Baslaromyces pinophilus and application thereof
By screening and identifying *Pseudomonas pineophilus* HY25, the problem of unstable control effects of Fusarium biocontrol agents in natural environments was solved, achieving effective control of Fusarium root rot in tobacco and promoting tobacco growth, with significant antagonistic and growth-promoting effects.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-31
- Publication Date
- 2026-04-03
AI Technical Summary
Existing Fusarium biocontrol agents have unstable control effects in natural environments, making it difficult to provide biocontrol bacteria resources that have good antagonistic effects and are easy to reproduce.
The pine-loving bacterium HY25 was screened out, identified by morphological and molecular marker methods, and its control effect and growth-promoting characteristics against Fusarium root rot in tobacco were studied. Whole genome analysis was conducted, and it was applied to the green control of Fusarium root rot in tobacco.
Pine-loving bacterium HY25 has a 75% control effect on tobacco Fusarium root rot, promotes tobacco growth, increases plant height, fresh weight and leaf area, and has the ability to solubilize phosphorus, potassium and fix nitrogen.
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Figure CN121780338A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of biological control technology, and more specifically to a pine-loving bacterium and its applications. Background Technology
[0002] Fusarium is both saprophytic and parasitic, widely distributed in nature, and has a wide host range. Nearly 81% of important economic crops can be infected and harmed by Fusarium.
[0003] The biocontrol microorganisms of Fusarium are mainly divided into three categories: bacteria, actinomycetes, and fungi. The main biocontrol bacteria include rhizobia, pseudomonads, Bacillus, Bacillus-like bacteria, and myxococci; the main biocontrol actinomycetes include Streptomyces and Nocardia; and the main biocontrol fungi include Trichoderma, non-pathogenic Fusarium, arbuscular mycorrhizal fungi, Paecilomyces, and yeasts.
[0004] Biocontrol microorganisms can inhibit or kill Fusarium through direct mechanisms such as competition, hyperparasitism, lysis, and antagonism. They can also enhance the host's resistance to Fusarium by inducing host resistance or promoting growth.
[0005] Despite significant progress in the development of biocontrol agents for Fusarium, the complexity of the natural environment often leads to unstable control effects in the field, which slows down the promotion of biological control of Fusarium root rot.
[0006] Therefore, how to provide a biocontrol bacterial resource for controlling Fusarium with good antagonistic effect, strong stability and easy propagation is a technical problem that urgently needs to be solved by those skilled in the art. Summary of the Invention
[0007] In view of this, the present invention provides a pine-loving bacterium and its application.
[0008] This invention analyzes the biocontrol potential of *Pseudomonas pineophilus* against *Fusarium oxysporum* through the following pathways: (1) screening strains from the tobacco rhizosphere that effectively inhibit *Fusarium oxysporum* and accurately identifying the selected strains using morphological and molecular marker (ITS) methods; (2) evaluating its disease control effect against *Fusarium oxysporum* root rot and its growth-promoting properties in tobacco; (3) studying its mechanism of action in controlling *Fusarium oxysporum* root rot and promoting tobacco growth; and (4) conducting whole-genome analysis of the strain. The research results will provide a theoretical basis and excellent strain resources for the green control of *Fusarium oxysporum* root rot.
[0009] To solve the above-mentioned technical problems, the present invention adopts the following technical solution: A type of pine-loving bacterium, named HY25, is classified as follows: Talaromyces pinophilus It was deposited on December 15, 2025, at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 20252900, and the deposit address is Wuhan University, Wuhan, China.
[0010] The above-mentioned *Pseudomonas aeruginosa* is used to promote tobacco growth and prevent tobacco root rot.
[0011] Furthermore, the pathogen causing the tobacco root rot is Fusarium tumefaciens.
[0012] Furthermore, the *Fusarium* species are *Fusarium solani*, *Fusarium oxysporum*, *Fusarium graminearum*, *Fusarium spp.*, *Fusarium moniliforme*, *Fusarium rosenbergii*, *Fusarium thunbergii*, and *Fusarium moniliforme*.
[0013] Furthermore, the growth promotion is aimed at improving the agronomic traits of tobacco.
[0014] Furthermore, the agronomic traits include plant height, stem diameter, fresh and dry weight of aboveground and root parts, number of leaves, and maximum leaf area.
[0015] Furthermore, it is used for phosphorus solubilization, potassium solubilization, and nitrogen fixation.
[0016] As can be seen from the above technical solution, compared with the prior art, the present invention has the following beneficial effects: This invention screened and obtained a strain of *Pseudomonas pineophilus* HY25, which showed an inhibition rate of up to 70.99% in plate confrontation. Pot experiments showed that it had a control efficacy of 75% against Fusarium root rot in tobacco. Furthermore, *Pseudomonas pineophilus* HY25 originates from the tobacco rhizosphere and is more suitable for the tobacco rhizosphere environment. *Pseudomonas pineophilus* HY25 not only has good control effects against Fusarium root rot in tobacco, but also has the ability to solubilize phosphorus, potassium, and fix nitrogen. It significantly increases the plant height, fresh weight, and maximum leaf area of tobacco plants, making it a biocontrol fungus with good potential. Attached Figure Description
[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.
[0018] Figure 1 This is the result of the confrontation between strain HY25 and Fusarium oxysporum in Example 1 of the present invention.
[0019] Figure 2 The morphological characteristics of strain HY25 in Example 2 of this invention on PDA medium.
[0020] Figure 3 This is a phylogenetic tree of strain HY25 and related closely related species constructed based on the ITS sequence in Example 2 of the present invention.
[0021] Figure 4The culture medium in Example 4 of this invention was used to verify the phosphorus-solubilizing, nitrogen-fixing, and potassium-solubilizing abilities of *Pseudomonas sylvestris* HY25.
[0022] Figure 5 The images show the appearance of tobacco seedlings under different treatments in Example 5 of this invention. A represents tobacco seedlings inoculated with Fusarium oxysporum, B represents tobacco seedlings inoculated with Fusarium oxysporum and Bacillus hyssodon HY25, and C represents tobacco seedlings as a blank control. Detailed Implementation
[0023] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.
[0024] The tested Fusarium strain was Fusarium solanum (Tobacco Fusarium). Fusarium solani ACCC 36234, Fusarium oxysporum ( F. oxysporum ACCC 39331, Fusarium graminearum ( F. graminearum ACCC 39334, Shared Fusarium ( F. commune ACCC 39357, Fusarium rosenbergii ( F. incarnatum ACCC 31942, Fusarium oxysporum ( F. chlamydosporum ACCC 32577, Fusarium moniliformes ( F. proliferatum ACCC 39253, all purchased from the China Agricultural Microbial Culture Collection Center (accc.org.cn).
[0025] Test culture media and reagents: Potato Dextrose Agar (PDA) medium; Modified Ashbee nitrogen-fixing medium (KH2PO4 0.2g, NaCl 0.2g, MgSO4 0.2g, CaCO3 5g, K2SO4 0.1g, Glucose 10g, distilled water 1L, Agar 18g); Monkina Inorganic Phosphorus Medium and Monkina Organic Phosphorus Medium; Modified potassium-solubilizing medium (sucrose 10.0g, (NH4)2SO4 0.5g, Na2HPO4 2.0g, MgSO4·7H2O 0.2g, NaCl 0.2g, CaCO3 0.1g, potassium feldspar 5.0g, agar 15.0g, distilled water 1L, pH 7.0~7.5).
[0026] Excel 2019 was used for data statistics and organization, Prism was used for graphing, SPSS 26.0 was used for Least Significant Difference (LSD) analysis of variance at the 0.05 level, and MEGA 11 was used for neighbor-joining method to construct phylogenetic trees.
[0027] Example 1 Isolation and screening of biocontrol bacteria The tobacco root and stem samples were collected in July 2024 from Huayuan County, Xiangxi Tujia and Miao Autonomous Prefecture, Hunan Province. A total of 19 samples were collected from different plots using a five-point sampling method. The tobacco root and stem samples, along with the rhizosphere soil, were placed in sealed bags and stored at 4°C for later use.
[0028] (1) Isolation and purification of biocontrol bacteria Tobacco rhizomes were collected and their surfaces were cleaned with sterile water. Under aseptic conditions, the cleaned rhizomes were cut and surface-sterilized with 75% ethanol. The roots were then cut into 2-3 cm segments with a sterile blade, soaked in 75% ethanol for 30 seconds to 1 minute, rinsed three times with sterile water, and placed on PDA culture dishes in a laminar flow hood. The dishes were incubated upside down at 28°C. Observation was continued for 1-3 days. When colonies appeared on the solid culture medium, colonies of varying morphology, color, and size were picked and inoculated onto new solid culture medium for further cultivation. Single-morphological strains were isolated. After colonies grew on the new solid culture medium, the culture was purified twice more to isolate multiple single strains. A total of 39 strains were isolated and purified from 19 collected rhizosphere samples. The purified single strains were inoculated onto PDA solid slant culture medium using an inoculation needle and temporarily stored at 4°C.
[0029] (2) Confrontation culture test between biocontrol bacteria and Fusarium oxysporum To screen fungal strains with antagonistic activity against *Fusarium oxysporum*, seven *Fusarium oxysporum* species were used as target bacteria, and the plate confrontation method was employed for screening antagonistic bacteria. *Fusarium oxysporum* mycelia were inoculated into freshly prepared PDA medium 2.5 cm from the edge. Mycelia of the fungi to be screened were picked up with an inoculation needle and inoculated 2.5 cm from the *Fusarium oxysporum* mycelia. A control of inoculating only *Fusarium oxysporum* was used. The medium was incubated at 28°C for 7 days, with three replicates. The diameter of the *Fusarium oxysporum* mycelia was measured, and the average inhibitory effect was calculated.
[0030] Inhibition rate (%) = (Growth radius of control pathogen - Growth radius of treated pathogen) / (Growth radius of control pathogen - Radius of inoculated bacterial cake) × 100.
[0031] The results are shown in Table 1. Figure 1 As shown.
[0032] Table 1. Inhibition rate of strain HY25 against seven Fusarium species
[0033] The results of confrontation culture between the strain and seven species of Fusarium oxysporum showed that strain HY25, isolated from the rhizome samples of tobacco from Huayuan County, Xiangxi Tujia and Miao Autonomous Prefecture, Hunan Province, had a significant inhibitory effect on Fusarium oxysporum. Figure 1 The inhibition rates against the seven tested Fusarium species ranged from 34.91% to 70.99%, with the highest inhibition rate against Fusarium graminearum at 70.99% (Table 1).
[0034] Example 2 Identification of strains (1) Morphological identification The purified strain HY25 was inoculated onto PDA plates and incubated upside down at 28°C in the dark for 7 days. The morphology of the colonies was observed, including colony morphology on both sides of the culture medium surface. Results are as follows: Figure 2 As shown.
[0035] (2) Molecular identification Using extracted DNA as a template, the ITS sequence of strain HY25 was amplified using universal primers ITS1 (5'-TCCGTAGGTGAACCTGCGG-3', SEQ ID No. 1) / ITS4 (5'-TCCTCCGCTTATTGATATGC-3', SEQ ID No. 2). The PCR amplification products were detected by 1% agarose gel electrophoresis, and those that passed the tests were sent to Sangon Biotech (Shanghai) Co., Ltd. for gene sequencing.
[0036] The ITS sequence of HY25 is as follows:
[0037] The ITS sequence of the target strain obtained from sequencing was compared with the NCBI database using BLAST. The analysis results showed that strain HY25 was similar to *Pseudomonas pineophilus*. Talaromyces pinophilus The sequence similarity of FJ48 was 100.00%. Homologous sequences were imported into MEGA 11 software to construct a joint phylogenetic tree using the neighbor-to-neighbor method. Figure 3 It can be seen that strain HY25 is similar to *Pseudomonas pineophilus*. T. pinophilus They cluster together on the same branch.
[0038] Based on morphological and molecular identification, strain HY25 was identified as *Pinococcus pyogenes*.
[0039] Example 3 HY25, a fungus known as *Pseudomonas pineae*, is classified as follows: Talaromyces pinophilus It was deposited on December 15, 2025, at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 20252900, and the deposit address is Wuhan University, Wuhan, China.
[0040] Example 4 Determination of the growth-promoting ability of *Pseudomonas sylvestris* HY25 (1) Determination of phosphorus solubility Prepare solid culture media containing inorganic and organic phosphorus of *Montagna spp.*. Using a sterile punch, inoculate 2 mm diameter mycelial discs of *Montagna spp.* HY25 into the center of the medium and culture at 28°C for 7 days. The presence of a circular transparent ring around the fungal colony indicates phosphorus solubilization ability.
[0041] (2) Determination of nitrogen fixation and potassium solubilization capacity The *Pseudomonas syringae* HY25 strain was inoculated onto modified Assumption nitrogen-fixing medium and modified potassium-solubilizing medium, respectively, and cultured at 28°C for 7 days. The growth status and morphological characteristics of the strain were observed. If the experimental results showed that the fungus could still grow in the absence of exogenous nitrogen and potassium, it could be preliminarily identified as a nitrogen-fixing bacterium and a potassium-solubilizing bacterium.
[0042] *Pinococcus pyogenes* HY25 was inoculated onto modified Assumption nitrogen-fixing medium, Monkina inorganic phosphorus medium, Monkina organic phosphorus medium, and modified potassium-solubilizing medium, with uninoculated medium serving as a control. The results showed that ( Figure 4 *Pseudomonas aeruginosa* grows normally on Assumption nitrogen-fixing medium, with a distinct clear zone forming around each colony, indicating its nitrogen-fixing ability. It also grows normally on Monkina inorganic phosphorus medium, with a phosphate-solubilizing zone around each colony, indicating its ability to dissolve inorganic phosphorus. Furthermore, it grows normally on Monkina organic phosphorus medium, indicating its ability to decompose and utilize organic phosphorus. Finally, it grows normally on modified potassium-solubilizing medium, indicating its potassium-solubilizing ability.
[0043] Example 5 Experiment on growth promotion and disease prevention of *Pseudomonas syringae* HY25 in pots (1) Determination of tobacco growth trait indicators Eighteen healthy tobacco seedlings with similar growth at the 5-6 leaf stage were selected and transplanted into 12.5cm diameter round pots for 7 days to allow them to recover. Two treatments were included: a negative control group (CK) receiving 10mL of root irrigation water; and a treatment group (biocontrol bacteria) receiving a root irrigation concentration of 1×10⁻⁶. 7 10 mL of fermentation broth of *Bacillus pineophilus* HY25 (CFU / mL) was added. Plant height, stem diameter, fresh and dry weight of aboveground and root parts, number of leaves, maximum leaf area, and root-to-shoot ratio were measured 14 days after inoculation.
[0044] Leaf area (cm²) 2 = 0.6345 × leaf length (cm) × leaf width (cm).
[0045] Root-to-shoot ratio = Fresh weight of roots / Fresh weight of aboveground parts.
[0046] Table 2. Growth-promoting activity of *Bacillus hystericus* HY25
[0047] When tobacco seedlings (Yunyan 87) were inoculated with *Bacillus pineophilus* HY25, their plant height, stem diameter, number of leaves, maximum leaf area, aboveground fresh weight / dry weight, and underground dry weight were significantly higher than those of the control, while their root-to-shoot ratio was significantly lower, indicating that it has a good growth-promoting effect on tobacco seedlings.
[0048] (2) Biocontrol effect of *Fusarium oxysporum* HY25 against *Fusarium oxysporum* 2 mm diameter mycelial cakes of *Hylocereus undatus* HY25 were obtained using a sterile punch and inoculated onto PDA medium. After incubation at 28°C for 10 days, the spores were washed off with sterile water to obtain a spore suspension. The spore concentration was adjusted to 1 × 10⁻⁶ using a hemocytometer. 7 Tobacco plants with 5-6 leaves were transplanted and allowed to recover for 7 days before being potted. Three treatments were included: a positive control group (Fusarium oxysporum) treated with a concentration of 1×10⁻⁶ cells / mL. 7 Root drenching was performed using *Fusarium oxysporum* spore suspension at a concentration of 1 × 10⁶ / mL; negative control group (CK): root drenching was performed using water; treatment group (Fusarium + biocontrol bacteria): root drenching was performed with *Bacillus hyssodon* suspension HY25 for 7 days, followed by treatment with 1 × 10⁶ / mL solution of *Fusarium oxysporum* spore suspension. 7 The roots were treated with a Fusarium spore suspension of 1 / mL. After 14 days, a disease survey was conducted according to the "Classification and Survey Methods for Tobacco Diseases and Pests" (GB / T23222—2008), and the disease index and relative control efficacy of each treatment group were calculated.
[0049] The disease grading standards shall be implemented in accordance with the "Grading and Survey Methods for Tobacco Diseases and Pests, GB / T 23222-2008": Level 0: Asymptomatic; Grade 1: 1-25% of leaves wilted; Grade 3: 26-50% of leaves wilted; Level 5: 51-75% of leaves wilted; Level 7 death.
[0050] Disease index = ∑ (disease grade × number of plants) / (highest disease grade × total number of plants) × 100.
[0051] Relative efficacy (%) = (CK disease index - treatment disease index) / CK disease index × 100%.
[0052] Tobacco seedling growth under different treatments, as follows Figure 5 The root rot disease severity index was 13.89 for the treatment with *Fusarium oxysporum* HY25, while it was 55.56 for the treatment with only *Fusarium oxysporum* HY25. The control efficacy of *Fusarium oxysporum* HY25 against *Fusarium* root rot was 75%.
[0053] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.
[0054] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims
1. A type of pine-loving bacterium, characterized in that, Named HY25, its classification is named Talaromyces pinophilus It was deposited on December 15, 2025, at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M20252900, and the deposit address is Wuhan University, Wuhan, China.
2. The application of the *Pseudomonas pineophilus* as described in claim 1 in promoting tobacco growth and preventing tobacco root rot.
3. The application as described in claim 2, characterized in that, The pathogen causing tobacco root rot is Fusarium tumefaciens.
4. The application as described in claim 3, characterized in that, The *Fusarium* species mentioned are *Fusarium solanum*, *Fusarium oxysporum*, *Fusarium graminearum*, *Fusarium spp.*, *Fusarium moniliforme*, *Fusarium rosenbergii*, *Fusarium thunbergii*, and *Fusarium moniliforme*.
5. The application as described in claim 2, characterized in that, The term "growth promotion" refers to improving the agronomic traits of tobacco.
6. The application as described in claim 5, characterized in that, The agronomic traits include plant height, stem diameter, fresh and dry weight of aboveground and root parts, number of leaves, and maximum leaf area.
7. The application as described in claim 2, characterized in that, It is used for phosphorus solubilization, potassium solubilization, and nitrogen fixation.