Stenotrophomonas maltophilia capable of degrading lignin and resisting bacteria and application of stenotrophomonas maltophilia

Abstract

The invention discloses stenotrophomonas maltophilia with lignin degradation and antibacterial effects and application of the stenotrophomonas maltophilia, and relates to the field of microorganisms. The stenotrophomonas maltophilia mjt3 is preserved in the China General Microbiological Culture Collection Center on January 20, 2026, the preservation date is January 20, 2026, and the preservation number is CGMCC No.37470. The stenotrophomonas maltophilia mjt3 can be used for preparing a stenotrophomonas maltophilia strain. The stenotrophomonas maltophilia mjt3 disclosed by the invention is a multifunctional biocontrol strain with both lignin degradation capability and pathogenic bacterium inhibition activity. The strain inhibits the growth of pathogenic bacteria through a plurality of mechanisms such as antibacterial substance generation, ecological niche competition and extracellular enzyme secretion, degrades agricultural wastes, not only can effectively control the infection of pathogenic bacteria such as alternaria alternata, but also can obviously reduce the use of chemical pesticides, improve the soil micro-ecology and improve the soil quality. And an eco-friendly solution is provided for realizing green prevention and control of diseases, cyclic utilization of resources and environmental protection.

Description

Technical Field

[0001] This invention relates to the field of microbiology, specifically to a maltophilic oligotrophozoite with both lignin-degrading and antibacterial properties and its applications. Background Technology

[0002] Alternaria alternata is a widely distributed plant pathogenic fungus that infects various economic crops such as tobacco, tomato, and citrus, causing severe leaf spot disease. This pathogen primarily infects leaves, forming characteristic brown concentric ring-shaped lesions, leading to leaf tissue necrosis and premature leaf drop, severely impacting crop yield and quality. Under warm and humid conditions, Alternaria alternata spreads rapidly and is difficult to control, often causing significant economic losses once an outbreak occurs. While traditional chemical control methods have some effectiveness, long-term use can lead to increased pathogen resistance, excessive pesticide residues, and environmental pollution, necessitating a safer and more effective control strategy.

[0003] Meanwhile, agricultural and forestry production generates a large amount of lignin-rich waste annually. Lignin, due to its complex aromatic structure, is difficult to degrade, becoming a key bottleneck restricting the resource utilization of agricultural waste. Utilizing microorganisms to degrade lignin offers advantages such as being environmentally friendly and cost-effective, making it an important pathway to achieving the recycling of agricultural waste.

[0004] To address the aforementioned challenges, developing multifunctional biocontrol strains that combine lignin degradation capabilities with pathogen inhibition activity has become a key direction for modern sustainable agriculture. These strains inhibit pathogen growth through multiple mechanisms, including producing antimicrobial substances, competing for ecological niches, and secreting extracellular enzymes, while simultaneously degrading agricultural waste. This not only effectively controls the infection of pathogens such as Alternaria, but also significantly reduces the use of chemical pesticides and improves soil microecology, providing an eco-friendly solution for achieving green disease control, resource recycling, and environmental protection. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention develops a multifunctional microorganism possessing both lignin degradation properties and inhibitory activity against plant pathogenic fungi, aiming to meet the needs of modern agricultural green pest control and agricultural waste resource utilization. This invention provides a microbial strain with significant biocontrol potential: *Stenotrophomonas maltophilia* (…). 嗜麦芽窄食单胞菌 a) mjt3, this strain can effectively degrade lignin and is effective against Alternaria ( 链格孢 It exhibits a strong inhibitory effect.

[0006] In a first aspect, the present invention provides a *Stenotrophomonas maltophilia* strain that simultaneously possesses lignin-degrading and antibacterial properties. 嗜麦芽窄食单胞菌 mjt3, the maltophilic oligotrophomonas ( 窄食单胞菌属 嗜麦芽mjt3 is deposited at the China General Microbiological Culture Collection Center (CGMCC) on January 20, 2026, with accession number CGMCC No. 37470.

[0007] In a second aspect, the present invention provides a microbial preparation comprising live cells, inactivated cells, fermentation broth, metabolites, or combinations thereof of the aforementioned Stenotrophomonas maltophilia mjt3.

[0008] Furthermore, the microbial preparation also includes an agriculturally or industrially acceptable carrier.

[0009] Furthermore, the carrier includes at least one of a solid carrier or a liquid carrier.

[0010] Optionally, the solid carrier is selected from at least one of peat moss, diatomaceous earth, kaolin, bentonite, straw powder, and wheat bran; the liquid carrier is selected from at least one of water, mineral oil, and vegetable oil.

[0011] In a third aspect, the present invention provides the use of the aforementioned Stenotrophomonas maltophilia mjt3 or the aforementioned microbial preparation in any of the following aspects: A1. Application in the preparation of products containing degradable lignocellulose materials; A2. Application in the preparation of products for the prevention and control of plant diseases or the inhibition of food spoilage bacteria.

[0012] Furthermore, the lignocellulosic material described in A1 is selected from at least one of agricultural waste, forestry waste, kitchen waste, or industrial lignocellulosic residue. In one embodiment of the present invention, the lignocellulosic material is agricultural waste.

[0013] Furthermore, the plant disease described in A2 is caused by pathogenic microorganisms. Optionally, the plant disease is caused by fungi or bacteria; optionally, the disease is at least one of black spot, wilt, gray mold, or soft rot.

[0014] In one embodiment of the present invention, the disease is black spot disease, preferably, the disease is Alternaria alternata (Alternaria). 链格孢 Dark spots caused by )

[0015] In a fourth aspect, the present invention provides a method for treating agricultural waste, comprising the following steps: mixing the aforementioned maltophilic oligotrophomonas mjt3 or the aforementioned microbial preparation with lignocellulose waste, and carrying out fermentation treatment under suitable conditions.

[0016] Furthermore, the suitable conditions are: temperature 20-30℃, processing time 2-5 days.

[0017] In a fifth aspect, the present invention provides a method for preventing and controlling plant diseases, comprising the following steps: applying the aforementioned Stenotrophomonas maltophilia mjt3 or the aforementioned microbial preparation to the surface of plants, plant seeds, plant growth media or agricultural products.

[0018] In a sixth aspect, the present invention provides a method for culturing the aforementioned maltophilic oligotrophozoites mjt3, the method comprising culturing the aforementioned maltophilic oligotrophozoites mjt3 at 20-30°C and 150-200 rpm.

[0019] Furthermore, the culture medium is at least one of LB medium and broth medium.

[0020] The beneficial effects of the present invention include, but are not limited to: The maltophilic oligotrophomonas described in this invention ( 嗜麦芽窄食单胞菌 MJT3 is a multifunctional biocontrol strain that combines lignin degradation and pathogen inhibition activity, avoiding the cumbersome operation of strains with single-function effects. This strain inhibits pathogen growth through multiple mechanisms, including producing antimicrobial substances, competing for ecological niches, and secreting extracellular enzymes, while simultaneously degrading agricultural waste. It not only effectively controls the infection of pathogens such as Alternaria, but also significantly reduces the use of chemical pesticides and improves soil microecology, providing an eco-friendly solution for achieving green disease control, resource recycling, and environmental protection. Attached Figure Description

[0021] The accompanying drawings, which are included to provide a further understanding of the invention and form part of this invention, illustrate exemplary embodiments of the invention and are used to explain the invention, but do not constitute an undue limitation of the invention. In the drawings: Figure 1 This is a colony diagram of Stenotrophomonas maltophilia mjt3 isolated in an embodiment of the present invention.

[0022] Figure 2 In this embodiment of the invention, a phylogenetic tree was constructed in MEGA 11 software using a similar bacterial sequence that has a high degree of similarity to the amplified sequence of Stenotrophomonas maltophilia mjt3 strain.

[0023] Figure 3 This is a schematic diagram illustrating the antagonistic effect of Stenotrophomonas maltophilia mjt3 against pathogenic fungi in an embodiment of the present invention. Figure 3 AC represents the results for control bacteria 1, control bacteria 2, and mjt3, respectively.

[0024] Figure 4 This is a schematic diagram illustrating the lignin degradation results of *Stenotrophomonas maltophiliae* mjt3 in an embodiment of the present invention, wherein... Figure 4 AC represents the results for control bacteria 1, control bacteria 2, and mjt3, respectively. Detailed Implementation

[0025] The present invention is described in detail below with reference to the embodiments, but the present invention is not limited to these embodiments. Unless otherwise specified, the raw materials and catalysts in the embodiments of the present invention are all purchased through commercial channels.

[0026] This invention aims to develop a multifunctional microorganism possessing both lignin degradation capabilities and inhibitory activity against plant pathogenic fungi, to meet the needs of modern agricultural green pest control and agricultural waste resource utilization. This invention provides a microbial strain, *Stenotrophomonas maltophilia* mjt3, with significant biocontrol potential. This strain can effectively degrade lignin and inhibit *Alternaria alternata* (…). 链格孢 It exhibits a strong inhibitory effect.

[0027] 1. Isolation and culture of Stenotrophomonas maltophilia The *Hydroxytrophozoites* species described in this invention were obtained through screening in a forest at the Tobacco Research Institute, Chinese Academy of Agricultural Sciences, Qingdao City, Shandong Province, China in January 2024. The specific procedures are as follows: Add 10 g of soil from the humus layer of fallen leaves and branches in the tobacco forest to 90 mL of sterile ultrapure water, shake at 28℃ and 180 rpm for 30 min, mix thoroughly, and then let stand. Take 1 mL of the suspension and dilute it serially (10... -1 10 -2 10 -3 10 -4 10 -5 The diluted suspension was spread at a rate of 100 μL onto Solarbio Agar (NA) solid medium and incubated at 28°C for 5 days. Colonies on the NA solid medium were then streaked five times for purification. The purified single strain was then cultured on NA solid medium, and the colony morphology on the medium was smooth, raised, pale yellow, and round (e.g., ...). Figure 1 As shown in the image, the strain number is mjt3, which is deposited at the China General Microbiological Culture Collection Center (CGMCC) on January 20, 2026, with accession number CGMCC No. 37470. Single colonies of the purified strain were picked using an inoculation loop and inoculated into Solarbio nutrient broth (NB) medium. After shaking at 28°C and 180 rpm for 24 h, the bacterial solution was mixed thoroughly with 50% glycerol at a 1:1 ratio and stored at -80°C for later use. When the mjt3 strain is needed, the preserved bacterial solution is inoculated into NA medium using the streak plate method and incubated statically at 28°C. Once the colonies have grown well, the strain is activated.

[0028] 2. Identification of Stenotrophomonas maltophilia Genomic DNA was extracted from strain mjt3. Following the instructions for using Novizan's 2×Taq Plus Master Mix II DNA polymerase, PCR amplification was performed using the extracted mjt3 genomic DNA as a template and 16S rRNA universal primers. The PCR products were then sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing.

[0029] The sequences of the universal primers for 16S rRNA are as follows: upstream primer (27F): 5'-AGAGTTTGATCCTGGCTCAG-3' (SEQ ID NO.1), downstream primer (1492R): 5'-TACGGTTACCTTGTTACGACTT-3' (SEQ ID NO.2). The PCR amplification system is shown in Table 1, and the PCR amplification program used is shown in Table 2.

[0030] Table 1 PCR amplification system

[0031] Table 2 PCR amplification program

[0032] The 16S rRNA amplification sequence of strain mjt3 is as follows:

[0033] Gene sequence alignment analysis using EzBioCloud (https: / / www.ezbiocloud.net / ) and NCBI Blast revealed that strain mjt3 shared 99.86% similarity with Stenotrophomonas maltophilia MTCC 434(T). Phylogenetic trees were constructed in MEGA 11 software using amplified sequences of similar bacteria with high sequence similarity to strain mjt3. Figure 2 As shown, strain mjt3 belongs to Stenotrophomonas maltophilia ( 嗜麦芽窄食单胞菌 ).

[0034] 3. Antagonistic pathogenic fungi of Stenotrophomonas maltophilia mjt3 Oat agar (OA): Used for pathogen activation. Formula: 30.0g oat flakes, 15.0-20.0g agar, 1000mL distilled water, pH natural (approximately 6.5±0.2).

[0035] Nutrient broth medium (NB): used for activation of biocontrol bacteria MJT3. Prepare according to the standard formula (10g peptone, 3g beef extract, 5g sodium chloride, 1000mL distilled water, pH 7.2-7.4), dispense into shake flasks, and sterilize at 121℃ for 20 minutes.

[0036] Basal culture medium for confrontation culture: PDA medium was used. PDA formula: 200g peeled potato, 20g glucose, 15-20g agar, 1000mL distilled water, natural pH.

[0037] Control bacterium 1: Stenotrophomonas maltophilia, 嗜麦芽窄食单胞菌 Purchased from China Industrial Microbial Culture Collection Center, strain number CICC 24002; Control bacterium 2: Stenotrophomonas maltophilia, 嗜麦芽窄食单胞菌 Purchased from the China Industrial Microbial Culture Collection Center, strain number CICC 25363.

[0038] Red star pathogenic bacteria (Alternaria alternata, 链格孢 Alternaria (CX06) was cultured in the dark at 25°C for 7 days on oat flocculation medium (OA) until vigorous mycelial growth or sporulation began, obtaining activated Alternaria. Mycelial cakes were collected using a 5 mm punch and inoculated into the center of the medium. Single colonies of MJT3 were picked and inoculated into 250 mL Erlenmeyer flasks containing 50 mL of NB medium. The cultures were then incubated at a suitable temperature (28°C) on a shaker (200 rpm). Odionation (OD) was measured periodically. 600 Value, to determine its logarithmic growth phase (usually OD). 600The value was 0.4. The logarithmic-phase bacterial suspension was transferred to sterile centrifuge tubes and centrifuged at low speed (4000 rpm, 10 min), discarding the supernatant (i.e., the old culture medium). The bacterial pellet was resuspended by pipetting with an equal volume or appropriate amount of sterile physiological saline or sterile water to prepare the experimental bacterial suspension. The logarithmic-phase MJT3 bacterial suspension was inoculated into the wells of the bacterial cake next to the *Acer rubrum* pathogen (50 μL). It was incubated statically in the dark at 25℃ for 5 days, and the antibacterial effect was observed daily (the pathogen strain is preserved in the National Agricultural Environmental Microbial Germplasm Resource Bank).

[0039] Replace the mjt3 strain with control strains 1 and 2, and repeat the above experiment as a positive control.

[0040] Depend on Figure 3 It is evident that strain mjt3 significantly inhibited the growth of CX06, indicating that it had an antagonistic effect on CX06. Control strain 1 had no significant inhibitory effect on CX06, while control strain 2 had an antagonistic effect on CX06, but the effect was significantly worse than that of mjt3.

[0041] 4. Maltodactylophilic oligotrophomonas mjt3 degrades lignin The activation steps for mjt3 are the same as in Example 3. A lignin degradation screening medium consisting of 10 g / L yeast extract, 20 g / L glucose, 18 g / L agar, and 0.1 g / L aniline blue indicator was used to test the lignin degradation of the strains. OD was collected via spot inoculation. 600 2.5 µL of a bacterial suspension with a pH of approximately 0.4 was inoculated into the center of a plate containing an indicator and incubated at 28 °C. After 48 h, the presence or absence of aniline blue color was observed to assess the lignin degradation capacity of the strain.

[0042] Observation indicators: Under natural light or a white background, observe the color changes of the culture medium around and below the colonies.

[0043] Positive result (+): The blue color around the colony disappears and a clear yellow halo appears.

[0044] Negative result (-): The area around and below the colony remains uniformly blue, with no color change.

[0045] Weak positive: A light-colored area appears below the colony, but it is not completely transparent.

[0046] Depend on Figure 4 It is evident that strain MJT3 effectively removed the indicator color, exhibiting a distinct yellow halo, demonstrating its ability to degrade lignin. In contrast, strain 2 showed no significant decolorization and lacked the ability to degrade lignin. Strain 1 exhibited slight decolorization, but its performance was significantly inferior to that of MJT3.

[0047] The above description is merely an embodiment of the present invention, and the scope of protection of the present invention is not limited to these specific embodiments, but is determined by the claims of the present invention. For those skilled in the art, the present invention can have various modifications and variations. Any modifications, equivalent substitutions, improvements, etc., made within the technical concept and principle of the present invention should be included within the scope of protection of the present invention.

Claims

1. A maltodactylophilic oligotrophomonas with both lignin-degrading and antibacterial properties ( Stenotrophomonas maltophilia mjt3, characterized in that, The maltophilic oligotrophomonas ( Stenotrophomonas maltophilia mjt3 is deposited at the China General Microbiological Culture Collection Center (CGMCC) on January 20, 2026, with accession number CGMCC No. 37470.

2. A microbial preparation, characterized in that, The microbial preparation comprises live cells, inactivated cells, fermentation broth, metabolites, or combinations thereof of Stenotrophomonas maltophilia mjt3 as described in claim 1.

3. The microbial preparation according to claim 2, characterized in that, The microbial preparation also includes agriculturally or industrially acceptable carriers.

4. The use of the Stenotrophomonas maltophilia mjt3 as described in claim 1 or the microbial preparation as described in any one of claims 2-3 in any of the following aspects: A1. Application in the preparation of products containing degradable lignocellulose materials; A2. Application in the preparation of products for the prevention and control of plant diseases or the inhibition of food spoilage bacteria.

5. The application according to claim 4, characterized in that, The lignocellulose material described in A1 is selected from at least one of agricultural waste, forestry waste, kitchen waste, or industrial lignocellulose residue.

6. The application according to claim 4, characterized in that, The plant diseases described in A2 are caused by pathogenic microorganisms.

7. A method for treating agricultural waste, characterized in that, The process includes the following steps: mixing the maltophilic oligotrophosome mjt3 as described in claim 1 or the microbial preparation as described in any one of claims 2-3 with lignocellulose waste and fermenting it under suitable conditions.

8. The method according to claim 7, characterized in that, The suitable conditions are: temperature 20-30℃, processing time 2-5 days.

9. A method for preventing and controlling plant diseases, characterized in that, The process includes the following steps: applying the Stenotrophomonas maltophilia mjt3 as described in claim 1 or the microbial preparation as described in any one of claims 2-3 to the surface of plants, plant seeds, plant growth media or agricultural products.

10. A method for culturing *Stenotrophomonas maltophilia* mjt3 as described in claim 1, characterized in that, The method includes culturing the maltophilic oligotrophoblast mjt3 as described in claim 1 at 20-30°C and 150-200 rpm.

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