Paenibacillus fumusaculi XE201 and application thereof

Abstract

The application discloses a strain of Azomonotrichon indicum XE201 and application thereof, relates to the technical field of plant disease biological control, and discloses a strain of Azomonotrichon indicum XE201 and application thereof. The Azomonotrichon indicum XE201 has been preserved in the China Center for Type Culture Collection on October 16, 2023, the preservation address is Wuhan, China, and the preservation number is CCTCC NO: M 20231909. The Azomonotrichon indicum XE201 has a significant antagonistic effect on a tobacco bacterial wilt pathogen Ralstonia solanacearum, and the inhibition rate reaches 65.71%. The germination rate of tobacco seeds treated by the Azomonotrichon indicum XE201 reaches 95.53%, the growth of tobacco plants treated by the bacteria is better, and the Azomonotrichon indicum XE201 can significantly promote the germination of tobacco seeds and the growth of tobacco plants.

Description

Technical Field

[0001] This invention relates to the field of plant disease biocontrol technology, and particularly to a strain of *Microbacterium neemophilus* (…). Microbacterium azadirachtae XE201 and its applications. Background Technology

[0002] tobacco( Nicotianatabacum L Tobacco, belonging to the Solanaceae family and the Nicotiana genus, is an annual herbaceous plant and an important economic crop in my country. Tobacco bacterial wilt is caused by *Ralstonia solanacearum* (…). Ralstonia solanacearum Tobacco bacterial wilt is a devastating soil-borne bacterial disease caused by [unspecified pathogen]. It is widely distributed, particularly severe in tropical and subtropical regions. In China, it is prevalent in tobacco-growing areas south of the Yangtze River, and in recent years, its reach has expanded to northern tobacco-growing provinces such as Shandong, Henan, Shaanxi, and Liaoning, with the disease becoming increasingly serious. Tobacco bacterial wilt affects tobacco yield and quality, and in severe cases, can lead to total crop failure, causing enormous economic losses to my country's tobacco production annually.

[0003] Currently, the main control measures for tobacco bacterial wilt include breeding disease-resistant varieties, chemical control, agricultural control, and biological control. Chemical control is the most commonly used method for controlling tobacco bacterial wilt, but excessive chemical control not only leads to drug resistance in pathogens and reduces control effectiveness, but also poses a series of adverse effects, such as threats to the environment and humans. Biological control has the characteristics of long-term effectiveness, high safety, and environmental friendliness. Utilizing beneficial microorganisms to control tobacco bacterial wilt is one of the important means. Previous researchers have conducted sporadic screening of biocontrol bacteria and growth-promoting bacteria for tobacco bacterial wilt. Developing more types and more effective biocontrol bacteria and growth-promoting bacteria for tobacco diseases is of great significance to tobacco production. Summary of the Invention

[0004] To address the shortcomings of the existing technology, this invention provides a strain of *Microbacterium neemophilus* (…). Microbacterium azadirachtae XE201 and its applications, this *Microbacterium neemophilus* ( Microbacterium azadirachtae XE201 can effectively promote the germination of tobacco seeds and the growth of tobacco plants. Simultaneously, it achieves a 65.71% inhibition rate against bacterial wilt, specifically through the following technologies:

[0005] In a first aspect, the present invention provides a strain of *Microbacterium neemophilus* (…). Microbacterium azadirachtae XE201, this bacterium was deposited at the China Center for Type Culture Collection (CCTCC), Wuhan, China, on October 16, 2023, with accession number CCTCC NO: M 20231909.

[0006] Furthermore, the aforementioned *Microbacterium azadirachtinum* ( Microbacterium azadirachtaeXE201 is used to promote tobacco seed germination, promote tobacco plant growth, and prevent tobacco bacterial wilt.

[0007] Furthermore, the aforementioned *Microbacterium azadirachtinum* ( Microbacterium azadirachtae Single colonies of XE201 are pale yellow, slightly transparent, moist, and have smooth edges.

[0008] A second aspect of the present invention provides the above-mentioned *Azadirachtinum* (… Microbacterium azadirachtae Application of XE201 in promoting tobacco seed germination.

[0009] Furthermore, the above applications include: using *Microbacterium azadirachtinum* (… Microbacterium azadirachtae Tobacco seeds were soaked in a bacterial suspension of XE201 for 30-40 minutes; the concentration of the bacterial suspension was 2×10⁻⁶. 8 CFU / mL.

[0010] A third aspect of the present invention provides the above-mentioned *Azadirachtinum* (… Microbacterium azadirachtae Application of XE201 in promoting tobacco plant growth.

[0011] Furthermore, the above application includes: applying the *Microbacterium azadirachtinum* (*Microbacterium tumefaciens*) at a dosage of 20 mL / strain. Microbacterium azadirachtae The bacterial suspension of XE201 was inoculated into tobacco plants; the concentration of the bacterial suspension was 2 × 10⁻⁶. 8 CFU / mL; the inoculation method is root drenching.

[0012] A fourth aspect of the present invention provides *Microbacterium azadirachtinum* (… Microbacterium azadirachtae Application of XE201 in the prevention and control of tobacco bacterial wilt.

[0013] Furthermore, the above application includes: applying the *Microbacterium azadirachtinum* (*Microbacterium tumefaciens*) at a dosage of 20 mL / strain. Microbacterium azadirachtae The bacterial suspension of XE201 was inoculated into tobacco plants; the concentration of the bacterial suspension was 2 × 10⁻⁶. 8 CFU / mL; the inoculation method is spraying or root irrigation.

[0014] In a fifth aspect, the present invention provides a microbial agent for promoting tobacco seed germination, promoting tobacco plant growth, and preventing tobacco bacterial wilt, the microbial agent comprising the aforementioned *Microbacterium neemata* (…). Microbacterium azadirachtae )XE201.

[0015] Furthermore, the aforementioned *Microbacterium azadirachtinum* ( Microbacterium azadirachtae The concentration of XE201 in the bacterial agent is OD 600 0.3.

[0016] Compared with the prior art, the advantages of the present invention are:

[0017] 1. The *Azadirachtinum* microbacterium provided by this invention ( Microbacterium azadirachtae XE201 is effective against Ralstonia solanacearum, the pathogen of tobacco bacterial wilt. Ralstonia solanacearum It has a significant antagonistic effect, with an antibacterial rate of 65.71%.

[0018] 2. The germination rate of tobacco seeds treated with this *Azadirachtinum* XE201 was as high as 95.53%, which can significantly promote the germination of tobacco seeds.

[0019] 3. Tobacco plants treated with this bacterium grow better and can effectively promote the growth of tobacco plants. Attached Figure Description

[0020] Figure 1 This is a single colony morphology result of strain XE201 in the example;

[0021] Figure 2 This is a phylogenetic tree diagram of strain XE201 in the examples;

[0022] Figure 3 This is a graph showing the antibacterial effect of strain XE201 against Ralstonia solanacearum in the examples.

[0023] Figure 4 The graph shows the detection results of the XE201 strain producing ferrophile in the example.

[0024] Figure 5 The figure shows the effect of strain XE201 on tobacco seed germination in the example. Detailed Implementation

[0025] The technical solution of the present invention will be clearly and completely described below. 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.

[0026] Example

[0027] 1. Isolation and culture of bacterial strains

[0028] The XE201 strain of this invention was collected on August 12, 2022, from a field in Enshi Prefecture, Hubei Province, where bacterial wilt was severe. The isolation and culture method is as follows:

[0029] The resuspended soil sample solution was serially diluted in physiological saline (0.85%, NaCl) and spread onto PDA, LB, 1 / 5 LB, and NA media, and incubated at 28°C for 24 h. The number of colonies on each medium was recorded. Based on the growth characteristics of the colonies, such as color, size, elevation, transparency, hardness, and edge regularity, single colonies were picked and streaked three times on the corresponding medium to obtain pure culture strains.

[0030] Single colonies of strain XE201 obtained from isolation and culture are shown below. Figure 1 As shown, single colonies of the strain are pale yellow, slightly transparent, moist, and have smooth edges.

[0031] 2. Identification of strain species

[0032] (1) Pick the purified bacterial culture with a 10 μL pipette tip into a 2 mL centrifuge tube, add 100 μL of 1×TEBuffer (10 mmol / L Tris.HCl; 1 mmol / L EDTA; pH 8.0), boil for 10 min and immediately place in a -20℃ refrigerator overnight, centrifuge at 12000 rpm for 10 min, and take the supernatant to obtain the DNA of the strain.

[0033] (2) Using genomic DNA as a template, the bacterial 16S rRNA gene (as shown in SEQ ID NO:1) was amplified. The PCR system and reaction conditions are shown in Table 1. After the reaction, the PCR products were analyzed by agarose gel electrophoresis. The obtained fragments were sequenced. A phylogenetic tree was constructed using MEGA software.

[0034] Table 1 PCR reaction system and reaction

[0035]

[0036] The nucleotide sequences of the primer pairs in the above PCR system are as follows:

[0037] Primer F: 5'-agagtttgatcctggctcag-3' (as shown in SEQ ID NO:2);

[0038] Primer R: 5'-ggttaccttgttacgactt-3' (as shown in SEQ ID NO:3).

[0039] (3) The sequence obtained from sequencing was compared with the Ez-Taxon website and the NCBI website to determine the taxonomic status of the strain.

[0040] 16S rDNA sequencing yielded a 1398 bp gene sequence fragment. Sequence alignment confirmed that XE201 is related to the type strain *Microbacterium neemophilus*. Microbacterium azadirachtae The highest similarity was found in DSM 23848. A phylogenetic tree constructed based on the 16S rDNA sequence is shown below. Figure 2 As shown, by Figure 2 It is evident that XE201 clustered with Azadirachtinibacillus, indicating that XE201 belongs to Azadirachtinibacillus.

[0041] 3. Preservation of microbial strains

[0042] The purified strain was streaked on an agar slant, and then 5 mL of sterile glycerol preservation solution (20% glycerol) was added. The bacterial growth was vortexed down using a vortex mixer, marked, and finally the bacterial mixture was stored in an ultra-low temperature freezer at -80℃ (at least 3 backups of each strain).

[0043] 4. Detection of antibacterial ability against Ralstonia solanacearum

[0044] Take 10 µL of bacterial suspension (OD) 600 =0.3), inoculated in the center of a 9cm diameter NA solid culture medium, and after the bacterial solution was fully absorbed, the culture dish was placed in an inverted incubator at 28 ℃ for 72 h. The pathogen, tobacco bacterial wilt (Tobacco Bacterium wilt), was then introduced. Ralstonia solanacearum ) Prepared into OD 600 The bacterial suspension was prepared at a concentration of 0.3 g / mL. The bacterial suspension was then sprayed onto NA medium, and the culture dishes were transferred to a 28°C incubator and incubated upside down for 24 hours. The antibacterial activity of the strains was then assessed.

[0045] Strain XE201 against tobacco bacterial wilt ( Ralstonia solanacearum The test results of the antibacterial ability of ) are as follows Figure 3 As shown, the results indicate that strain XE201 is effective against Ralstonia solanacearum, the pathogen of tobacco bacterial wilt (Ralstonia solanacearum). Ralstonia solanacearum The strain XE201 exhibited significant antagonistic effects, with an inhibition rate of 65.71%. This indicates that strain XE201 can be used to control tobacco bacterial wilt. Specifically, the strain XE201 suspension was inoculated into tobacco plants at a dosage of 20 mL / plant; the concentration of the suspension was 2 × 10⁻⁶. 8 CFU / mL; inoculation methods include spraying or root drenching.

[0046] 5. Detection of Ferrophilic Production Capacity

[0047] The CAS detection medium formula is as follows: 10 mL / L 20% sucrose solution, 30 mL / L 10% acid-hydrolyzed casein, 1 mL / L 1 mmol / L CaCl2, 20 mL / L 1 mmol / L MgSO4, and 15 g / L agar. At approximately 60℃, slowly add 50 mL each of phosphate buffer and CAS staining solution, and autoclave at 121℃ for 20 min. CAS staining solution: 1 mmol / L CAS (chrome azure), 0.1 mmol / L FeCl3, and 4 mmol / L HDTMA (hexadecyltrimethylammonium bromide). Phosphate buffer: 24.27 g / L Na2HPO4·12H2O, 5.905 g / L NaH2PO4·2H2O, 0.75 g / L KH2PO4, 2.50 g / L NH4Cl, 1.25 g / L NaCl, pH 6.8. Dilute 10-fold before use.

[0048] 10 μL of a concentration of 2×10 7 The CFU / mL bacterial suspension spot NA is located in the center of the CAS identification medium. The plate is transferred to a 28℃ constant temperature incubator and continuously observed. The appearance of a yellow halo indicates the production of heptaphilin.

[0049] The test results of the ability to produce heptaphilum are as follows: Figure 4 As shown, a yellow halo appeared on the CAS plate, indicating that strain XE201 can produce ferrophosphate, which means that strain XE201 can promote the root growth and development of tobacco by synthesizing and secreting ferrophosphate (see the article "Screening and Identification of Ferrophilic Producing Bacteria and Their Growth-Promoting Effect on Tested Plants").

[0050] 6. Effects on tobacco seed germination

[0051] Tobacco seed disinfection: Soak tobacco seeds (K326) in 15% H2O2 for 10 min, then in 75% ethanol for 30 s, and rinse three times with sterile water.

[0052] The bacterial strain was inoculated into NB liquid medium and cultured overnight at 28°C and 180 rpm. The cells were collected by centrifugation at 8000 rpm for 5 min, the supernatant was discarded, and the suspension was resuspended in sterile water to adjust the concentration to 2 × 10⁻⁶. 7 CFU / mL; after disinfection, tobacco seeds were soaked in a prepared bacterial suspension for 30 min, and control tobacco seeds were soaked in sterile water for 30 min; double-layer sterile filter paper was placed in a petri dish and moistened with 2 mL of sterile water; 100 tobacco seeds were evenly dotted on the filter paper, and the petri dish was placed in a 28℃ greenhouse for incubation, keeping the filter paper moist, and the seed germination was observed.

[0053] The results of the seed germination analysis are as follows: Figure 5As shown in the figure, "**" represents a highly significant difference (p < 0.01). After treatment with XE201 bacterial suspension, the seed germination rate reached 95.53%, while the germination rate of tobacco seeds treated with sterile water (control) was only 87.78%, indicating that XE201 bacterial suspension treatment can significantly promote the germination of tobacco seeds.

[0054] 7. Effects on tobacco growth

[0055] The bacterial strain was inoculated into NB liquid medium and cultured overnight at 28°C and 180 rpm. The cells were collected by centrifugation at 8000 rpm for 5 min, the supernatant was discarded, and the suspension was resuspended in sterile water to adjust the concentration to 2 × 10⁻⁶. 7 CFU / mL, each tobacco plant was irrigated with 20 mL of bacterial solution 14 days and 21 days after transplanting, while the control group was irrigated with 20 mL of sterile water. After 28 days, the differences in plant height, leaf length, leaf width and number of leaves between the treatment group and the control group were measured.

[0056] The growth-promoting effects of strain XE201 on tobacco are shown in Table 2 below. Compared with the control group, the physiological indicators of tobacco plants after root drenching with strain XE201 were significantly improved (p<0.01 or p<0.001). Plant height increased by approximately 0.43 cm, leaf length by approximately 1.36 cm, leaf width by approximately 0.82 cm, and the number of leaves increased by approximately one. This indicates that root drenching with strain XE201 suspension can promote tobacco plant growth and has a good growth-promoting effect.

[0057] Table 2. Growth-promoting effects of strain XE201 on tobacco.

[0058]

[0059] Note: "**" in the table indicates highly significant differences. p <0.01); "***" indicates extremely significant difference ( p <0.001).

[0060] The above examples confirmed that strain XE201 is *Microbacterium azadirachtinum* (…). Microbacterium azadirachtae The antibacterial ability of strain XE201 was determined to be effective against Ralstonia solanacearum (Ralstonia solanacearum). Ralstonia solanacearum The strain XE201 exhibited significant antagonistic effects, with an inhibition rate of 65.71%. Ferrophilic production capacity testing revealed that strain XE201 possessed the ability to produce ferophiles. Furthermore, treatment with strain XE201 significantly promoted tobacco seed germination, achieving a germination rate as high as 95.53%, approximately 8 percentage points higher than that of tobacco seeds treated with sterile water. Growth-promoting experiments showed that treatment with strain XE201 significantly promoted tobacco plant growth, with significant increases in physiological indicators such as plant height, leaf length, and leaf width.

[0061] The above detailed embodiments describe the implementation of the present invention; however, the present invention is not limited to the specific details described in the above embodiments. Within the scope of the claims and technical concept of the present invention, various simple modifications and changes can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

Claims

1. A strain of *Microbacterium neemophilus* ( Microbacterium azadirachtae XE201, characterized in that, The *Microbacterium neemata* XE201 was deposited on October 16, 2023, at the China Center for Type Culture Collection (CCTCC), Wuhan, China, with accession number CCTCC NO: M 20231909.

2. The *Azadirachtinum* strain according to claim 1 (… Microbacterium azadirachtae The application of XE201 in promoting tobacco seed germination is characterized by, Tobacco seeds were soaked in a bacterial suspension of *Microbacterium neemophilus* XE201 for 30-40 minutes; the concentration of the bacterial suspension was 2 × 10⁻⁶. 8 CFU / mL.

3. A *Azadirachtinum* strain according to claim 1 (… Microbacterium azadirachtae The application of XE201 in promoting tobacco plant growth is characterized by, The bacterial suspension of *Microbacterium neemophilus* XE201 was inoculated into tobacco plants at a dosage of 20 mL / plant; the concentration of the bacterial suspension was 2 × 10⁻⁶. 8 CFU / mL; the inoculation method is root drenching.

4. A *Microbacterium neemata* strain according to claim 1 (… Microbacterium azadirachtae The application of XE201 in the control of tobacco bacterial wilt is characterized by, The bacterial suspension of *Microbacterium neemophilus* XE201 was inoculated into tobacco plants at a dosage of 20 mL / plant; the concentration of the bacterial suspension was 2 × 10⁻⁶. 8 CFU / mL; the inoculation method is spraying or root irrigation.

5. A fungicide for promoting tobacco seed germination, promoting tobacco plant growth, and controlling tobacco bacterial wilt, characterized in that, Includes the *Azadirachtinibacillus* XE201 as described in claim 1.

6. The microbial agent according to claim 5, characterized in that, The concentration of *Microbacterium neemophilus* XE201 in the inoculum was OD. 600 =0.3.

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