Bifidobacterium dendriforme QHHQ09-2, a bacterial agent and application thereof

Abstract

The application belongs to the technical field of microorganisms, and specifically discloses a kind of Microbacterium arborescens QHHQ09-2, a microbial agent thereof and application. The Microbacterium arborescens QHHQ09-2 of the application has a preservation number of CGMCC No.33708. The Microbacterium arborescens has the functions of IAA production, nitrogen fixation and alkali tolerance, can promote plant growth and development, slow down the decomposition or transfer of chlorophyll in plant leaves, delay plant senescence, and effectively inhibit Gibberella zeae.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a tree-shaped microbacterium QHHQ09-2, its inoculum, and its applications. Background Technology

[0002] Microbacterium is a genus of Gram-positive, non-spore-forming rod-shaped bacteria belonging to the family Microbacteriaceae in the phylum Actinobacteria. Microbacteria are distributed in various environments, including saline-alkali lands, marine environments, soils, and plants, with over 140 species recorded and new species continuously being reported. Currently, most functional studies on microbacteria focus on their ability to degrade pollutants and tolerate heavy metals; some strains can promote plant growth through nitrogen fixation, phosphorus solubilization, and the production of plant growth hormones, and show good potential for improving the tolerance of crops to abiotic stresses such as salt and heavy metals.

[0003] Therefore, as an important microbial resource, microbes can be effectively applied to the development of microbial fertilizers and inoculants by enhancing their diversity and exploring and identifying their potential functions. This application can not only promote the ecological balance of agriculture, but also serve as an important means to promote the development of sustainable and green agriculture. Summary of the Invention

[0004] In view of the above, it is necessary to provide a strain that has the functions of producing IAA (indoleacetic acid), fixing nitrogen and being alkali tolerant, which can promote plant growth and development, slow down the decomposition or transfer of chlorophyll in plant leaves, delay plant senescence, and effectively inhibit the pathogen of Fusarium graminearum in corn.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0006] This invention provides a *Microbacterium arborescens* QHHQ09-2, which is classified as *Microbacterium arborescens*. It is deposited at the China General Microbiological Culture Collection Center (CGMCC) at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, on March 3, 2025, with accession number CGMCC No. 33708.

[0007] Furthermore, the dendritic microbacterium QHHQ09-2 has the functions of producing IAA, fixing nitrogen and / or being alkali resistant.

[0008] The present invention also provides a microbial agent comprising the above-mentioned dendritic microbacterium QHHQ09-2 and / or its fermentation products.

[0009] The present invention also provides the application of the above-mentioned dendritic microbacterium QHHQ09-2 or the above-mentioned microbial agent in promoting plant growth.

[0010] Furthermore, the promotion of plant growth includes increasing plant height, stem diameter, dry weight, and / or fresh weight.

[0011] The present invention also provides the application of the above-mentioned dendritic microbacterium QHHQ09-2 or the above-mentioned bacterial agent in slowing down the decomposition or transfer of chlorophyll in plant leaves.

[0012] Furthermore, the plants include chili peppers and lettuce.

[0013] The present invention also provides the application of the above-mentioned dendritic microbacterium QHHQ09-2 or its agent in inhibiting Fusarium graminearum, the causal agent of maize scab.

[0014] Furthermore, the inhibition of *Fusarium graminearum* includes inhibiting the mycelial growth and spore germination of *Fusarium graminearum*.

[0015] The present invention also provides the application of the above-mentioned dendritic microbacterium QHHQ09-2 or its agent in the preparation of drugs for preventing and controlling animal and plant diseases caused by Fusarium graminearum.

[0016] The present invention has the following beneficial effects:

[0017] 1. The Microbacterium arborescens QHHQ09-2 of this application can produce IAA, has nitrogen-fixing and alkali-resistant functions, which helps to promote plant growth and development, thereby improving crop yield and quality.

[0018] 2. The dendritic microbacterium QHHQ09-2 of this application and the microbial agent prepared using the dendritic microbacterium QHHQ09-2 can be applied to plant cultivation, effectively promoting plant growth, while slowing down the decomposition or transfer of chlorophyll in old leaves of plants, thereby improving photosynthesis of plant leaves and delaying plant senescence.

[0019] 3. The Microbacterium arborescens QHHQ09-2 of this application can effectively inhibit Fusarium graminearum, including inhibiting the mycelial growth and spore germination of Fusarium graminearum. Therefore, its application as a drug for the prevention and control of plant and animal diseases caused by Fusarium graminearum can improve the disease resistance of plants and animals. Attached Figure Description

[0020] Figure 1This is a colony diagram of the *Microbacterium dendriticum* strain QHHQ09-2 on a plate.

[0021] Figure 2 This is a microscopic image of the *Microbacterium dendriticum* strain QHHQ09-2 of this invention.

[0022] Information on the preservation of biological materials

[0023] The strain information deposited in this application is: Microbacterium arborescens QHHQ09-2, classified as Microbacterium arborescens, deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, on March 3, 2025, with accession number CGMCC No. 33708. Detailed Implementation

[0024] To make the technical problems, technical solutions and advantages of the present invention clearer, a detailed description will be given below in conjunction with specific embodiments.

[0025] All features disclosed in this specification, or all steps in all disclosed methods or processes, may be combined in any way, except for mutually exclusive features and / or steps.

[0026] Unless otherwise stated, each feature disclosed in this specification (including any appended claims and abstract) is merely one example of a series of equivalent or similar features.

[0027] Example 1

[0028] This embodiment provides a *Microbacterium arborescens* strain QHHQ09-2, which was isolated from silt at a depth of 20 meters at the bottom of Qinghai Lake. The specific isolation and screening method is as follows:

[0029] (1) Isolation and purification of strains

[0030] Soil samples were collected from silt at a depth of 20 meters at the bottom of Qinghai Lake, placed in sterile bags, and brought back to the laboratory for storage at 4℃. 10g of fresh soil sample was weighed and added to 90mL of sterile physiological saline, shaken at 180rpm for 30min, and 1ml of the liquid was added to a liquid culture medium with cellulose as the sole carbon source and incubated at 15℃ and 180rpm for one week. After enrichment, the supernatant was diluted to 10⁻⁶. -3 10 -4 10 -5Three gradients were used, with 100 μL of liquid from each dilution spread onto cellulase screening solid medium. After incubation at 15°C for 4 days, single colonies grew. After colony growth, the strain was purified three times on cellulase screening plates using the streak method to obtain a single bacterial strain, numbered QHHQ09-2.

[0031] (2) Physiological and biochemical identification of colonies

[0032] Refer to Bergey's Manual of Bacteriological Identification (9th Edition) to observe the morphology, color, etc., of the colonies grown by the strain on the surface of LB medium. Figure 1 Colony morphology diagram and Figure 2 Microscopic examination shows that the strain was cultured on nutrient agar at 30°C for 48 hours. The colonies were round, raised, smooth, with regular edges, and dark orange in color. Microscopic examination revealed that it was a Gram-positive bacillus that did not produce spores.

[0033] (3) Sequence analysis of the strain's 16S rRNA

[0034] The strain QHHQ09-2 was sent to the sequencing and identification department of Shanghai Sangon Biotech for sequencing identification. The sequencing results are as follows:

[0035]

[0036] The sequencing results were compared and analyzed in the NCBI ribosome database. The 16S rDNA sequence analysis results showed that the strain of this invention had 100% homology with Microbacterium arborescens, that is, the strain is Microbacterium arborescens.

[0037] The microorganisms in this embodiment were preserved as follows:

[0038] Microbacterium arborescens QHHQ09-2, classified as Microbacterium arborescens, is deposited at the China General Microbiological Culture Collection Center (CGMCC) at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. The deposit date is March 3, 2025, and the accession number is CGMCCNo. 33708.

[0039] Example 2

[0040] This embodiment measures the ability of Microbacterium arborescens QHHQ09-2 in Example 1 to produce IAA. The specific measurement method is as follows:

[0041] The Salkowski colorimetric method was used. The strain was inoculated into LB liquid medium containing L-tryptophan (100 mg / L) and cultured at 30°C and 200 rpm for 24 h using a shaker. The bacterial suspension was centrifuged at 4°C and 5000 rpm for 10 minutes. 1 mL of the supernatant was added to 3 mL of Salkowski colorimetric reagent, with an uninoculated LB liquid medium and the mixture of colorimetric reagent serving as a blank control. After incubation at room temperature in the dark for 30 min, observation was performed; a red color indicated the ability to secrete IAA. Standard curves were prepared by using IAA standard solutions of different concentrations. The OD of the reaction solution was measured after reacting under the above conditions. 530 Calculate the concentration of IAA in the supernatant.

[0042] The calculated IAA production capacity of *Microbacterium dendriticum* QHHQ09-2 is 24.93 ± 2.54 mg / L.

[0043] Example 3

[0044] This embodiment measures the nitrogen fixation capacity of Microbacterium arborescens QHHQ09-2 from Example 1 above. The specific measurement method is as follows:

[0045] Preparation method of Asbhy nitrogen-free medium: Weigh 10g glucose, 0.2g dipotassium hydrogen phosphate, 0.2g sodium chloride, 0.2g magnesium sulfate monohydrate, 0.2g potassium sulfate, and 5g calcium carbonate, dissolve them in 1000mL of water, and add 18±2g agar. Autoclave at 121℃ for 20min.

[0046] The four-point inoculation method was used to inoculate the *Microbacterium arborescens* QHHQ09-2 obtained in Example 1 onto Asbey nitrogen-free culture plates, with three plates inoculated repeatedly. After inoculation, the plates were placed in a 30°C incubator for 7 days, and the bacterial growth and the formation of clear zones around the colonies were observed daily during the incubation period.

[0047] Results: *Microbacterium dendriticum* QHHQ09-2 produced a clear zone when cultured on Asbey nitrogen-free medium, indicating that the strain possesses nitrogen-fixing ability. The size of the clear zone was measured using the cross-crossing method, and the ratio of the clear zone diameter (D) to the colony diameter (d) was calculated to be D / d = 4.73 ± 0.21, indicating that *Microbacterium dendriticum* QHHQ09-2 has a strong nitrogen-fixing ability.

[0048] Example 4

[0049] This embodiment tests the alkali resistance of Microbacterium arborescens QHHQ09-2 from Example 1 above. The specific testing method is as follows:

[0050] Microbacterium arborescens (QHHQ09-2) was inoculated into liquid LB medium and cultured at 30℃ and 200 rpm for 22 h to prepare a seed culture. The concentration of the seed culture was adjusted to an OD600 of 0.5, and then 2% was inoculated into beef extract peptone medium prepared with NaOH at four pH gradients (8, 9, 10, and 11), in triplicate. The cultures were incubated at 37℃ and 180 rpm for 2–5 days, and growth was observed and data recorded. OD600 was measured every 24 h. 600 The alkali tolerance of the strain was determined by measuring the alkali value for 5 days. The results are shown in Table 1 below.

[0051] Table 1. Alkali resistance test results of *Microbacterium dendriticum* QHHQ09-2

[0052]

[0053] As pH increased, the absorbance of the strain showed a decreasing trend, stabilizing after the fourth day. Alkali tolerance tests revealed that *Microbacterium arborescens* QHHQ09-2 could grow at pH 8–10 (in the table, + indicates normal growth, more + signs indicate faster reproduction and growth, and - indicates inhibited growth), indicating that the strain possesses strong alkali tolerance.

[0054] Example 5

[0055] This embodiment provides a microbial agent, the preparation method of which includes the following steps:

[0056] (1) Microbacterium arborescens QHHQ09-2 was activated on LB agar plates, and single colonies were picked and cultured on LB slant agar plates at 30°C to obtain activated strains;

[0057] (2) The activated strain was inoculated into LB liquid medium and cultured with shaking at 30℃ and 160r / min for 18h to obtain seed culture;

[0058] (3) The seed culture was inoculated into LB medium at a volume ratio of 1:60 and cultured at 30℃ and 150r / min for 2 days to obtain the fermentation broth;

[0059] (4) After solid-liquid separation of the fermentation broth, cells, spores and fermentation broth can be obtained;

[0060] (5) Concentrate the fermentation broth to obtain a concentrated solution;

[0061] (6) The above-mentioned bacterial cells, spores and concentrated liquid are mixed to obtain the bacterial agent.

[0062] Example 6

[0063] This embodiment provides a method for using the microbial agent from Example 5 in chili pepper cultivation, as detailed below:

[0064] At the 4-5 leaf stage of the chili pepper, root application of a 500-fold dilution of the microbial agent described in Example 5 was initiated, with an interval of 7 days between applications, for a total of 3 treatments. Each application used 200 ml of water per pot. The first root application used a 1000-fold dilution of compound fertilizer (N+P2O5+K2O, 20-20-20, Nanning Hanhe Biotechnology Co., Ltd.) as the stock solution, while the second and third treatments used tap water as the stock solution.

[0065] Example 7

[0066] This embodiment provides a method for using the microbial agent from Example 5 in chili pepper cultivation, as detailed below:

[0067] At the 4-5 leaf stage of the chili pepper, root application of an 800-fold dilution of the microbial agent described in Example 5 was initiated, with applications every 7 days for a total of 3 treatments. Each application used 200 ml of water per pot. The first root application used a 1000-fold dilution of compound fertilizer (N+P2O5+K2O, 20-20-20, Nanning Hanhe Biotechnology Co., Ltd.) as the stock solution, while the second and third treatments used tap water as the stock solution.

[0068] Comparative Example 1

[0069] This comparative example provides a method for growing chili peppers. The difference between this comparative example and Example 7 is that the first treatment in this comparative example only involves root application of compound fertilizer, while the second and third treatments only involve root application of tap water. All other operations are exactly the same.

[0070] Seven days after the third treatment, chili peppers from Examples 6 and 7 and Comparative Example 1 were collected. The following indicators were investigated on the collected chili peppers. The investigation indicators and methods are shown in Table 2, and the investigation results are shown in Table 3.

[0071] Table 2 Survey Indicators and Methods

[0072]

[0073] Table 3. Effects of different treatments on various indicators of chili peppers

[0074]

[0075] As shown in Table 2, the treatments in Examples 6 and 7 significantly increased the plant height, stem diameter, and above-ground and below-ground dry and fresh weight of chili peppers, indicating that the application of the microbial agent of the present invention can effectively promote the growth of chili peppers. Furthermore, the SPDA values ​​of Examples 6 and 7 were significantly higher than those of Comparative Example 1, indicating that the microbial agent treatment of the present invention can slow down the decomposition or transfer of chlorophyll in older leaves, improve photosynthesis in chili pepper leaves, and delay plant senescence.

[0076] Example 8

[0077] This embodiment provides a method for using the microbial agent from Example 5 in lettuce cultivation, as detailed below:

[0078] At the 5-6 leaf stage of lettuce, root application of a 500-fold dilution of the microbial agent described in Example 5 was initiated, with applications every 7 days for a total of 3 treatments. Each treatment used 200 ml of water per pot. The first root application used a 1000-fold dilution of compound fertilizer (N+P2O5+K2O, 20-20-20, Nanning Hanhe Biotechnology Co., Ltd.) as the stock solution, while the second and third treatments used tap water as the stock solution.

[0079] Example 9

[0080] This embodiment provides a method for using the microbial agent from Example 5 in lettuce cultivation, as detailed below:

[0081] At the 5-6 leaf stage of lettuce, root application of the microbial agent described in Example 5 at an 800-fold dilution was initiated, with one application every 7 days for a total of 3 treatments. Each treatment used 200 ml of water per pot. The first root application used a 1000-fold dilution of compound fertilizer (N+P2O5+K2O, 20-20-20, Nanning Hanhe Biotechnology Co., Ltd.) as the stock solution, while the second and third treatments used tap water as the stock solution.

[0082] Comparative Example 2

[0083] This comparative example provides a method for growing lettuce. The difference between this comparative example and Example 7 is that the first treatment in this comparative example only involves root application of compound fertilizer, while the second and third treatments only involve root application of tap water. All other operations are exactly the same.

[0084] Seven days after the third treatment, lettuce samples were collected from Examples 8 and 9 and Comparative Example 2. The collected lettuce was investigated for the following indicators. The investigation indicators and methods are shown in Table 4, and the investigation results are shown in Table 5.

[0085] Table 4 Survey Indicators and Methods

[0086]

[0087] Table 5. Results of the effects of different treatments on various indicators of lettuce.

[0088]

[0089] As shown in Table 4, the dry weight and fresh weight of the whole lettuce in Examples 6 and 7 were increased to varying degrees compared with Comparative Example 2. This indicates that the application of the microbial agent of the present invention can effectively promote lettuce growth and increase its yield.

[0090] Example 10

[0091] This embodiment measures the inhibitory effect of Microbacterium arborescens QHHQ09-2 on Fusarium graminearum in Example 1 above. The measurement method is as follows:

[0092] 1) Preparation of Microbacterium arborescens bacterial culture: Microbacterium arborescens QHHQ09-2 was inoculated on LB slant medium and cultured at 30℃ for 18h to obtain activated Microbacterium arborescens; the activated strain was inoculated into LB liquid medium and cultured at 30℃ and 160r / min for 18h to obtain seed culture; the seed culture was transferred to LB medium at a volume ratio of 1:60 and cultured at 30℃ and 150r / min for 2d to obtain Microbacterium arborescens bacterial culture.

[0093] 2) Cultivation of *Fusarium graminearum*: Under aseptic conditions, the *Fusarium graminearum* strain preserved in slant agar was inoculated into PDA solid medium and cultured at 28 ℃ for 7 days until a large number of mycelia grew, thus obtaining activated *Fusarium graminearum*. The activated *Fusarium graminearum* was then inoculated into PDA slant agar and cultured at 28 ℃ for 10 days. 10 mL of 0.05% Tween-80 (V / V) was added, and the mycelia were removed by filtration through sterile gauze. The spore concentration was adjusted to 1×10⁻⁶ using sterile water. 7 The concentration of cfu / mL was used to obtain a Fusarium graminearum spore solution, which was then stored at -20 ℃ for later use.

[0094] 3) Determination of the effect of Microbacterium arborescens QHHQ09-2 on the mycelial growth of Fusarium graminearum.

[0095] 10 μL of the described dendritic microbacterial culture was inoculated at four different locations 3 cm from the center of the PDA medium. Sterile water was used as the control group. The culture was incubated at 37 ℃ for 1 day. Subsequently, 10 μL of *Fusarium graminearum* spores was inoculated at the center of the medium. After incubation at 28 ℃ for 4 days, the colony diameter was measured using calipers (measured twice using the cross-hatching method, and the average value was taken), and the inhibition rate was calculated using the following formula:

[0096] Inhibition rate (%) = [(control colony diameter - treated colony diameter) / control colony diameter] × 100.

[0097] The measurement results are shown in Table 6 below.

[0098] Table 6. Effects of Microbacterium dendriticum QHHQ09-2 on the mycelial growth of Fusarium graminearum.

[0099]

[0100] As shown in Table 6, the *Microbacterium arborescens* QHHQ09-2 of this invention exhibits a high inhibition rate of 70.2% against the growth of *Fusarium graminearum*. This indicates that *Microbacterium arborescens* QHHQ09-2 of this invention has a good inhibitory effect on the mycelial growth of *Fusarium graminearum*.

[0101] 4) Determination of the effect of Microbacterium arborescens QHHQ09-2 on the spore germination of Fusarium graminearum in maize.

[0102] An equal volume of *Microbacterium dendriticum* bacterial suspension and *Fusarium graminearum* spore suspension were mixed and incubated at 28°C. Spore germination was examined at 12, 18, 24, 36, and 48 h, and the spore germination inhibition rate was calculated. Sterile water was used as a control instead of the bacterial suspension.

[0103] Spore germination inhibition rate / % = (control spore germination rate - treatment spore germination rate) / control spore germination rate × 100.

[0104] The measurement results are shown in Table 7 below.

[0105] Table 7. Effects of Microbacterium dendriticum QHHQ09-2 on spore germination of Fusarium graminearum in maize.

[0106]

[0107] As shown in Table 7, the inhibition rate of *Microbacterium arborescens* spore germination by *Microbacterium arborescens* QHHQ09-2 gradually increased with increasing culture time, showing a basically linear upward trend. At 48 hours of culture, the inhibition rate reached as high as 81.4%. This indicates that *Microbacterium arborescens* QHHQ09-2 of this invention has a good inhibitory effect on *Microbacterium arborescens* spore germination.

[0108] Fusarium graminearum (Gibberella zeae Schwein. Petch), in its asexual stage, is the main pathogen causing Fusarium head blight in maize in my country. It primarily contaminates cereals and produces fungal toxins, posing a threat to human and animal health. The *Gibberella graminearum* strain QHHQ09-2 of this invention exhibits good inhibitory effects on the mycelial growth and spore germination of *Gibberella graminearum*. Therefore, *Gibberella graminearum* strain QHHQ09-2 can be used in the preparation of drugs to control plant and animal diseases caused by *Gibberella graminearum*, for example, in the preparation of drugs to control Fusarium head blight in maize.

Claims

1. A Microbacterium arborescens QHHQ09-2, characterized in that, The preservation number of the tree-shaped microbacterium is CGMCC No. 33708.

2. A microbial agent comprising the tree-shaped microbacterium QHHQ09-2 according to claim 1.

3. The use of the Bifidobacterium dendriforme QHHQ09-2 of claim 1 or the bacterial agent of claim 2 in promoting plant growth, characterized in that, The plants are pepper and lettuce.

4. The use according to claim 3, wherein the compound is ###0002### The promoting plant growth comprises increasing plant height, stem diameter, dry weight and / or fresh weight.

5. The tree-shaped microbacterium QHHQ09-2 according to claim 1 or the microbial agent according to claim 2 for use in inhibiting Gibberella zeae.

6. Use according to claim 5, characterized in that, The inhibiting Gibberella zeae comprises inhibiting hyphal growth and spore germination of Gibberella zeae.

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