Next Article in Journal
From Folk Medicine to Pharmacology: A Systematic Review of the Anti-Inflammatory Evidence for Hymenaea spp. (Fabaceae)
Previous Article in Journal
In Vitro Regeneration, Acclimatization, Phytochemical Profiling, and Antioxidant Properties of Hong Hoen Sirirugsa (Globba sirirugsae Saensouk & P.Saensouk)
Previous Article in Special Issue
Investigating the Biocontrol and Plant Growth-Promoting Potential of Pseudomonas yamanorum for Sustainable Management of Tomato Early Blight (Alternaria alternata)
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Plants
Volume 14
Issue 22
10.3390/plants14223546
plants-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Endophytic Pseudomonas koreensis A1 of Bletilla striata as a Plant Growth Promoter and Biocontrol Agent Against Rice Sheath Blight

by
Jian-Wei Jiang
1,†,
Yue Qiu
1,†,
Jing-Xue Luo
1,
Jia-Le Liu
1,
Hua-Jian Feng
1,
Yi Zhou
1,* and
Sheng Cheng
1,2,*
1
College of Agriculture, Yangtze University, Jingzhou 434025, China
2
School of Pharmacy, Xinyang Agriculture and Forestry University, Xinyang 464000, China
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Plants 2025, 14(22), 3546; https://doi.org/10.3390/plants14223546
Submission received: 21 October 2025 / Revised: 16 November 2025 / Accepted: 18 November 2025 / Published: 20 November 2025
(This article belongs to the Special Issue Biological Control of Plant Diseases Caused by Pathogenic Microorganisms—2nd Edition)
Browse Figures
Versions Notes

Abstract

Rice sheath blight caused by Rhizoctonia solani is a devastating global rice disease. This study aimed to isolate biocontrol bacteria from the medicinal plant Bletilla striata for managing the disease. Strain A1 demonstrated the strongest antagonistic activity, with a 91.92% inhibition rate against R. solani in vitro. It also exhibited a broad antifungal spectrum against ten plant pathogenic fungi. Morphological and molecular (16S rRNA and recA genes) analysis identified strain A1 as Pseudomonas koreensis. In detached leaf assays, lesion length was significantly reduced. Pot and field trials showed control efficacies of 65.54% and 72.53%, respectively, comparable to the chemical agent Jinggangmycin. Strain A1 secreted extracellular enzymes (protease, β-1,3-glucanase), siderophores, and auxin (IAA), and possessed phosphate-solubilizing and nitrogen-fixing capabilities. The strain significantly enhanced the activities of key defense enzymes (POD, PAL, PPO, CAT, SOD) in rice. Furthermore, both its sterile culture filtrate and the corresponding crude ethyl acetate extract exhibited strong, direct suppression of R. solani growth. LC-MS analysis identified potential antifungal compounds, including Pseudomonic Acid, Artemisinin, and Tetradecane, in the extract. In conclusion, P. koreensis A1 is a promising biocontrol and plant growth-promoting candidate for sustainable management of rice sheath blight.

1. Introduction

As a primary staple food for more than half of the global population, rice (Oryza sativa L.) is a critically important cereal crop worldwide [1]. Rice sheath blight, caused by Rhizoctonia solani, is a devastating disease capable of infecting rice plants throughout their growth cycle, leading to yield losses exceeding 50% [2,3]. The severity of this disease necessitates effective control strategies to mitigate crop loss and prevent epidemics [4]. Currently, chemical control using Jinggangmycin is the primary method. However, prolonged use of chemical fungicides has led to reduced efficacy, pathogen resistance, and environmental pollution. Consequently, there is an urgent need for environmentally friendly alternatives to control R. solani. Biological control, being eco-friendly and less prone to inducing resistance, is increasingly recognized as the best future alternative to chemical pesticides.
To date, biological control of rice sheath blight has primarily focused on bacteria, especially endophytic bacteria [5]. For instance, Bacillus sp. [6], Paenibacillus sp. [2] and Burkholderia sp. [7] have been studied and proven effective for field control. Nevertheless, current biocontrol agents have not consistently performed well under field conditions. The discovery of novel, stable, and effective biocontrol strains therefore remains a critical priority.
Pseudomonas species are ubiquitous in various environments, including soil, water, plant rhizospheres, sediments, and even extreme habitats. They have simple nutritional requirements and play significant roles in plant health as rhizosphere or endophytic bacteria [8]. Pseudomonas protects plants from pathogens by producing diverse bioactive metabolites, such as siderophores, antibiotics, hydrolases, volatiles, and phytoanticipins, and through competition [9]. Additionally, they can enhance disease resistance by promoting plant growth and inducing defense-related enzymes [9,10]. For example, volatile organic compounds (VOCs) emitted by P. fluorescens ZX effectively suppress tomato gray mold [11]. The antimicrobial activity of Pseudomonas sp. B17-12 against tomato gray mold and fusarium wilt is likely due to volatile compounds like tetradecane [12]. Pseudomonas fluorescens strain VSMKU3054 significantly increases defense enzyme levels such as peroxidase (POX), phenylalanine ammonia-lyase (PAL), lipoxygenase (LOX), and total phenolic content in tomato plants, while moderately enhancing polyphenol oxidase (PPO) activity [9].
Endophytic bacteria are important sources of biocontrol agents. They function through various mechanisms, including antagonistic metabolite production, niche competition, plant growth promotion, and induced systemic resistance [13]. Studies indicate that many endophytic bacteria from medicinal plants possess antagonistic effects against plant pathogens [14]. For instance, Mohamad et al. [15] isolated over 100 endophytic bacteria from Glycyrrhiza glabra, several of which showed effective antifungal activity against eight different plant pathogenic fungi. Bacillus amylolyticus G146, isolated from Fraxinus spp, exhibited strong control against cotton verticillium wilt [16]. This suggests that medicinal plant endophytes provide valuable resources for developing novel biocontrol agents.
Bletilla striata, an Orchidaceae family medicinal plant primarily distributed in East Asia, exhibits pharmacological activities including wound healing, antimicrobial, anticancer, antioxidant, and antiviral properties [17,18]. The low disease prevalence in B. striata suggests it may host a unique endophytic microbiome with great biocontrol potential. Therefore, this study aimed to: isolate and identify endophytic bacteria from B. striata with high inhibitory effect against R. solani; evaluate the biocontrol effectiveness of P. koreensis A1 against rice sheath blight; and elucidate its potential mechanisms of action, including the production of antifungal compounds and induction of host defense responses.

2. Results

2.1. Isolation, Screening and Identification of Biocontrol strain A1

Sixty-two endophytic bacterial strains were isolated from Bletilla striata roots. Eleven showed antagonistic activity against Rhizoctonia solani in dual-culture assays, with inhibition rates ranging from 42.42% to 92.28% (Figure 1A,B). Strain A1, with the highest inhibition rate (92.28%, Figure 1B), was selected for further study. Phylogenetic analysis based on 16S rDNA and recA gene sequences showed that strain A1 grouped within the P. koreensis cluster (Figure 2). Sequences were deposited in GenBank under accession numbers PV195001 (16S rDNA) and PV282236 (recA).

2.2. Antifungal Spectrum of Pseudomonas koreensis A1

Strain A1 displayed a broad-spectrum antifungal activity against all ten plant pathogenic fungi tested. The inhibition rates varied, with the highest observed against Verticillium dahlia (98.45%) and the lowest against Bipolaris sorokiniana (61.28%). The average inhibition rate for the other eight pathogens was 82.35%, demonstrating its potential as a broad-spectrum biocontrol agent (Figure 3).

2.3. Biocontrol Efficacy of P. koreensis A1 Against Rice Sheath Blight

In the detached leaf assay, A1 fermentation broth significantly alleviated disease symptoms, reducing lesion area to 11.94%, comparable to that for the Jinggangmycin treatment (14.16%) and significantly lower than that for the LB control (62.06%) (Figure 4A,C). Strain A1 efficacy was further validated in greenhouse and field trials. In the greenhouse, A1 fermentation broth provided 82.90% control efficacy. Notably, under field conditions, A1 control efficacy reached 72.53%, statistically comparable to Jinggangmycin (69.63%) and significantly higher than the control (Table 1). These results confirm the strong potential of strain A1 for practical application.

2.4. Strain A1 Promotes Rice Seedling Growth

Application of A1 fermentation broth significantly enhanced rice seedling growth under greenhouse conditions. Compared to the LB-treated control, A1-treated seedlings showed increases of 5.10% in stem length, 10.61% in root length, 38.55% in fresh weight, and 22.91% in dry weight (Figure 5A–E).

2.5. Antifungal Activity of Culture Filtrate and Crude Extract

The sterile culture filtrate (SCF) of strain A1 exhibited dose-dependent inhibition against R. solani mycelial growth, with inhibitory activity progressively increasing at higher SCF concentrations (Figure 6A,F). Notably, 100% growth inhibition was achieved at 40% (v/v) SCF concentration (Figure 6A,F). Microscopic observation revealed that SCF treatment caused severe hyphal morphological abnormalities, including shrinkage, swelling, and distortion (Figure 6B–E). Treatment with protease K did not significantly reduce SCF antifungal activity (Figure 6G), suggesting the active components are not primarily proteinaceous. The ethyl acetate crude extract of A1 fermentation broth also exhibited potent dose-dependent antifungal activity against R. solani, achieving an inhibition rate of 96% at 600 μg/mL (Figure 7).

2.6. LC-MS Analysis of the Antifungal Crude Extract

LC-MS analysis of the crude extract identified several compounds with known antimicrobial properties, including Artemisinin, Pseudomonic Acid, Tetradecane, 2-Undecanone, and 2-Nonanone (Table 2 and Supplementary Figure S1). These compounds likely contribute to the direct antagonistic activity of strain A1.

2.7. Plant Growth-Promoting and Biochemical Traits of P. koreensis A1

Strain A1 exhibited multiple plant growth-promoting traits. It produced siderophores, auxin (IAA) (Figure 8E), and ammonia (Figure 8J), and showed abilities to solubilize inorganic phosphate, release potassium, and fix nitrogen (Figure 8A,B,D). It also secreted several extracellular enzymes, including protease and β-1,3-glucanase, but not cellulase or amylase (Figure 8F–I). Catalase activity was confirmed (Figure 8C).

2.8. Strain A1 Primes Defense-Related Enzymes in Rice

Application of A1 fermentation broth significantly induced systemic resistance in rice plants. At 72 h post-treatment, activities of key defense-related enzymes—polyphenol oxidase (PPO), catalase (CAT), phenylalanine ammonia-lyase (PAL), superoxide dismutase (SOD), and peroxidase (POD)—were significantly enhanced by 375%, 200%, 52.6%, 50%, and 275%, respectively, compared to the control (Figure 9).

3. Discussion

The search for potent biocontrol agents from unique ecological niches is a promising strategy for sustainable agriculture. Medicinal plants often possess antiparasitic, antibiotic, antioxidant, and anticancer activities [19]. Endophytes within these plants can synthesize bioactive secondary metabolites similar to their hosts, making medicinal plants valuable reservoirs for biological control agents [14,20,21,22]. This study isolated an endophytic bacterium, Pseudomonas koreensis A1, from the medicinal plant Bletilla striata, demonstrating remarkable potential for controlling rice sheath blight and promoting plant growth. Previous studies reported P. koreensis as a biocontrol agent against diseases caused by Bipolaris sorokiniana, Fusarium solani and Alternaria alternata [23,24,25]. To our knowledge, this is the first report of P. koreensis isolated from B. striata exhibiting inhibitory effects against rice sheath blight, expanding the host range and biocontrol potential of this species.
Pseudomonas koreensis A1 inhibited eight pathogenic fungi with inhibition rates exceeding 70%, indicating broad-spectrum potential. Furthermore, it showed high in vitro inhibition of R. solani and provided significant control in detached leaf, greenhouse, and field experiments, with efficacy comparable to Jinggangmycin. Thus, P. koreensis A1 demonstrates significant potential for development as a biopesticide. The slightly reduced field efficacy compared to the greenhouse is common, attributable to complex environmental factors like microbial competition, fluctuating weather, and soil variability [26]. Nevertheless, field efficacy exceeding 72% underscores its practical utility.
The biocontrol mechanism of P. koreensis A1 appears multifaceted. Our LC-MS analysis identified several antifungal compounds in the crude extract, including Pseudomonic Acid, Artemisinin, and volatile organic compounds such as Tetradecane and 2-Nonanone. Pseudomonic Acid (also known as Mupirocin), a clinically important antibiotic, exhibits broad-spectrum bactericidal activity against both Gram-positive (e.g., Staphylococcus aureus, including methicillin-resistant strains) and Gram-negative bacteria (e.g., Escherichia coli) [27]. Its primary mechanism involves the reversible inhibition of isoleucyl-tRNA synthetase, which disrupts bacterial protein synthesis [28]. While its role in antifungal activity is less defined, it is plausible that the compound targets a similar pathway in fungi or exerts its effect through alternative mechanisms, such as disrupting fungal membrane integrity. Artemisinin exhibits antifungal efficacy against a range of fungi, including Aspergillus niger, Fusarium spp., Trichoderma spp. and A. flavus [29,30]. A key mechanism of this growth inhibition is the induction of oxidative stress via reactive oxygen species generation [31]. Meanwhile, volatile compounds like Tetradecane and 2-Nonanone are frequently associated with membrane permeability alterations and inhibition of hyphal growth and sporulation in fungi. Tetradecane synergistically enhances antifungal activity against Candida albicans [32,33]. 2-Undecanone and 2-Nonanone combat fungi by inhibiting biofilm formation, blocking yeast-hypha transition, and interacting with Hwp1 protein [34,35]. The diverse arsenal of bioactive molecules produced by A1 enables a concerted action against R. solani, simultaneously compromising its cellular integrity, metabolic processes, and redox homeostasis. This multi-targeted attack results in a synergistic reduction in fungal pathogenicity and invasiveness, underpinning the strain’s broad-spectrum antifungal activity.
Biocontrol bacteria can produce extracellular enzymes to suppress pathogen growth [36,37,38]. In addition to direct antifungal effects, P. koreensis A1possesses an array of plant growth-promoting traits, including secretion of extracellular enzymes, siderophores, IAA, and ammonia, and abilities to solubilize phosphate and potassium and fix nitrogen. These traits directly translated into enhanced rice seedling growth, establishing P. koreensis A1 as a plant growth-promoting rhizobacterium (PGPR). Healthier plants are inherently more disease-resistant.
Moreover, P. koreensis A1 acts as an elicitor of plant innate immunity. We demonstrated significant and simultaneous upregulation of five key defense enzymes (POD, PAL, PPO, CAT, SOD) in A1-treated rice plants. By scavenging intracellular reactive oxygen species (ROS), these enzymes bolster plant immunity, thereby activating defense signaling pathways dependent on salicylic acid (SA), jasmonic acid (JA), and ethylene (ET) [39,40]. This “priming” of defense responses prepares the plant for a more robust and rapid defense upon pathogen challenge [41,42]. The coordinated increase in these enzymes, involved in lignin synthesis, phenolic compound production, and ROS scavenging, creates a hostile environment for invading pathogens like R. solani [43,44]. In conclusion, the synergistic combination of multiple antagonistic and plant growth-promoting mechanisms makes P. koreensis A1 a robust and promising multi-functional bio-agent.
It is important to acknowledge a key limitation of this study. While our in vitro and detached leaf assays provide compelling evidence for the direct antifungal activity of A1 and its metabolites, the experimental design does not allow us to definitively dissect the relative contributions of direct inhibition versus induced host immunity in the biocontrol efficacy observed in plant. In planta, it is highly plausible that both mechanisms operate synergistically. The direct antagonism reduces the pathogen load, while the simultaneously induced systemic resistance (as evidenced by the primed defense enzymes) enhances the plant’s innate ability to confine the infection. Disentangling this complex interplay, for example, through gene knockout mutants of specific bacterial traits or the use of plant signaling mutants, represents a fascinating and necessary direction for future research to fully unravel the mechanistic basis of this promising biocontrol agent.

4. Materials and Methods

4.1. Isolation of Endophytic Bacteria from Bletilla striata Roots

Healthy B. striata specimens were collected from the Yangtze University Industrial Park, Jingzhou City, Hubei province. Roots were thoroughly washed with running water and externally sterilized by sequential submersion in 75% ethanol (2 min), 4% sodium hypochlorite (3 min), and 75% ethanol (1 min), followed by five rinses with aseptic distilled water. The effectiveness of surface disinfection was verified by plating the last rinse water on Luria–Bertani agar (LA). Sterilized root tissues were aseptically ground with sterile quartz sand and serially diluted. Aliquots (100 μL) of dilutions (104 to 106) were spread on LA plates and incubated at 28 °C for 3 days. Morphologically distinct colonies were purified and subsequently preserved at −80 °C in 15% glycerol for long-term storage.

4.2. Evaluation of Antifungal Activity Against Rhizoctonia solani

Antagonistic activity was evaluated using a dual-culture assay. A 6-mm-diameter mycelial plug from a 3-day-old R. solani culture was placed at the center of a fresh potato dextrose agar (PDA) plate. Eleven bacterial isolates from purified plates were streaked as two parallel lines at a distance of 20 mm from the fungus. Plates inoculated with R. solani alone served as negative control group. All plates were incubated at 28 °C for 2 days, after which radial fungal growth was measured. The inhibition rate was calculated as: [(Rc − Rt)/Rc] × 100, where Rc is radial growth in the control, and Rt is radial growth in the treatment.

4.3. Molecular Identification of Strain A1

Under culture conditions of LB medium, 28 °C, and shaking at 130 rpm, strain A1 was grown overnight. Genomic DNA was extracted using a bacterial genome extraction kit (Tsingke Biotechnology Co., Ltd., Wuhan, China). The 16S rRNA and recA genes were amplified with primer pairs 27F/1492R and recA-F/recA-R [45]. Purified PCR products were sequenced by Tsingke Biotechnology Co., Ltd. (Wuhan, China). Obtained sequences were analyzed using the NCBI BLAST web server (https://blast.ncbi.nlm.nih.gov/Blast.cgi, accessed on 20 October 2025) against the GenBank database. The maximum likelihood (ML) and neighbor joining (NJ) method in MEGA 7.0 was used to infer phylogenetic trees, assessing node confidence with 1000 bootstrap replications [44].

4.4. Assessment of Antifungal Spectrum

Strain A1, exhibiting the strongest antibacterial activity against R. solani, was selected for the antifungal spectrum test against ten plant pathogenic fungi: Bipolaris sorokiniana, Phytophthora infestans, Fusarium oxysporum f. sp. vasinfectum, F. fujikuroi, Alternaria solani, F. graminearum, Sclerotinia sclerotiorum, Verticillium dahlia, A. solani and P. nicotianae as preserved in the Fungi Herbarium of Yangtze University (Jingzhou, China). The dual-culture assay was performed as described in Section 4.2, and inhibition rates were calculated.

4.5. Antifungal Activity of Sterile Culture Filtrate

Strain A1 was grown in LB medium for 7 days at 28 °C with shaking at 200 rpm. The culture was centrifuged and filter-sterilized (0.22 μm pore size) to obtain the sterile culture filtrate (SCF). The SCF was incorporated into PDA at concentrations of 10%, 20%, 30%, and 40% (v/v) [46]. To test sensitivity to proteolysis, the SCF was treated with protease K (100 μg/mL) at 28 °C for 1 h before being added to PDA at 10% (v/v). A 6-mm mycelial plug of R. solani was positioned centrally on each plate, and mycelial growth was measured after incubation at 28 °C. The inhibition rate was assessed following the protocol in Section 4.2.

4.6. Detached Leaf Assay

Healthy, five-week-old rice leaves were cut into 5 cm segments and surface-disinfected with 2.5% (v/v) sodium hypochlorite for 30 s, subsequently rinsed three times with sterile water. Leaf segments were divided into three treatment groups and immersed for 1 h in 100 mL of either: A1 fermentation broth (OD600 = 1.0), Jinggangmycin (0.5 mg/mL), or LB medium. Each leaf segment was then inoculated at the center with a 6-mm mycelial plug obtained from a 2-day-old colony R. solani culture. Treated leaves were maintained at 28 °C with high humidity for 5 days, after which lesion length was measured.

4.7. Greenhouse Assessment of Biocontrol Efficacy

Seeds of rice cultivar ‘Xudao No. 9’ were surface-disinfected and germinated on sterile moist filter paper at 28 °C. Uniformly germinated seeds were transplanted into plastic pots containing sterilized soil and grown in a greenhouse at 28 °C, 90% relative humidity, and with a photoperiod of 16 h light and 8 h darkness. Upon reaching the 4–5 leaf stage, plants were grouped into three treatments: (1) R. solani + A1 fermentation broth (1.0 × 108 CFU/mL), (2) R. solani + Jinggangmycin (0.5 mg/mL), and (3) R. solani + LB medium. Treatments were applied as foliar sprays twice at a one-week interval. Twenty-four hours after the first spray, R. solani sclerotia were attached to leaf sheaths near the plant base using sterile insect needles. Plants were enclosed transparent plastic bags for 48 h to sustain elevated humidity levels. Disease severity was assessed 14 days post-inoculation using a 0–9 scale standard (IRRI, 1980). The disease index and relative control efficacy were calculated as follows:
Disease index = [∑(Number of diseased plants in a grade × Disease grade)/(Total number of plants × Highest disease grade)] × 100.
Control efficacy (%) = [(Disease index of control − Disease index of treated group)/Disease index of control] × 100.

4.8. Field Trial for Control of Rice Sheath Blight

A field trial was conducted in Jingzhou, Hubei Province, in a site with a history of severe rice sheath blight. Rice cultivation followed local conventional practices. The area experiences frequent and severe natural sheath blight occurrence, with recent incidence rates reaching 70% and disease indices between 30 and 40. The experimental field was divided into 12 plots (30 m2 each) using a randomized block design, with 45 cm spacing between plots. Two treatments were applied: A1 fermentation broth (1 × 108 CFU/mL) and LB medium. At the early onset of sheath blight, treatments were applied via foliar spray. Disease severity was assessed 10 days post-treatment by randomly selecting 20 plants per plot. Disease evaluation and control efficacy calculations followed Section 4.7.

4.9. Antifungal Activity of Crude Extract

Antifungal active compounds were extracted following Mandal et al. [47]. A1 was inoculated into 100 mL LB broth and cultivated at 28 °C with shaking at 150 rpm for 24 h. Then, 10 mL of the fermented broth was transferred into 1 L LB medium and cultivated under the same conditions for one week. To obtain the supernatant, the fermented broth was subjected to centrifugation at 10,000 rpm for 20 min. The supernatant was combined with an equal volume of ethyl acetate, vigorously shaken in a separatory funnel, and then allowed to stand for phase separation. Following three rounds of extraction, the combined organic phases were concentrated using a rotary evaporator at 35 °C. The dried extract was weighed and dissolved in mass spectrometry-grade methanol to 20 mg/mL. The preparation was filtered through a 0.22 µm filter for subsequent experiments. The crude extract was incorporated into PDA medium to achieve final concentrations of 50, 100, 200, and 500 µg/mL. A mycelial plug of R. solani was aseptically transferred to the center of each PDA plate, with three replicates per concentration, and after incubation at 28 °C for two days, the mycelial diameter was measured for the subsequent determination of the inhibition rate.

4.10. LC-MS Analysis of Antifungal Metabolites

Components of the lipopeptide active substances were analyzed using high-resolution liquid chromatography-mass spectrometry (LC-MS). The crude extract from Section 4.9 was diluted to 5 mg/mL with methanol, sterilized by filtration through a 0.22-μm membrane into dedicated sample vials. Chromatography employed a Kinetex® F5 column (100 mm × 2.1 mm, 2.6 μm), with ultrapure water as mobile phase A and LC-MS grade acetonitrile as mobile phase B. Gradient elution was: 0–2 min, 5% B; 2–8 min, 5–60% B; 8–20 min, 60–95% B; 20–25 min, 95% B; 25–25.01 min, 95–5% B; and 25.01–27 min, 5% B. The injection volume was 10 μL, flow rate 300 μL/min, and detection wavelength 254 nm. Mass spectrometry used an ESI ion source in positive and negative ion modes. Ion spray voltage was 5000 V, ion source temperature 500 °C, column temperature 40 °C, and mass scanning range (m/z) 100 to 1250. Initial LC-MS data were interpreted using SCIEX OS 1.7.0 software. By comparing data with existing database information, the main components of the lipopeptide active substances were identified.

4.11. Characterization of Plant Growth-Promoting Properties

4.11.1. Biosynthesis of Siderophores

Siderophore production was determined using the Chrome Azurol S (CAS) agar plate assay [48]. A 6 mm sterile filter disc was positioned at the center of the CAS plate, and 10 μL of strain A1 inoculum was inoculated onto it. Formation of orange halos indicated siderophore production.

4.11.2. Phosphorus Solubilization

Phosphate solubilization ability was assessed using NBRIP agar medium [49]. Aseptic technique was used to place a 6-mm filter paper disc in the center of the NBRIP plate, inoculated with 10 μL of strain A1 suspension, and incubated in the dark at 28 °C for 7 days. The appearance of a clear zone demonstrated phosphate solubilization.

4.11.3. Nitrogen Fixation

Nitrogen-fixing capacity was evaluated by streaking a fresh A1 colony from an LA plate onto a nitrogen-free medium [50] and incubating at 25 °C for 7 days. Colony growth was observed post-incubation.

4.11.4. Extracellular Enzyme Activity

Activities of extracellular enzymes (protease, chitinase, cellulase) were determined using skim milk agar medium [51], carboxymethyl cellulose agar (CMC) [52], and colloidal chitin solid medium [53], respectively. The A1 bacterial suspension was inoculated onto these media. After incubation at 28 °C for 7 days, clear zones around colonies indicated positive enzymatic reactions.

4.11.5. Indole-3-Acetic Acid (IAA) Production

Auxin production was assessed using the Salkowski method [50]. Strain A1 was cultured in LB medium with 10 g/L tryptophan at 28 °C and 200 rpm for 2 days. After centrifugation (10,000 rpm, 10 min), 2 mL supernatant was mixed with 2 mL Salkowski reagent (0.5 M FeCl3 in 35% HClO4). The mixture was maintained in darkness for 30 min. A color change to pink indicated IAA production.

4.12. Measurement of Defense-Related Enzyme Activities

Rice plants at the tillering stage received four treatments: A1 fermentation broth alone; LB medium. Leaf samples were collected 72 h after the final treatment. Activities of POD, PAL, PPO, CAT, and SOD were measured using commercial assay kits (Solarbio, Beijing, China) according to the manufacturer’s instructions.

4.13. Statistical Analysis

Statistical analyses were performed in SPSS 23.0 (IBM, Armonk, NY, USA). We conducted one-way ANOVA to evaluate overall variability across treatments. Where ANOVA indicated significance (p < 0.05), Duncan’s test was employed for pairwise comparisons, as it offers high detection power for experiments with multiple groups. Data are presented as mean ± standard error.

5. Conclusions

This study isolated an endophytic bacterial strain A1 with high antagonistic activity against Rhizoctonia solani from Bletilla striat, identified as Pseudomonas koreensis. The strain exhibited broad-spectrum antifungal activity, significant control efficacy against rice sheath blight in pot and field trials, and pronounced growth-promoting effects on rice. Its biocontrol mechanisms involve production of antifungal metabolites, multiple plant growth-promoting traits, and induction of host defense responses. Therefore, P. koreensis A1 represents a highly promising multi-functional biocontrol agent for the sustainable management of rice sheath blight.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/plants14223546/s1, Figure S1: LC-MS profile of antimicrobial compounds from P. koreensis A1. The analysis identified several antifungal metabolites in the crude extract, including Pseudomonic Acid (A), Artemisinin (B), 2-Nonanone (C), 2-Undecanone (D), and Tetradecane (E).

Author Contributions

Conceptualization, J.-W.J., S.C. and Y.Z.; Methodology, J.-W.J., Y.Q., J.-X.L., J.-L.L., H.-J.F. and S.C.; Visualization, J.-W.J., Y.Q. and J.-L.L.; Writing, original draft, J.-W.J., Y.Q. and S.C.; Writing, review and editing, J.-W.J., Y.Q., J.-X.L., J.-L.L., H.-J.F., S.C. and Y.Z.; Investigation, Y.Q., J.-X.L., J.-L.L. and H.-J.F.; Funding acquisition, Y.Z.; Data curation, J.-W.J., S.C. and Y.Z. All authors have read and agreed to the published version of the manuscript.

Funding

The authors acknowledge financial support from the Key Agricultural Technology Research Project of Hubei Province (Grant No. HBNYHXGG2024-03) for the research and publication of this article.

Data Availability Statement

Data are contained within the article and Supplementary Materials.

Conflicts of Interest

The authors declare that there are no potential conflicts of interest that could have influenced the work reported in this paper.

References

  1. Gross, B.L.; Zhao, Z. Archaeological and genetic insights into the origins of domesticated rice. Proc. Natl. Acad. Sci. USA 2014, 111, 6190–6197. [Google Scholar] [CrossRef]
  2. Peng, D.; Li, S.; Wang, J.; Chen, C.; Zhou, M. Integrated biological and chemical control of rice sheath blight by Bacillus subtilis NJ-18 and jinggangmycin. Pest Manag. Sci. 2014, 70, 258–263. [Google Scholar] [CrossRef] [PubMed]
  3. Prasad, B.; Eizenga, G.C. Rice sheath blight disease resistance identified in Oryza spp. accessions. Plant Dis. 2008, 92, 1503–1509. [Google Scholar] [CrossRef] [PubMed]
  4. Slaton, N.A.; Cartwright, R.D.; Meng, J.; Gbur, E.E.; Norman, R.J. Sheath blight severity and rice yield as affected by nitrogen fertilizer rate, application method, and fungicide. Agron. J. 2003, 95, 1489–1496. [Google Scholar] [CrossRef]
  5. Tao, H.; Li, X.; Huo, H.; Cai, Y.; Cai, A. Bacillus velezensis Y6, a potential and efficient biocontrol agent in control of rice sheath blight caused by Rhizoctonia solani. Microorganisms 2024, 12, 1694. [Google Scholar] [CrossRef]
  6. Shrestha, B.K.; Karki, H.S.; Groth, D.E.; Jungkhun, N.; Ham, J.H. Biological control activities of rice-associated Bacillus sp. strains against sheath blight and bacterial panicle blight of rice. PLoS ONE 2016, 11, e0146764. [Google Scholar] [CrossRef]
  7. Meng, X.-J.; Galileya Medison, R.; Cao, S.; Wang, L.-Q.; Cheng, S.; Tan, L.-T.; Sun, Z.-X.; Zhou, Y. Isolation, identification, and biocontrol mechanisms of endophytic Burkholderia vietnamiensis C12 from Ficus tikoua Bur against Rhizoctonia solani. Biol. Control 2023, 178, 105132. [Google Scholar] [CrossRef]
  8. Alattas, H.; Glick, B.R.; Murphy, D.V.; Scott, C. Harnessing Pseudomonas spp. for sustainable plant crop protection. Front. Microbiol. 2024, 15, 1485197. [Google Scholar] [CrossRef] [PubMed]
  9. Suresh, P.; Shanmugaiah, V.; Rajagopal, R.; Muthusamy, K.; Ramamoorthy, V. Pseudomonas fluorescens VSMKU3054 mediated induced systemic resistance in tomato against Ralstonia solanacearum. Physiol. Mol. Plant Pathol. 2022, 119, 101836. [Google Scholar] [CrossRef]
  10. Sun, D.; Zhuo, T.; Hu, X.; Fan, X.; Zou, H. Identification of a Pseudomonas putida as biocontrol agent for tomato bacterial wilt disease. Biol. Control 2017, 114, 45–50. [Google Scholar] [CrossRef]
  11. Yue, Y.; Wang, Z.; Zhong, T.; Guo, M.; Huang, L.; Yang, L.; Kan, J.; Zalán, Z.; Hegyi, F.; Takács, K.; et al. Antifungal mechanisms of volatile organic compounds produced by Pseudomonas fluorescens ZX as biological fumigants against Botrytis cinerea. Microbiol. Res. 2023, 267, 127253. [Google Scholar] [CrossRef]
  12. Wei, X.-H.; Nie, Q.-W.; Medison, R.G.; Zheng, T.-W.; Meng, X.-J.; Sun, Z.-X.; Zhou, Y. Evaluation of Pseudomonas koreensis B17-12 as a potential biological control agent against postharvest diseases of tomato. Physiol. Mol. Plant Pathol. 2024, 132, 102311. [Google Scholar] [CrossRef]
  13. Eljounaidi, K.; Lee, S.K.; Bae, H. Bacterial endophytes as potential biocontrol agents of vascular wilt diseases—Review and future prospects. Biol. Control 2016, 103, 62–68. [Google Scholar] [CrossRef]
  14. Stierle, A.; Strobel, G.; Stierle, D. Taxol and taxane production by taxomyces andreanae, an endophytic fungus of pacific yew. Science 1993, 260, 214–216. [Google Scholar] [CrossRef]
  15. Mohamad, O.A.A.; Li, L.; Ma, J.B.; Hatab, S.; Xu, L.; Guo, J.W.; Rasulov, B.A.; Liu, Y.H.; Hedlund, B.P.; Li, W.J. Evaluation of the antimicrobial activity of endophytic bacterial populations from chinese traditional medicinal plant licorice and characterization of the bioactive secondary metabolites produced by Bacillus atrophaeus against Verticillium dahliae. Front. Microbiol. 2018, 9, 924. [Google Scholar] [CrossRef]
  16. Liu, L.; Galileya Medison, R.; Zheng, T.-W.; Meng, X.-J.; Sun, Z.-X.; Zhou, Y. Biocontrol potential of Bacillus amyloliquefaciens YZU-SG146 from Fraxinus hupehensis against Verticillium wilt of cotton. Biol. Control 2023, 183, 105246. [Google Scholar] [CrossRef]
  17. He, X.; Wang, X.; Fang, J.; Zhao, Z.; Huang, L.; Guo, H.; Zheng, X. Bletilla striata: Medicinal uses, phytochemistry and pharmacological activities. J. Ethnopharmacol. 2017, 195, 20–38. [Google Scholar] [CrossRef] [PubMed]
  18. Xu, D.; Pan, Y.; Chen, J. Chemical constituents, pharmacologic properties, and clinical applications of Bletilla striata. Front. Pharmacol. 2019, 10, 1168. [Google Scholar] [CrossRef]
  19. Chota, A.; George, B.P.; Abrahamse, H. Potential treatment of breast and lung cancer using Dicoma anomala, an african medicinal plant. Molecules 2020, 25, 4435. [Google Scholar] [CrossRef]
  20. Alharthi, F.; Althagafi, H.A.; Jafri, I.; Oyouni, A.A.A.; Althaqafi, M.M.; Al-Hijab, L.Y.A.; Al-Hazmi, N.E.; Elagib, S.M.; Naguib, D.M. Phytochemical composition and bioactivities of some hydrophytes: Antioxidant, antiparasitic, antibacterial, and anticancer aroperties and mechanisms. Plants 2024, 13, 2148. [Google Scholar] [CrossRef]
  21. Pandey, S.S.; Jain, R.; Bhardwaj, P.; Thakur, A.; Kumari, M.; Bhushan, S.; Kumar, S. Plant probiotics—Endophytes pivotal to plant health. Microbiol. Res. 2022, 263, 127148. [Google Scholar] [CrossRef] [PubMed]
  22. Sharma, H.; Rai, A.K.; Dahiya, D.; Chettri, R.; Nigam, P.S. Exploring endophytes for in vitro synthesis of bioactive compounds similar to metabolites produced in vivo by host plants. AIMS Microbiol. 2021, 7, 175–199. [Google Scholar] [CrossRef]
  23. Rafikova, G.F.; Korshunova, T.Y.; Minnebaev, L.F.; Chetverikov, S.P.; Loginov, O.N. A new bacterial strain, Pseudomonas koreensis IB-4, as a promising agent for plant pathogen biological control. Microbiology 2016, 85, 333–341. [Google Scholar] [CrossRef]
  24. Yu, X.; Ai, C.; Xin, L.; Zhou, G. The siderophore-producing bacterium, Bacillus subtilis CAS15, has a biocontrol effect on Fusarium wilt and promotes the growth of pepper. Eur. J. Soil Biol. 2011, 47, 138–145. [Google Scholar] [CrossRef]
  25. Chen, L.; Shi, H.; Heng, J.; Wang, D.; Bian, K. Antimicrobial, plant growth-promoting and genomic properties of the peanut endophyte Bacillus velezensis LDO2. Microbiol. Res. 2019, 218, 41–48. [Google Scholar] [CrossRef]
  26. Cernava, T. How microbiome studies could further improve biological control. Biol. Control 2021, 160, 104669. [Google Scholar] [CrossRef]
  27. Dorotkiewicz-Jach, A.; Markwitz, P.; Drulis-Kawa, Z. The In vitro anti-Pseudomonal activity of Cu2+, strawberry furanone, gentamicin, and lytic phages alone and in combination: Pros and cons. Int. J. Mol. Sci. 2021, 22, 9830. [Google Scholar] [CrossRef] [PubMed]
  28. Zivkovic, I.; Gruic-Sovulj, I. Exploring mechanisms of mupirocin resistance and hyper-resistance. Biochem. Soc. Trans. 2024, 52, 1109–1120. [Google Scholar] [CrossRef] [PubMed]
  29. Librizzi, V.; Malacrinò, A.; Li Destri Nicosia, M.G.; Barger, N.; Luzzatto-Knaan, T.; Pangallo, S.; Agosteo, G.E.; Schena, L. Extracts from environmental strains of Pseudomonas spp. effectively control fungal plant diseases. Plants 2022, 11, 436. [Google Scholar] [CrossRef]
  30. Knudsmark, J.K.; Duke, S.O.; Cedergreeen, N. Potential ecological roles of artemisinin produced by Artemisia annua L. J. Chem. Ecol. 2014, 40, 100–117. [Google Scholar] [CrossRef]
  31. Zhu, P.; Yue, C.; Zeng, X.; Chen, X. Artemisinin Targets Transcription Factor PDR1 and Impairs Candida glabrata Mitochondrial Function. Antioxidants 2022, 11, 1855. [Google Scholar] [CrossRef]
  32. Carballeira, N.M.; O’Neill, R.; Parang, K. Racemic and optically active 2-methoxy-4-oxatetradecanoic acids: Novel synthetic fatty acids with selective antifungal properties. Chem. Phys. Lipids 2005, 136, 47–54. [Google Scholar] [CrossRef] [PubMed]
  33. Ayaz, Z.; Zainab, B.; Rashid, U.; Darwish, N.M.; Gatasheh, M.K.; Abbasi, A.M. In silico screening of synthetic and natural compounds to inhibit the binding capacity of heavy metal compounds against EGFR Protein of lung cancer. BioMed Res. Int. 2022, 2022, 2941962. [Google Scholar] [CrossRef] [PubMed]
  34. Azzollini, A.; Boggia, L.; Boccard, J.; Sgorbini, B.; Lecoultre, N.; Allard, P.M.; Rubiolo, P.; Rudaz, S.; Gindro, K.; Bicchi, C.; et al. Dynamics of metabolite induction in fungal co-cultures by metabolomics at both volatile and non-volatile levels. Front. Microbiol. 2018, 9, 72. [Google Scholar] [CrossRef] [PubMed]
  35. Rajasekharan, S.K.; Shemesh, M. The Bacillary postbiotics, including 2-undecanone, suppress the virulence of pathogenic microorganisms. Pharmaceutics 2022, 14, 962. [Google Scholar] [CrossRef]
  36. Meng, X.; Luo, Y.; Zhao, X.; Fu, Y.; Zou, L.; Cai, H.; Zhou, Y.; Tu, M. Isolation, identification, and biocontrol mechanisms of endophytic Burkholderia arboris DHR18 from rubber tree against red root rot disease. Microorganisms 2024, 12, 1793. [Google Scholar] [CrossRef]
  37. Chetan, K.K.; Varma, P.K.; Chandrasekhar, V.; Kumar, P.A.; Vasanthi, V. Plant, bacteria and fungi crosstalk: Direct and indirect biocontrol mechanisms of sugarcane rhizoplane Pseudomonas species against Fusarium wilt. Rhizosphere 2024, 31, 100952. [Google Scholar] [CrossRef]
  38. Macagnan, D.; Romeiro, R.d.S.; Pomella, A.W.V.; deSouza, J.T. Production of lytic enzymes and siderophores, and inhibition of germination of basidiospores of Moniliophthora (ex Crinipellis) perniciosa by phylloplane actinomycetes. Biol. Control 2008, 47, 309–314. [Google Scholar] [CrossRef]
  39. Shine, M.B.; Yang, J.W.; El-Habbak, M.; Nagyabhyru, P.; Fu, D.Q.; Navarre, D.; Ghabrial, S.; Kachroo, P.; Kachroo, A. Cooperative functioning between phenylalanine ammonia lyase and isochorismate synthase activities contributes to salicylic acid biosynthesis in soybean. New Phytol. 2016, 212, 627–636. [Google Scholar] [CrossRef]
  40. Richter, H.; Lieberei, R.; Strnad, M.; Novák, O.; Gruz, J.; Rensing, S.A.; von Schwartzenberg, K. Polyphenol oxidases in Physcomitrella: Functional PPO1 knockout modulates cytokinin-dependent development in the moss Physcomitrella patens. J. Exp. Bot. 2012, 63, 5121–5135. [Google Scholar] [CrossRef]
  41. Yang, Z.; Zhi, P.; Chang, C. Priming seeds for the future: Plant immune memory and application in crop protection. Front. Plant Sci. 2022, 13, 961840. [Google Scholar] [CrossRef]
  42. Conrath, U.; Beckers, G.J.; Langenbach, C.J.; Jaskiewicz, M.R. Priming for enhanced defense. Annu. Rev. Phytopathol. 2015, 53, 97–119. [Google Scholar] [CrossRef] [PubMed]
  43. Zhang, J.; Chen, L.; Fu, C.; Wang, L.; Liu, H.; Cheng, Y.; Li, S.; Deng, Q.; Wang, S.; Zhu, J.; et al. Comparative transcriptome analyses of gene expression changes triggered by Rhizoctonia solani AG1 IA infection in resistant and susceptible rice varieties. Front. Plant Sci. 2017, 8, 1422. [Google Scholar] [CrossRef] [PubMed]
  44. Yang, X.; Yan, S.; Li, Y.; Li, G.; Zhao, Y.; Sun, S.; Su, J.; Cui, Z.; Huo, J.; Sun, Y.; et al. Defense-related enzyme activities and metabolomic analysis reveal differentially accumulated metabolites and response pathways for sheath blight resistance in rice. Plants 2024, 13, 3554. [Google Scholar] [CrossRef] [PubMed]
  45. Spilker, T.; Baldwin, A.; Bumford, A.; Dowson, C.G.; Mahenthiralingam, E.; LiPuma, J.J. Expanded multilocus sequence typing for Burkholderia species. J. Clin. Microbiol. 2009, 47, 2607–2610. [Google Scholar] [CrossRef]
  46. Xu, Y.; Wang, L.; Liang, W.; Liu, M. Biocontrol potential of endophytic Bacillus velezensis strain QSE-21 against postharvest grey mould of fruit. Biol. Control 2021, 161, 104711. [Google Scholar] [CrossRef]
  47. Mandal, S.M.; Sharma, S.; Pinnaka, A.K.; Kumari, A.; Korpole, S. Isolation and characterization of diverse antimicrobial lipopeptides produced by Citrobacter and Enterobacter. BMC Microbiol. 2013, 13, 152. [Google Scholar] [CrossRef]
  48. Shin, S.H.; Lim, Y.; Lee, S.E.; Yang, N.W.; Rhee, J.H. CAS agar diffusion assay for the measurement of siderophores in biological fluids. J. Microbiol. Methods 2001, 44, 89–95. [Google Scholar] [CrossRef]
  49. Nautiyal, C.S. An efficient microbiological growth medium for screening phosphate solubilizing microorganisms. FEMS Microbiol. Lett. 1999, 170, 265–270. [Google Scholar] [CrossRef]
  50. Liaqat, F.; Eltem, R. Identification and characterization of endophytic bacteria isolated from in vitro cultures of peach and pear rootstocks. 3 Biotech 2016, 6, 120. [Google Scholar] [CrossRef]
  51. Yang, J.-H.; Zhang, W.-W.; Zhuang, Y.-Q.; Xiao, T. Biocontrol activities of bacteria from cowdung against the rice sheath blight pathogen. J. Plant Dis. Protect. 2017, 124, 131–141. [Google Scholar] [CrossRef]
  52. Zheng, T.-W.; Liu, L.; Nie, Q.-W.; Hsiang, T.; Sun, Z.-X.; Zhou, Y. Isolation, identification and biocontrol mechanisms of endophytic bacterium D61-A from Fraxinus hupehensis against Rhizoctonia solani. Biol. Control 2021, 158, 104621. [Google Scholar] [CrossRef]
  53. Guo, X.; Chen, D.-D.; Peng, K.-S.; Cui, Z.-W.; Zhang, X.-J.; Li, S.; Zhang, Y.-A. Identification and characterization of Bacillus subtilis from grass carp (Ctenopharynodon idellus) for use as probiotic additives in aquatic feed. Fish. Shellfish Immunol. 2016, 52, 74–84. [Google Scholar] [CrossRef] [PubMed]
Plants 14 03546 g001
Figure 1. (A) Dual-culture assay of eleven selected endophytic bacterial strains of Bletilla striata against Rhizoctonia solani. (B) Inhibition rates of the eleven selected endophytic bacterial strains against R. solani. Data are presented as mean ± standard error, with different letters above bars denoting significant differences (p < 0.05, Duncan’s test).
Figure 1. (A) Dual-culture assay of eleven selected endophytic bacterial strains of Bletilla striata against Rhizoctonia solani. (B) Inhibition rates of the eleven selected endophytic bacterial strains against R. solani. Data are presented as mean ± standard error, with different letters above bars denoting significant differences (p < 0.05, Duncan’s test).
Plants 14 03546 g001
Plants 14 03546 g002
Figure 2. Phylogenetic analysis of strain A1, based on 16S rDNA (A) and recA (B) gene sequences, was conducted using the maximum likelihood (ML) and neighbor joining (NJ) method in MEGA 7.0. Bootstrap values of ML/NJ from 1000 replicates are shown at nodes. Phaseolibacter flectens ATCC 12,775 (NR_024706) and Metapseudomonas lalkuanensis strain PE08 (CP043311) were chosen as the outgroup.
Figure 2. Phylogenetic analysis of strain A1, based on 16S rDNA (A) and recA (B) gene sequences, was conducted using the maximum likelihood (ML) and neighbor joining (NJ) method in MEGA 7.0. Bootstrap values of ML/NJ from 1000 replicates are shown at nodes. Phaseolibacter flectens ATCC 12,775 (NR_024706) and Metapseudomonas lalkuanensis strain PE08 (CP043311) were chosen as the outgroup.
Plants 14 03546 g002
Plants 14 03546 g003
Figure 3. Antifungal spectrum of strain A1 against ten plant pathogenic fungi. Inhibition zones of Bipolaris sorokiniana (A), Phytophthora infestans (B), Fusarium oxysporum f. sp. vasinfectum (C), F. fujikuroi (D), Alternaria solani (E), F. graminearum (F), Sclerotinia sclerotiorum (G), A. solani (H), P. nicotianae (I), and Verticillium dahlia (J) are visible in dual-culture plates. (K) Bar chart shows corresponding inhibition rates. Data are presented as mean ± standard error, with different letters above bars denoting significant differences (p < 0.05, Duncan’s test).
Figure 3. Antifungal spectrum of strain A1 against ten plant pathogenic fungi. Inhibition zones of Bipolaris sorokiniana (A), Phytophthora infestans (B), Fusarium oxysporum f. sp. vasinfectum (C), F. fujikuroi (D), Alternaria solani (E), F. graminearum (F), Sclerotinia sclerotiorum (G), A. solani (H), P. nicotianae (I), and Verticillium dahlia (J) are visible in dual-culture plates. (K) Bar chart shows corresponding inhibition rates. Data are presented as mean ± standard error, with different letters above bars denoting significant differences (p < 0.05, Duncan’s test).
Plants 14 03546 g003
Plants 14 03546 g004
Figure 4. Biocontrol efficacy of Pseudomonas koreensis A1 against rice sheath blight in detached leaf and greenhouse assays. (A) Detached leaf bioassay: Lesion length on rice leaves treated with A1 fermentation broth, Jinggangmycin or LB medium after inoculation with R. solani. (B) Greenhouse pot assay: Disease index of rice plants treated with A1 fermentation broth, Jinggangmycin, or LB medium, following challenge with R. solani. (C) The Disease index of the rice leaves. (D) The Lesion length relative of the rice plants. Data are presented as mean ± standard error, with different letters above bars denoting significant differences (p < 0.05, Duncan’s test).
Figure 4. Biocontrol efficacy of Pseudomonas koreensis A1 against rice sheath blight in detached leaf and greenhouse assays. (A) Detached leaf bioassay: Lesion length on rice leaves treated with A1 fermentation broth, Jinggangmycin or LB medium after inoculation with R. solani. (B) Greenhouse pot assay: Disease index of rice plants treated with A1 fermentation broth, Jinggangmycin, or LB medium, following challenge with R. solani. (C) The Disease index of the rice leaves. (D) The Lesion length relative of the rice plants. Data are presented as mean ± standard error, with different letters above bars denoting significant differences (p < 0.05, Duncan’s test).
Plants 14 03546 g004
Plants 14 03546 g005
Figure 5. Plant growth-promoting effects of P. koreensis A1 on rice seedlings. (A) Phenotype of seedlings after treatment with A1 fermentation broth or LB medium. Stem length (B), root length (C), fresh weight (D), and dry weight (E) of rice seedlings. Data are presented as mean ± standard error, with different letters above bars denoting significant differences (p < 0.05, Duncan’s test).
Figure 5. Plant growth-promoting effects of P. koreensis A1 on rice seedlings. (A) Phenotype of seedlings after treatment with A1 fermentation broth or LB medium. Stem length (B), root length (C), fresh weight (D), and dry weight (E) of rice seedlings. Data are presented as mean ± standard error, with different letters above bars denoting significant differences (p < 0.05, Duncan’s test).
Plants 14 03546 g005
Plants 14 03546 g006
Figure 6. Inhibitory effect of the sterile culture filtrate (SCF) from P. koreensis A1 on the mycelial growth and morphology of R. solani. (A) Dose-dependent inhibition of R. solani by different concentrations of SCF after incubation at 28 °C for 2 days. (B–D) Microscopic observations (40× magnification) of abnormal hyphal morphology induced by SCF treatment, including shrinkage (B), deformation (C), and swelling (D). (E) Normal hyphal growth in the LB medium control. (F) Bar chart quantifying the inhibition rate of SCF at different concentrations. Data are shown as mean ± standard error. Data are presented as mean ± standard error, with different letters above bars denoting significant differences (p < 0.05, Duncan’s test). (G) Bar chart comparing the inhibition rate of 10% SCF with or without protease K treatment, showing that the antifungal activity was largely unaffected by protease. Data are presented as mean ± standard error. The absence of significant differences (p ≥ 0.05) between columns was indicated by “ns” according to Duncan’s test.
Figure 6. Inhibitory effect of the sterile culture filtrate (SCF) from P. koreensis A1 on the mycelial growth and morphology of R. solani. (A) Dose-dependent inhibition of R. solani by different concentrations of SCF after incubation at 28 °C for 2 days. (B–D) Microscopic observations (40× magnification) of abnormal hyphal morphology induced by SCF treatment, including shrinkage (B), deformation (C), and swelling (D). (E) Normal hyphal growth in the LB medium control. (F) Bar chart quantifying the inhibition rate of SCF at different concentrations. Data are shown as mean ± standard error. Data are presented as mean ± standard error, with different letters above bars denoting significant differences (p < 0.05, Duncan’s test). (G) Bar chart comparing the inhibition rate of 10% SCF with or without protease K treatment, showing that the antifungal activity was largely unaffected by protease. Data are presented as mean ± standard error. The absence of significant differences (p ≥ 0.05) between columns was indicated by “ns” according to Duncan’s test.
Plants 14 03546 g006
Plants 14 03546 g007
Figure 7. Antifungal activity of P. koreensis A1 crude lipopeptide extract against R. solani. (A) Dose-dependent inhibition of R. solani by different concentrations of crude lipopeptide extracts from P. koreensis A1 after incubation at 28 °C for 2 days. (B) Inhibition rate calculated based on radial growth compared to the control. Data are presented as mean ± standard error, with different letters above bars denoting significant differences (p < 0.05, Duncan’s test).
Figure 7. Antifungal activity of P. koreensis A1 crude lipopeptide extract against R. solani. (A) Dose-dependent inhibition of R. solani by different concentrations of crude lipopeptide extracts from P. koreensis A1 after incubation at 28 °C for 2 days. (B) Inhibition rate calculated based on radial growth compared to the control. Data are presented as mean ± standard error, with different letters above bars denoting significant differences (p < 0.05, Duncan’s test).
Plants 14 03546 g007
Plants 14 03546 g008
Figure 8. Functional characterization of P. koreensis A1 for plant growth-promoting and biocontrol traits. Qualitative assays confirm the strain’s ability to: solubilize potassium (A) and phosphate (B); produce catalase (C), IAA (E), siderophores (G), protease (F), and ammonia (J); and grow on a nitrogen-free medium (D), indicating nitrogen fixation ability. In contrast, activities for cellulase (H) and amylase (I) were not detected.
Figure 8. Functional characterization of P. koreensis A1 for plant growth-promoting and biocontrol traits. Qualitative assays confirm the strain’s ability to: solubilize potassium (A) and phosphate (B); produce catalase (C), IAA (E), siderophores (G), protease (F), and ammonia (J); and grow on a nitrogen-free medium (D), indicating nitrogen fixation ability. In contrast, activities for cellulase (H) and amylase (I) were not detected.
Plants 14 03546 g008
Plants 14 03546 g009
Figure 9. Activities of defense-related enzymes in rice seedlings 72 h after treatment with strain A1. The activities of SOD (A), CAT (B), POD (C), PPO (D), and PAL (E) were significantly increased in the A1-treated group compared to control (LB medium). Data are presented as mean ± standard error, with different letters above bars denoting significant differences (p < 0.05, Duncan’s test).
Figure 9. Activities of defense-related enzymes in rice seedlings 72 h after treatment with strain A1. The activities of SOD (A), CAT (B), POD (C), PPO (D), and PAL (E) were significantly increased in the A1-treated group compared to control (LB medium). Data are presented as mean ± standard error, with different letters above bars denoting significant differences (p < 0.05, Duncan’s test).
Plants 14 03546 g009
Table 1. Biocontrol activity of strain A1 fermentation broth and Jinggangmycin against rice sheath blight in field trials.
Table 1. Biocontrol activity of strain A1 fermentation broth and Jinggangmycin against rice sheath blight in field trials.
TreatmentDisease IndexControl (%)
Control (LB)12.72 ± 0.95 a-
Jinggangmycin3.58 ± 1.13 b69.63%
A1 (108 cfu/mL)2.94 ± 0.52 b72.53%
Data are presented as mean ± standard error, with different letters above bars denoting significant differences (p < 0.05, Duncan’s test).
Table 2. Identification of antimicrobial compounds in the crude extract of P. koreensis A1 by LC-MS analysis.
Table 2. Identification of antimicrobial compounds in the crude extract of P. koreensis A1 by LC-MS analysis.
Material TypePeak TimeMass/Charge
Artemisinin6.035–6.071281.1394
Pseudomonic Acid7.278–7.314499.2913
Tetradecane7.314–7.349197.2275
2-Undecanone7.830–7.866169.1598
2-Nonanone8.046–8.082141.1285
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Jiang, J.-W.; Qiu, Y.; Luo, J.-X.; Liu, J.-L.; Feng, H.-J.; Zhou, Y.; Cheng, S. Endophytic Pseudomonas koreensis A1 of Bletilla striata as a Plant Growth Promoter and Biocontrol Agent Against Rice Sheath Blight. Plants 2025, 14, 3546. https://doi.org/10.3390/plants14223546

AMA Style

Jiang J-W, Qiu Y, Luo J-X, Liu J-L, Feng H-J, Zhou Y, Cheng S. Endophytic Pseudomonas koreensis A1 of Bletilla striata as a Plant Growth Promoter and Biocontrol Agent Against Rice Sheath Blight. Plants. 2025; 14(22):3546. https://doi.org/10.3390/plants14223546

Chicago/Turabian Style

Jiang, Jian-Wei, Yue Qiu, Jing-Xue Luo, Jia-Le Liu, Hua-Jian Feng, Yi Zhou, and Sheng Cheng. 2025. "Endophytic Pseudomonas koreensis A1 of Bletilla striata as a Plant Growth Promoter and Biocontrol Agent Against Rice Sheath Blight" Plants 14, no. 22: 3546. https://doi.org/10.3390/plants14223546

APA Style

Jiang, J.-W., Qiu, Y., Luo, J.-X., Liu, J.-L., Feng, H.-J., Zhou, Y., & Cheng, S. (2025). Endophytic Pseudomonas koreensis A1 of Bletilla striata as a Plant Growth Promoter and Biocontrol Agent Against Rice Sheath Blight. Plants, 14(22), 3546. https://doi.org/10.3390/plants14223546

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop