylase [38]. Biocontrol antagonists were inoculated into corresponding chromogenic mediums. (Amylase detection medium; cellulase detection medium; protease detection medium. The specific formula of the medium can be found in Supplement I.) All experiments were carried out at 28 °C for 3 d and observed conformational changes; these were recorded via photos.

2.3.3. IAA Production

The generation of IAA by P. polymyxa YF and B. amyloliquefaciens HT strains was qualitatively and quantitatively determined using the colorimetric method, as detailed in previous studies [30,31]. Briefly, the cultures of P. polymyxa YF and B. amyloliquefaciens HT were placed in King’s medium with and without L-tryptophan (Macklin, Shanghai, China) for 2 d at 30 °C and 180 rpm. After centrifugation at 5000× g rpm for 5 min, the supernatant was mixed with Salkowski’s reagent (150 mL concentrated H₂SO₄ (Nanjing Reagent, Nanjing, China), 250 mL dH₂O, 7.5 mL 0.5 M FeCl₃·6H₂O (FuChen, Tianjin, China)) in a 1:1 ratio using brief vortexing. After incubation at 30 °C in the dark for 30 min, the absorbance of the mixture was measured at 530 nm using a spectrophotometer. The IAA concentration was quantified by comparing these readings against a standard IAA solution (Sigma Aldrich, Burlington, MA, USA).

2.4. Effect of P. polymyxa YF and B. amyloliquefaciens HT on Mycelial Morphology of R. solani

Five mm diameter plugs from R. solani cultures were inoculated into 100 mL Potato Dextrose Broth medium (composed of 200 g of potato, 20 g of glucose (Xilong, Guangzhou, China), and 1 L of distilled water). These cultures were incubated at 25 °C, agitated at 180 r/min for 4 d, and then mixed with 10% P. polymyxa YF or strain HT bacteria-free filtrates that had been previously incubated for 24 h. The non-inoculated medium filtrate was added to the control treatment. After maintaining for 4 d, scanning electron microscopy was used to observe the morphological changes of R. solani hyphae in each treatment group (SEM, ZEISS Gemini 500, Oberkochen, Germany). This experiment is in triplicate.

2.5. Effects of P. polymyxa YF and B. amyloliquefaciens HT on the Membrane Permeability of R. solani

The effect of the bacterial culture supernatant on the permeability of the R. solani cell membrane was studied by measuring electrical conductivity [39]. The hyphae of R. solani were cultured in 100 mL of PDB at 25 °C for 7 d. The obtained R. solani hyphae were centrifuged at 8000× g for 2 min and washed thrice with sterile distilled water. After that, 10% bacterial culture supernatant was added to the culture. Eventually, the supernatant was analyzed by a conductivity meter (INESA, Shanghai, China) within 24 h. The control was performed without the bacterial supernatant; this experiment was performed in triplicate.

2.6. Observation on Colonization of P. polymyxa YF Strain in Potato Plants

GFP-labeled P. polymyxa YF were constructed using GFP-marked vector pGFP4412 [40]. The colonization of P. polymyxa YF-GFP in the potato stems and roots was performed with minor modifications in the methods described [41]. P. polymyxa YF-GFP was incubated overnight in 100 mL LB liquid medium containing 100 μg/mL Amp (Rhawn, China) at 30 °C. The surfaces of the well-grown 5-week-old potato stems and roots were disinfected; then, they were transplanted into pots with sterilized soil. Next, they were treated with 100 mL P. polymyxa YF-GFP culture (10⁸ CFU/mL) and provided with 100 mL of water as a blank control for watering. Then, the plants were kept in a greenhouse with a temperature cycle of 25 °C/15 °C (day and night). To visualize the colonization of P. polymyxa YF-GFP in potato stems and roots, the potato stem and root tissue samples were randomly collected at 5, 10, 15, 20, and 25 d after inoculation with bacteria. The collected stems and roots were washed with water and cut into thin slices of about 1 mm for observation using confocal laser scanning microscopy (CLSM, Olympus FV3000, Olympus, Tokyo, Japan) with an excitation wavelength of 488 nm. To further determine the colonization rate of potato roots and stems, a portion of each stem and root sample (0.1 g) was ground in a mortar containing 0.9 mL of sterile distilled water, until a fine homogenate was obtained. After diluting the suspension, it was placed on a modified LB medium (100 μg/mL, Amp) and incubated at 30 °C for 24 h, followed by CFU counting [42].

2.7. Control Effect on R. solani and Growth Promotion for Potato of P. polymyxa YF in the Greenhouse

The Atlantic variety of potatoes from Ronghui market, Anning district, Lanzhou city, Gansu province, was selected. Fifteen healthy and plump potato tubers were used for sprouting potato seedlings. Wash the potato tubers, disinfect them by soaking in ethanol (70%) for 1 min and NaClO (5%) for 2 min, rinse them three times with excess sterile distilled water, and finally cut them into pieces with a sterile scalpel. Each potato tuber was placed individually in a plastic pot containing sterile soil and cultured for 5 weeks under natural conditions. The experimental design included 5 treatment groups: (1) drenching plant roots with 50 mL suspension of R. solani conidia (10⁵ conidia/mL); (2) applying 50 mL suspension of both R. solani conidia (10⁵ conidia/mL) and P. polymyxa YF suspension (10⁸ CFU/mL) to the roots; (3) drenched the roots of each plant with 50 mL of P. polymyxa YF suspension and, 48 h later, drenched them with 50 mL of R. solani conidial suspension (10⁵ conidia/mL); (4) a sole application of 50 mL P. polymyxa YF suspension; (5) drenched the roots of each plant with 50 mL of sterile water as a control. Two weeks post-treatment, we observed and recorded the symptoms and severity of disease manifestation in the R. solani-treated plants. In addition, the physiological parameters were also measured, including plant height (cm), root length (cm), stem thickness (mm), fresh weight (g), and dry weight (g). The disease incidence in each treatment group was quantified as a percentage of infected plants out of the total. Two weeks after inoculation, the phenotypes such as stems and leaves and browning were observed. These come as a result of the occurrence of the disease in the upper ground tissue; the extent to which the underground tissue is affected is assessed based on the characteristics of the potato black scurf after the seedlings are removed from the pot: rotten, the dark brown discoloration of the vascular tissue. Moreover, another result of the disease is that the plants die; in this case, the occurrence of the disease can be determined. To assess the pathogenic data obtained from the pathogenic test, we used one-way analysis of variance (ANOVA) and then Tukey’s HSD test (honest and significant differences) to determine the presence of any significant differences at the p < 0.05 level (SPSS Version 27.0) [43].

The severity of the disease affecting the potato seedlings was evaluated after treatment. According to the nonparametric marginal effect analysis of disease severity grade, based on 95% CI of $\widehat{p}$ij, the pathogenicity of all treatments differed from that of the control plants without inoculation. Disease severity was evaluated using a numerical rating scale from 0 to 9 [44]. Two weeks after inoculation, the disease severity of the underground part of the plant was classified and counted, and the following scores were assigned: 0 = no disease spots on leaves; 1 = disease spot area < 5% of total leaf area; 3 = disease spot area 6~10% of total leaf area; 5 = disease spot area 11~20% of total leaf area; 7 = disease spot area 21–50% of total leaf area; 9 = disease spot area > 50% of total leaf area [45]. The disease severity was calculated as a percentage of the total number of experimental plants for the underground plant part. Data were analyzed in IBM SPSS Statistics 27.0.

2.8. Potato Excised Tissues Experiment

Material varieties and sources are consistent with potted experiments. Specifically, healthy potatoes were selected, ensuring similarity in shape. The potato tubers were cut into fragments with an average size of 6.39 cm × 5.93 cm and a thickness of 1.5 cm. The potato tubers were washed and then disinfected by soaking in ethanol (70%) for 1 min and NaClO (5%) for 2 min; then, they were rinsed three times with excess sterile distilled water. Finally, they were cut into pieces with a sterile scalpel [46]. The slices were subjected to five treatments: (1) only sprayed with 100 μL of R. solani conidial suspension (10⁵ conidia/mL) on the surface of the tuber; (2) sprayed with 100 μL of R. solani conidial suspension (10⁵ conidia/mL) and 100 μL of P. polymyxa YF suspension (10⁸ CFU/mL) on the surface of the tuber; (3) sprayed with 100 μL for 48 h P. polymyxa YF suspension (10⁸ CFU/mL) and then sprayed with R. solani conidial suspension (10⁵ conidia/mL) on the surface of the tuber; (4) only sprayed with 100 μL of biocontrol bacteria (OD600 = 0.4) on the surface of the tuber; (5) sprayed with sterile dH₂O (control) on the surface of the tuber. The treated slices were placed in a Petri dish (90 mm) lined with sterile moist filter paper; then, they were incubated at 25 °C for 4 days. Lesion diameter was measured and calculated at 7 d post-inoculation. Disease incidence and severity were calculated using the method described in Section 2.7.

2.9. Production of Resistant Enzymes in Plants Induced by P. polymyxa YF

Enzymes involved in plat resistance, such as peroxidase (POD), polyphenol oxidase (PPO), catalase (CAT) and phenylalanine ammonia-lyase (PAL) were examined. The fermentation broth of R. solani and the cultures of P. polymyxa YF were prepared. Five treatment groups were established according to the descriptions provided in Section 2.7. After the potato plant reached the development stage of 7–8 compound leaves, a random sample of 0.5 g of root tissue from each treatment group was selected for analysis. The root tissues were then ground for the assays assessing the enzymatic activities of POD, PAL, CAT, and PPO; these were analyzed using commercial assay kits (AIDISHENG, Yancheng, China), following the manufacturer’s instructions [47,48]. The quantification of enzyme activities was achieved by recording the absorbance values using a spectrophotometer (UA-2800A, UNIC, Shanghai, China).

2.10. Detection of Synthesis Genes of Antimicrobial Lipopeptides of P. polymyxa YF and B. amyloliquefaciens HT

Using the primers designed in Table S1, the genes for iutirin, fengycin, suractin, polyketide synthase, and nonribosomal peptide synthesis were detected in the P. polymyxa YF and B. amyloliquefaciens HT. The primer sequences used in this study are described by Zhang et al. [33] (Table S1). The reaction system of the 25 μL PCR included 1.5 μL of the bacterial suspension (10⁸ CFU/mL), 1 μL of primer (10 μM), 12.5 μL 2× master mix with standard buffer (Sangon Biotech in Shanghai), and 10.5 μL nuclease-free water. The primer sequence was provided by Sangon Biotech in Shanghai. The reaction conditions were set: pre-denaturation at 94 °C for 7 min, denaturation at 94 °C for 45 s, annealing at 56 °C for 45 s, and extension at 72 °C for 120 s. This cycle was repeated 35 times. After the cycles, there was a final extension at 72 °C for 10 min and an infinite cycle at 4 °C. The PCR amplicons were analyzed by electrophoresis in 1% (w/v) agarose gel, and the sizes of the bands were determined using the 2 kb marker.

2.11. Identification of P. polymyxa YF Surfactin and Polymyxin B Sulfate Components by HPLC

P. polymyxa YF was inoculated into LB of 100 mL and cultured for 72 h; next, the bacterial suspension was centrifuged at 12,000× g rpm for 15 min. The collected supernatant was used for liquid phase analysis. The supernatant was filtered with a sterile filter membrane (0.22 μm); then, 50 mL of macroporous resin particles were added to every 100 mL supernatant; finally, the flask was shaken at room temperature for 4 h. The obtained mixture was shaken with a separatory funnel and then allowed to stand and separate. The separation was concentrated in a rotary evaporator with a condition of 40 °C and 60 rpm.

Samples and standard (10 µL) were injected into the HPLC, SHIMANZU Essentia LC-16 series device, consisting of an SPD-16 absorption and a C18 reversed-phase (ShimNex CS C18, 250 × 4.6 mm, 5 µm) column. For the crude extract of P. polymyxa YF and the standard sample of surfactin (purity ≥ 98%, Macklin-Aldrich Trading Co., Ltd., Shanghai, China), the mobile phase consisted of an acetonitrile–methanol solution (Damao, Tianjin, China) containing 0.1% TFA (Macklin, Shanghai, China) and water (90:10, v/v). The flow rate was maintained at 1 mL min⁻¹, and the chromatograms were detected at 205 nm. For the crude extract of P. polymyxa YF and polymyxin B sulfate (purity ≥ 96%, Med Chem Express Trading Co., Shanghai, China), the mobile phase was composed of acetonitrile and water (50:50, v/v), and the flow rate was 1 mL min⁻¹; the chromatograms were detected at 215 nm. The column temperature was 40 °C.

Accuracy was evaluated within and between the day data of the triple injections of each concentration and expressed as a relative error. The precision of the HPLC method was evaluated in terms of the relative standard deviation of the retention time, measured concentration, and the peak area of the studied solution within and between days. In this experiment, a 3-fold S/N ratio was used as the detection limit of the method. A polymyxin B sulfate and crude extract of P. polymyxa YF solution was formed, with a concentration of 1 mg mL⁻¹; then, a surfactin solution with the concentration of 5 mg mL⁻¹ was formed. With these, the injection was repeated, with a 10 µL injection volume, with 6 consecutive needles; this allowed us to investigate the precision of the instrument.

2.12. Statistical Analysis

The data were statistically analyzed using IBM SPSS Statistics 27.0 (IBM Corp., Armonk, NY, USA). All data are presented here as mean ± SEM. To detect the differences among the means in each group, one-way analysis of variance (ANOVA) was used, followed by Tukey’s HSD (honest significant difference) test at a significance level of p < 0.05.

3. Results

3.1. Effect of P. polymyxa YF and B. amyloliquefaciens HT against R. solani

In order to screen the biocontrol bacteria, which can effectively control potato black scurf caused by R. solani, six bacterial strains were evaluated for their antagonistic properties. Among them, compared with the control group, the P. polymyxa YF and B. amyloliquefaciens HT strains exhibited the highest inhibitory activity against R. solani, with inhibition rates of 61.33% and 60.56%, respectively (Turkey HSD test, p < 0.05) (Figure 1 and

Table 1. Evaluation of the inhibitory effect of six species of Bacillus against Rhizoctonia solani.

Bacillus Strains	Treatment Group Diameter (cm)	Control Diameter (cm)	Inhibition Ratio (%)
Bacillus subtilis	3.59 ± 0.07	9.00 ± 0.01	60.11 a
Paenibacillus polymyxa	3.48 ± 0.13	9.00 ± 0.01	61.33 a
Bacillus amyloliquefaciens	3.55 ± 0.09	9.00 ± 0.01	60.56 a
Bacillus megaterium	3.60 ± 0.10	9.00 ± 0.01	60.00 a
Bacillus mucilaginosus	3.78 ± 0.06	9.00 ± 0.01	58.00 b
Brevibacillus laterosporus	9.00 ± 0.01	9.00 ± 0.01	0.00 c

Note: ± represents the standard deviation of the three replicates (mean ± SD). According to the Tukey HSD test, different letters indicate significant differences at the 0.05 probability level.

). Due to their strong inhibitory effects, P. polymyxa YF and B. amyloliquefaciens HT were selected for further investigation.

The obtained cell-free culture filtrate (CF) was mixed with molten PDA medium (45 ± 5 °C) at final volume ratios of 10%, 20%, and 50% (v/v). Compared with the control, the inhibition of P. polymyxa YF was stronger than that of B. amyloliquefaciens HT in the three cases. Notably, at a 50% volume ratio, the inhibition rates of P. polymyxa YF and B. amyloliquefaciens HT on R. solani were 85.55% and 82.86%, respectively. At this concentration, both P. polymyxa YF and B. amyloliquefaciens HT CF completely prevent R. solani growth under the test conditions (Tukey HSD test, p < 0.05) (Figure 2 and

Table 2. Evaluation of the inhibitory effect of two biocontrol bacterial fermentation filtrates against R. solani.

Biocontrol Bacteria		Fermentation Broth Concentration
		10%	20%	50%
P. polymyxa YF	Control group	8.15 ± 0.06	7.95 ± 0.05	8.65 ± 0.02
	Treatment group	7.05 ± 0.04	4.35 ± 0.09	1.25 ± 0.07
	Inhibition rate (%)	13.50 c	45.28 b	85.55 a
B. amyloliquefaciens HT	Control group	7.85 ± 0.15	8.15 ± 0.16	8.75 ± 0.21
	Treatment group	7.50 ± 0.09	4.50 ± 0.18	1.50 ± 0.17
	Inhibition rate (%)	4.46 c	44.79 b	82.86 a

Note: ± represents the standard deviation of the three replicates (mean ± SD). According to the Tukey HSD test, different letters indicate significant differences at the 0.05 probability level.

). The efficacy of the filtrates was consistent with the results of the plate confrontation test, highlighting their potential as biocontrol agents against R. solani.

3.2. Effect of P. polymyxa YF and B. amyloliquefaciens HT on Mycelial Morphology of R. solani

As shown in Figure 3, the hyphae of R. solani treated with P. polymyxa YF showed morphological alteration. The hyphae displayed pronounced distortion, shrinkage, atrophy, and even breakage. The control group hyphae maintained a healthy appearance, with an abundant, smooth surface, and a uniform size with a normal physiological structure. In the B. amyloliquefaciens HT-treated group, the hyphae of R. solani also exhibited significant morphological differences compared to the control group, with significant deformation and wrinkling.

3.3. Effects of P. polymyxa YF and B. amyloliquefaciens HT Culture Supernatant on Membrane Permeabilization

The cell membrane permeability results showed a gradual increase in extracellular conductivity following the treatment with the biocontrol agents; meanwhile, those of the control group showed a marginal increase. Notably, the increase in conductivity was most rapid in the treatment groups and stabilized in 21 h. After 6 h of treatment, compared with the control group, the extracellular conductivity of P. polymyxa YF and B. amyloliquefaciens HT groups increased by 74.74 and 36.04 times, respectively. The results demonstrate that P. polymyxa YF and B. amyloliquefaciens HT could effectively disrupt the structural and functional integrity of R. solani cell membrane, contributing to their inhibitory effect on the growth and proliferation of R. solani (Figure 4).

3.4. Determination of Biological Characteristics of Strains P. polymyxa YF and B. amyloliquefaciens HT

As shown in Figure 5, the plates inoculated with P. polymyxa YF and B. amyloliquefaciens HT showed a protease hydrolysis circle, a cellulase hydrolysis circle, an amylase hydrolysis circle, and a yellow halo formed by iron ions (Figure 5). This indicates that P. polymyxa YF and B. amyloliquefaciens HT could have the ability to produce protease, cellulase, amylase, and siderophores. The pink color of the antagonistic bacterial supernatant and colorimetric solution mixed for 30 min showed that both P. polymyxa YF and B. amyloliquefaciens HT could produce IAA. Compared with the absence of tryptophan, the pink color of P. polymyxa YF and B. amyloliquefaciens HT became darker in the presence of tryptophan, indicating that tryptophan can promote the production of more IAA in P. polymyxa YF and B. amyloliquefaciens HT. In the presence of tryptophan, the amounts of IAA produced by P. polymyxa YF and B. amyloliquefaciens HT were 13.83 mg/L and 17.54 mg/L, respectively (Figure 6).

3.5. Observation on the Colonization of P. polymyxa YF in Potato Plants

After 5–10 d of inoculation, the colonization rate increased continuously, and the fluorescence intensity reached the strongest on the 10 d, indicating that a large number of antagonistic bacteria had penetrated the roots of potato, and the fluorescence intensity was stable at 15–20 d, indicating that P. polymyxa YF could be colonized continuously. The colonization rate was more than 10⁴ CFU/g. At 25 d, the fluorescence intensity darkened obviously and P. polymyxa YF no longer permeated continuously (Figure 7 and Figure 8A).

On day 5 of inoculation, the fluorescence intensity in the stem was the strongest, indicating that many antagonistic bacteria had penetrated the potato stem at this time. The fluorescence intensity was stable in the following 10–20 d, indicating that P. polymyxa YF could continue to colonize for 15 d, and the colonization rate was stable above 10⁴ CFU/g. At day 20, the fluorescence intensity darkened obviously, and P. polymyxa YF no longer permeated continuously (Figure 8B and Figure 9).

3.6. Control Effect on R. solani and Growth Promotion of P. polymyxa YF in the Greenhouse

In the pot experiment, only potato plants inoculated with R. solani showed symptoms of fusarium wilt, including severe discoloration of vascular bundles and wilted leaves. However, the vascular bundles of potato plants treated with P. polymyxa YF were only slightly discolored, indicating a low severity of black scurf (Figure 10). These results prove that strain P. polymyxa YF exhibited good control effectiveness against potato black scurf. In the pot experiment, the R. solani-treated seedlings had the highest incidence rate; the negative control had the largest median disease severity scores (8.80), $\widehat{p}$ij (0.88), and average grade $\stackrel{\mathrm{-}}{R}$ij (35.50) (Tukey’s HSD test, p < 0.05); the P. polymyxa YF prevention group and P. polymyxa YF treatment group had the smallest median disease severity scores (1.40 and 1.20), $\widehat{p}$ij (0.51 and 0.49), and average grade $\stackrel{\mathrm{-}}{R}$ij (21.00 and 20.00) (Tukey’s HSD test, p < 0.05); the corresponding control effects were 59.23% and 52.76%, respectively (Tukey’s HSD test, p < 0.05) (

Table 3. Evaluation of potted control effect of P. polymyxa YF on potato black scurf.

Treatment	MDRx	$\overline{\mathit{R}}$ijy	$\widehat{\mathit{p}}$ij	Control Effect (%)	$95\%\mathbf{CI}\mathbf{for}\widehat{\mathit{p}}$ij
					Lower	Upper
Blank Control	0.00	5.50	0.13	-	0.13	0.13
Negative Control	8.80	35.50	0.88	-	0.85	0.90
Prevention Group	1.40	21.00	0.51	59.23 ± 0.14 a	0.44	0.59
Treatment Group	1.20	20.00	0.49	52.76 ± 0.05 a	0.43	0.54

Note: The effects of R. solani on the disease severity were measured on a scale of zero to nine. Parameters include median, mean rank ($\stackrel{\mathrm{-}}{R}$ij), and relative treatment effect ($\widehat{p}$ij) with 95% confidence intervals (CI) for disease severity. Different letters indicate significant differences at the 0.05 probability level.

).

After 7 weeks, we determined the morphological indicators of the potato seedlings treated with P. polymyxa YF. Compared with the control, the plant height, stem thickness, root length, fresh weight, and dry weight of potato seedlings treated with P. polymyxa YF were significantly improved, increasing by 16.41%, 13.73%, 57.52%, 11.46%, and 56.03%, respectively (Tukey’s HSD test, p < 0.05). In the R. solani + P. polymyxa YF and P. polymyxa YF + R. solani treatment, compared with the treatment with R. solani, the plant height, root length, stem thickness, fresh weight, and dry weight also significantly increased (Figure 11) (Tukey’s HSD test, p < 0.05). The results demonstrate that P. polymyxa YF promotes the increase in the biomass of potato plants.

For the excised tissue inoculation, the experiment indicates that potato slices inoculated with R. solani showed typical hyphal growth on the surface of the tuber slices compared to the control group. With time, the treatment group’s hyphae continued accumulating on the surface of excised tissues. The surface is brown, and even the tuber tissue deteriorates and decays. In the treatment, compared with the control group, the mycelium growth of R. solani was more or less in the pathogen group, prevention group, and treatment group; among these, the pathogen group was the most, the treatment group was the second, and the prevention group was the least, indicating that the effect of P. polymyxa YF prevention was better than that of treatment. Moreover, the color of the tubers of P. polymyxa YF, prevention, and treatment groups are yellowish, and the symptoms of decay are significantly reduced (Figure 12). For the excised tissue inoculation, the negative control had the largest median disease severity scores (8.20), $\widehat{p}$ij (0.90), and average grade $\stackrel{\mathrm{-}}{R}$ij (54.50) (Tukey’s HSD test, p < 0.05); meanwhile, the P. polymyxa YF prevention group and P. polymyxa YF treatment group had the smallest median disease severity scores (4.00 and 5.80), $\widehat{p}$ij (0.43 and 0.68), and average grade $\stackrel{\mathrm{-}}{R}$ij (26.50 and 41.00) (Tukey’s HSD test, p < 0.05) (

Table 4. Evaluation of tuber control effect of P. polymyxa YF on potato black scurf.

Treatment	MDRx	$\overline{\mathit{R}}$ijy	$\widehat{\mathit{p}}$ij	Control Effect (%)	$95\%\mathbf{CI}\mathbf{for}\widehat{\mathit{p}}$ij
					Lower	Upper
Blank Control	0.00	5.50	0.08	-	0.08	0.08
Negative Control	8.20	54.50	0.90	-	0.85	0.95
YF Prevention Group	4.00	26.50	0.43	56.03 ± 0.43 a	0.33	0.54
YF Treatment Group	5.80	41.00	0.68	53.43 ± 0.24 ab	0.58	0.77
HT Prevention Group	3.40	22.40	0.37	50.50 ± 0.20 ab	0.26	0.47
HT Treatment Group	4.80	33.10	0.54	43.33 ± 0.18 b	0.43	0.65

Note: The mean and standard deviation (SD) of the data are shown (N = 3); different letters on the same line denote significant differences at the 0.05 level of p-value by the Kruskal–Wallis test and Dunn test as post hoc. The effects of R. solani on the disease severity of tubers were measured on a scale of zero to nine. Parameters include median, mean rank ($\stackrel{\mathrm{-}}{R}$ij), and relative treatment effect ($\widehat{p}$ij), with 95% confidence intervals (CI) for disease severity.

).

3.7. Detection of Antagonistic Enzyme Activities in Potato Plants

The POD, PPO, PAL, and CAT activities in the potato roots reflected the potatoes’ resistance to pathogens. Compared to the control, the enzyme activities of POD showed significant improvements in the P. polymyxa YF group, the treatment group, and the prevention group, which increased by 4.04, 3.95, and 3.38 times, respectively (Tukey HSD test, p < 0.05) (Figure 13A). Compared to the control, the enzyme activities of PPO showed significant improvements in the P. polymyxa YF group, the treatment group, and the prevention group, which increased by 0.54, 0.33, and 0.33 times, respectively (Tukey HSD test, p < 0.05) (Figure 13B). Compared to the control, the enzyme activities of CAT showed significant improvements in the P. polymyxa YF group, the treatment group, and the prevention group, which increased by 3.10, 2.81, and 2.30 times, respectively (Tukey HSD test, p < 0.05) (Figure 13C). Compared to the control, the enzyme activities of PAL showed significant improvements in the P. polymyxa YF group, the treatment group, and the prevention group, which increased by 0.46, 0.41, and 0.34 times, respectively (Tukey HSD test, p < 0.05) (Figure 13D).

After the roots of the potatoes were treated by strains P. polymyxa YF only, the content of POD, PPO, PAL, and CAT was significantly different (Tukey HSD test, p < 0.05). The results indicate that, even when potato seedlings are infected by R. solani, the strain of P. polymyxa YF can stimulate the production of defensive enzymes.

3.8. Detection of Synthesis Genes of Antimicrobial Lipopeptides of P. polymyxa YF and B. amyloliquefaciens HT

Using primer pairs designed to detect genes involved in the biosynthesis of iutirin, fengycin, suractin, polyketide synthase, and non-ribosomal peptide synthase, amplicons of the expected size were obtained (Figure S1). Through the inhibition test of the Oxford Cup, we obtained that the inhibition rates that the crude extract of P. polymyxa YF and standard of surfactin and polymyxin B sulfate had on R. solani: 35.26%, 51.87%, and 23.84 %, respectively (Figure 14). The liquid chromatography results showed that P. polymyxa YF produced surfactin and polymyxin B sulfate. At 205 nm, the peak value generated by YF coarse and Surfactin is 802,134 and 171,850; at 215 nm, the peak values produced by YF coarse and Polymyxin B Sulfate were 108,902 and 60,335, respectively. And there were no interfering peaks from the samples (Figure 15).

4. Discussion

R. solani is known for its broad host range of over 200 plant species, including important crops like rice, wheat, soybeans, potatoes, and tomatoes [7]. The potato black scurf caused by R. solani poses a significant challenge to global agriculture. Previous studies have acknowledged the potential of Bacillus species as biological control agents against R. solani. Yet, reports addressing their role in combating potato black scurf and exploring their biocontrol mechanisms are limited [49]. Our study is the first extensive exploration of the biological control efficacy of P. polymyxa YF and B. amyloliquefaciens HT against R. solani for potatoes.

After treating biocontrol bacteria P. polymyxa YF and B. amyloliquefaciens HT, the hyphae of R. solani in the antagonistic zone were observed by SEM, and a great deformation occurred. The bacteria-free filtrates of YF and SY89 caused the hyphae of the pathogenics to rupture and shrink, thereby inhibiting the mycelial growth of F. avenaceum [33]. The results of the report are consistent with those of this study. P. polymyxa YF and B. amyloliquefaciens HT inhibited the growth of pathogens by increasing the lipid peroxidation of the pathogen hyphae and decreasing the synthesis of the metabolism products [50].

Compared to the inoculated and uninoculated pathogens, P. polymyxa YF-treated seedlings significantly suppressed the growth of the pathogen and enhanced the plant’s growth and biomass. Under natural conditions, P. polymyxa YF could inhibit the disease and promote growth; these effects were more significant than those seen in the control. It was also observed that P. polymyxa YF was colonized more frequently in the potato rhizosphere. In greenhouse and field studies, B. subtilis MB14 and B. amyloliquefaciens MB101 can protect tomatoes from the pathogenic fungus (R. solani) [51,52]. Two B. subtilis strains, PCL1608 and PCL1612, producing iturin A, have exhibited the highest biocontrol and colonization capabilities [53]. The first step of bacterial cell attraction/adhesion is an efficient colonization phase and a further population decrease to reach a stable level at approximately 5 × 10⁶ cells per gram of roots [54]. Following rhizosphere and rhizoplane colonization, some soil-borne microorganisms can enter roots and establish subpopulations ranging from 10⁵ to 10⁷ CFU g⁻¹ FW [55]. The colonization process observed for P. polymyxa YF was similar to that reported for other plant-beneficial rhizobacteria. Plant-growth-promoting bacteria can be employed for their role in increasing the growth and yield of various crops such as wheat and rice. Among them, a series of laboratory experiments conducted on wheat under gnotobiotic (axenic) conditions demonstrated increases in root elongation (up to 17.3%), root dry weight (up to 13.5%), shoot elongation (up to 37.7%), and shoot dry weight (up to 36.3%) for the inoculated wheat seedlings. Bacillus pantothenticus P4 (MTCC 4695) and Pseudomonas pieketti Psd6 (MTCC 4715) increased rice grain yield by 55.5, 12.2, and 76.9% over the uninoculated control in micro-plot experiments [56,57].

Furthermore, P. polymyxa YF and B. amyloliquefaciens HT were found to amplify the genes of antimicrobial compounds, such as iturin, fengycin, surfactin, polyketide synthases, and nonribosomal polypeptide synthase biosynthesis genes. Antibiotics produced by Bacillus spp. play an essential role in the biocontrol of fungal diseases, and different types of metabolite production under in vitro assays showed that selected Bacillus spp. may be used in multiple modes of action against fungal pathogens [58]. A large number of antifungal peptides are reported by Bacillus spp. [59], including fengycin, surfactin, bacillomyci-ns D, F, and L, iturins A–E [60,61,62]. In addition, P. polymyxa YF also produces polymyxin B sulfate, which showed antibacterial activity both in vitro and in vivo, in this study. These antifungal peptides reduce the growth of many fungi, such as Rhizoctonia, Fusarium, Aspergillus, and Penicillium species [59]. In general, many antifungal peptides that produce bacteria or filamentous fungi have been reported to be biological control agents against different kinds of fungal diseases of plants [62,63,64,65]. Therefore, biocontrol bacteria could inhibit the growth of R. solani by secreting lipopeptides and other substances to achieve the purposes of prevention and control. These findings contribute significantly to our understanding of biocontrol effects and offer promising strategies for controlling potato black scurf and other fungal diseases in agriculture.

5. Conclusions

In summary, this study established P. polymyxa YF as a potent inhibitor of R. solani, the pathogen responsible for potato black scurf. Both P. polymyxa YF and B. amyloliquefaciens HT have demonstrated the ability to produce extracellular enzymes, contributing significantly to the inhibition of R. solani growth. These strains are characterized by their ability to produce iron carriers and IAA, alongside their proficiency in hydrolyzing cellulase, amylase, and protease. On this basis, the efficient antagonism caused by R. solani can significantly inhibit the growth of mycelium. Moreover, P. polymyxa YF effectively mitigates the incidence of potato black scurf and exerts a notable growth-promoting effect on plants. It can induce plant stress enzyme activity and potentially secrete antibacterial lipopeptides to achieve efficient prevention and control effects. This study represents the first instance of P. polymyxa YF being used as a biocontrol agent against potato black scurf, providing a foundation for developing novel and effective biocontrol strategies for this agricultural challenge.