Acidovorax delphinii htf4 and application thereof

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to an acidovorax Delf4 strain and application thereof. The invention discloses an acidovorax delphinii htf4, and the preservation number of the acidovorax delphinii htf4 is CGMCC No.34123. The acidovorax Delf4 disclosed by the invention has the capability of efficiently synthesizing indoleacetic acid (IAA), and the IAA yield of 75.74 mg / L can be realized without optimization in an NB culture medium containing 5mM L-tryptophan, which is obviously higher than that of other wild strains. In addition, the acidovorax delphinii htf4 disclosed by the invention has a remarkable growth promoting effect on plants (such as pepper). Pot experiments show that the acidovorax Delfsii htf4 can significantly increase the number of effective leaves, plant height and biomass of pepper, and has important application value in the field of microorganisms, especially in the fields of microbial plant growth promoters and green synthesis of IAA (Indoleacetic Acid).

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and particularly relates to a strain of Acidovorax delftii HTF4 and an application thereof. Background Art

[0002] The plant auxin indoleacetic acid (IAA) is one of the core endogenous hormones that regulates plant growth and development. It is directly involved in physiological processes such as cell elongation, vascular bundle differentiation and stress response, and plays an important role in promoting root development, enhancing stress resistance and increasing crop yield.

[0003] At present, indoleacetic acid is often prepared by chemical synthesis. However, traditional chemical synthesis methods have the following defects: on the one hand, highly toxic reagents such as cyanide and formaldehyde are required in the synthesis process, and the residues are easy to contaminate soil and water bodies, causing long-term harm to the environment; on the other hand, the biological activity of chemically synthesized IAA is low. For example, the plant response efficiency of its isomers (such as β-IAA) is significantly lower than that of natural α-IAA. In addition, the chemical synthesis process is complicated and the purification cost is high, which seriously limits its agricultural application. Therefore, the development of a new type of IAA synthesis method that is efficient, green, and low-cost to meet the huge demand of agricultural production has become a technical problem that needs to be solved urgently. Summary of the Invention

[0004] The present invention aims to provide a strain of Acidovorax delftii htf4 and its application. The Acidovorax delftii htf4 of the present invention can synthesize IAA efficiently, greenly and at low cost to meet the needs of agricultural production.

[0005] The invention provides a Delftia acidovorans htf4 strain, with a preservation number of CGMCC No.34123.

[0006] The present invention also provides a microbial agent, wherein the active ingredient of the microbial agent includes the Acidovorax delftii HTF4 according to claim 1.

[0007] The microbial agent described in the above technical solution further comprises agriculturally acceptable auxiliary materials.

[0008] Preferably, the agriculturally acceptable excipient comprises one or more of an adsorbent material, a liquid suspension matrix and an embedding material.

[0009] The present invention also provides the use of the Acidovorax delftii HTF4 described in the above technical solution or the microbial agent described in the above technical solution in producing indoleacetic acid and / or promoting crop growth.

[0010] The present invention also provides a method for producing indoleacetic acid, comprising the following steps:

[0011] The Acidovorax delftii htf4 described in the above technical solution is fermented and cultured using a culture medium containing tryptophan to obtain a fermentation liquid containing indoleacetic acid.

[0012] Preferably, the concentration of tryptophan in the tryptophan-containing culture medium is 0.1 mM to 10 mM.

[0013] The present invention also provides a method for promoting plant growth, comprising the following steps:

[0014] The planted plants are treated with the Acidovorax delftii htf4 described in the above technical solution or the microbial agent described in the above technical solution.

[0015] The dosage of the microbial agent in the above technical solution is 20-50 mL / plant, and the OD of the microbial agent is 600 The value is 0.3~1.0.

[0016] The plant described in the above technical solution is pepper.

[0017] The present invention provides a strain of Acidovorans Delftii htf4, with a deposit number of CGMCC No. 34123. The Delftia acidovorans htf4 described in the present invention can efficiently synthesize indoleacetic acid (IAA), with an indoleacetic acid yield significantly higher than that of other wild strains, and can reach a yield level of 75.74 mg / L without complex process optimization. In addition, the Delftia acidovorans htf4 described in the present invention showed a significant growth-promoting effect in pepper pot tests, effectively increasing the number of leaves, plant height, and aboveground and belowground biomass of the plants, demonstrating excellent plant growth-promoting ability. At the same time, as a wild-type strain, it has the advantages of good genetic stability, simple culture conditions, and ease of large-scale production. Therefore, it has important application value in the fields of microbial plant growth promoters and green biosynthesis of indoleacetic acid.

[0018] Biological deposit information

[0019] Delftia acidovorans htf4, biologically named Delftia acidovorans, is deposited in the General Microbiology Center of the China Culture Collection Administration of Microorganisms, abbreviated as CGMCC. The deposit date is April 8, 2025, and the address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit number is CGMCC No. 34123. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments are briefly introduced below.

[0021] Figure 1 Schematic diagram of the colony morphology of Acidovorax delftii htf4;

[0022] Figure 2 This is a schematic diagram of the phylogenetic tree of Acidovorax delftii htf4;

[0023] Figure 3 is the standard curve of IAA;

[0024] Figure 4 Comparison of the Salkowski color reaction status of culture supernatants of different strains;

[0025] Figure 5 This is a comparison of the growth status of pepper seedlings 15 days after treatment with Delft Acidovorax htf4 and the control group seedlings. DETAILED DESCRIPTION

[0026] The present invention provides a strain of Delftia acidovorans (HTF4), with a deposit number of CGMCC No. 34123. The HTF4 strain has the function of efficiently synthesizing indoleacetic acid, with an indoleacetic acid yield significantly higher than that of similar wild strains, and can reach a yield level of 75.74 mg / L without complex process optimization. In addition, the HTF4 strain showed a significant growth-promoting effect in pepper pot experiments, effectively increasing the number of leaves, plant height, and aboveground and belowground biomass of the plants, demonstrating excellent plant growth-promoting ability. At the same time, as a wild-type strain, it has the advantages of good genetic stability, simple culture conditions, and ease of large-scale production. It has important application value in the field of microbial plant growth promoters and green biosynthesis of indoleacetic acid. The colonies of the HTF4 strain are round, light yellow, translucent, convex, with neat edges, a smooth and moist surface, and a diameter of about 1 to 2 mm. The sequence of 16S rDNA of Acidovorax delftii htf4 of the present invention is shown in SEQ ID NO: 1:

[0027]

[0028] The present invention also provides a microbial agent, the active ingredients of which include the Acidovorax delftii HTF4 described in the above technical solution.

[0029] As an embodiment, the effective viable count of the microbial agent of the present invention is ≥ 10 billion CFU / g. As an embodiment, the OD of the microbial agent of the present invention is 600 As another embodiment, the OD of the microbial agent of the present invention is 0.3 to 1.0. 600 The value is 0.3. In one embodiment, the microbial agent of the present invention further comprises an agriculturally acceptable excipient. In one embodiment, the agriculturally acceptable excipient of the present invention comprises one or more of an adsorbent material, a suspending matrix, and an embedding material. In one embodiment, the adsorbent material of the present invention may be a solid culture medium. In one embodiment, the suspending matrix of the present invention may be a liquid culture medium.

[0030] As an embodiment, the preparation method of the microbial agent of the present invention includes the following steps: inoculating Delft Acidovorax HTF4 into a culture medium for culturing to obtain a Delft Acidovorax HTF4 culture; the culture medium includes a culture medium containing tryptophan; centrifuging the Delft Acidovorax HTF4 culture, collecting the bacterial cells and resuspending them to obtain the microbial agent.

[0031] As an embodiment, the culture temperature of the present invention may be 20°C to 37°C, and the culture time may be 24 to 96 hours. As another embodiment, the culture temperature of the present invention may be 28°C, and the culture time may be 24 hours. As an embodiment, the culture of the present invention may be in dark conditions, and the dark conditions may prevent the IAA with photooxidation characteristics from being oxidized and decomposed. As an embodiment, the culture speed of the present invention may be 150 to 200 r / min. As another embodiment, the culture speed of the present invention may be 180 r / min. As an embodiment, the culture medium of the present invention may be LB culture medium and / or NB culture medium. As an embodiment, the resuspension of the present invention includes: resuspending with sterile water and adjusting the OD 600 =0.3~1.0. As another embodiment, the resuspension of the present invention comprises: resuspension with sterile water and adjusting OD 600 =0.3. As an embodiment, the centrifugal speed of the present invention is 5000-10000 rpm, and the time is 5-10 min. As an embodiment, the centrifugal speed of the present invention is 8000 rpm, and the time is 5 min.

[0032] The present invention also provides the use of the Acidovorax delftii HTF4 described in the above technical solution or the microbial agent described in the above technical solution for producing indoleacetic acid and / or promoting crop growth and development. As an embodiment, the method for promoting plant growth described in the present invention can significantly improve plant growth indicators, including the number of effective leaves, plant height, above-ground dry weight, and below-ground dry weight. The Acidovorax delftii HTF4 described in the present invention or the microbial agent can efficiently synthesize indoleacetic acid and promote crop growth and development. Even without process optimization, the indoleacetic acid (IAA) yield can reach 75.74 mg / L, significantly higher than other control strains.

[0033] The present invention also provides a method for producing indoleacetic acid, comprising the following steps:

[0034] The Acidovorax delftii htf4 described in the above technical solution is fermented and cultured using a culture medium containing tryptophan to obtain a fermentation liquid containing indoleacetic acid.

[0035] The present invention utilizes a culture medium containing tryptophan to ferment the Acidovorax delftii HTF4 described in the above technical solution to produce a fermentation broth; the fermentation broth contains indoleacetic acid. In one embodiment, the tryptophan concentration in the culture medium containing tryptophan is 0.1 to 10 mM. In another embodiment, the tryptophan concentration in the culture medium containing tryptophan is 5 mM. In another embodiment, the culture medium containing tryptophan can be NB culture medium containing tryptophan. In one embodiment, the fermentation culture temperature can be 20 to 37°C, the rotation speed can be 150 to 200 rpm, and the duration can be 24 to 96 hours. In another embodiment, the fermentation culture temperature can be 28°C, the rotation speed can be 175 rpm, and the duration can be 96 hours.

[0036] In one embodiment, after obtaining the fermentation broth, the present invention centrifuges the fermentation broth and collects the supernatant to obtain indoleacetic acid. In one embodiment, the centrifugation speed of the present invention can be 5000-10000 rpm, and the time can be 5-20 minutes. In one embodiment, the centrifugation speed of the present invention can be 8000 rpm, and the time can be 10 minutes.

[0037] The present invention also provides a method for promoting plant growth, comprising the following steps:

[0038] The planted plants are treated with the Acidovorax delftii htf4 described in the above technical solution or the microbial agent described in the above technical solution.

[0039] As an embodiment, the plant of the present invention can be pepper. As an embodiment, the OD of the microbial agent of the present invention can be 600 The value is 0.3 to 1.0; the application amount of the microbial agent is 20 to 50 mL per plant. As another embodiment, the OD of the microbial agent of the present invention is 600 The value is 0.3; the application amount of the microbial agent is 30 mL / plant.

[0040] In one embodiment, the treatment method of the present invention includes root irrigation. In one embodiment, the root irrigation is performed twice. In one embodiment, the interval between two consecutive root irrigations is 6 days. In one embodiment, sterile water is added 3 days after the first root irrigation.

[0041] To further illustrate the present invention, the following describes in detail the strain of Acidovorax delftii htf4 and its applications provided by the present invention in conjunction with the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0042] Example 1

[0043] Isolation and identification of strains

[0044] (1) Isolation of strains

[0045] 10 g of litter and leaf humus was added to 90 mL of sterilized ultrapure water, shaken at 30°C and 180 rpm for 30 min, mixed thoroughly and then allowed to stand. 1 mL of the suspension was taken for gradient dilution. 70 μL of the diluted suspension was spread on NB solid medium and cultured in a 30°C incubator for 5 days. The colonies on the NB solid medium were streaked and purified repeatedly for 5 to 8 times. The purified single strain was cultured on the NB solid medium to obtain the following results: Figure 1 The colony shown is numbered htf4. The colony is round, light yellow, translucent, raised, with neat edges, a smooth and moist surface, and a diameter of about 1 to 2 mm.

[0046] (2) Identification of strains

[0047] The genomic DNA of strain htf4 was extracted, and according to the method of using Novagen 2×TaqPlus MasterMixⅡ DNA polymerase, the extracted genomic DNA was used as the amplification template and the 16S rDNA universal primer was used as the amplification primer. The sequences were: upstream primer (27F): 5'-AGAGTTTGATCCTGGCTCAG-3' (SEQ ID NO.2), downstream primer (1492R): 5'-TACGGTTACCTTGTTACGACTT-3' (SEQ ID NO.3).

[0048] The PCR products were sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing. The sequencing results of the strain 16SrDNA (SEQID NO.1) were compared in the NCBI and EzBioCloud databases, and strains with higher sequence similarity were selected to construct a phylogenetic tree using MEGA 11, as shown in Figure 1. Figure 2 The results showed that the strain htf4 was a Delftia acidovorans, which was biologically classified as Delftia acidovorans and deposited as a biological deposit.

[0049] Example 2

[0050] A microbial agent

[0051] The preserved htf4 strain was streaked on NA solid medium and cultured in the dark at 28°C for 24 hours. A typical single colony was picked and streaked for a second time for 24 hours to ensure the activity of the strain. A typical colony was inoculated into NB liquid medium and cultured at 28°C and 180 rpm for 24 hours. After the culture was completed, the cells were collected by centrifugation at 8000 rpm for 5 minutes, resuspended in sterile water and adjusted to OD 600 =0.3 working concentration. The microbial agent is obtained.

[0052] Example 3

[0053] Functional verification test of the plant growth hormone indoleacetic acid produced by Acidovorax delftii HTF4

[0054] 1. Comparative strains: 11 sample strains isolated from the same soil as htf4, as shown in Table 1. The identification process was the same as that of A. delftii htf4 in Example 1.

[0055] 2. Materials and equipment: Microplate reader, shaking table, 100 mL conical flask, medium-speed centrifuge, 50 mL centrifuge tube, pipette, NB nutrient broth, sterile distilled water, indoleacetic acid, FeCl3·5H2O, 98% (w / w) H2SO4.

[0056] 3. Experimental steps:

[0057] Take a 100 mL conical flask, add NB medium containing 5 mM L-tryptophan, and inoculate 100 μL of OD 600 = 0.3 bacterial suspension, cultured at 28 ° C, 175 rpm shaking conditions for 96 h;

[0058] The fully grown culture was centrifuged at 8000 rpm for 10 min, and the supernatant was collected;

[0059] Take 2 mL of the supernatant and react with 4 mL of Salkowski reagent (0.5 M FeCl3 / 98% H2SO4 / distilled water at a volume ratio of 1:30:50) for 30 min until the solution turns pink;

[0060] The absorbance of the reaction solution was measured at a wavelength of 530 nm using a microplate reader;

[0061] The culture medium without inoculation of the strain was used as blank control;

[0062] Preparation of standard curve: Take 0.1 mg / mL indoleacetic acid standard solution 0, 0.4, 0.8, 1.2, 1.6, 2.0 mL, make up to 2 mL with distilled water, add 4 mL of Salkowski reagent, react for 30 min, measure the absorbance at 530 nm, and obtain the data shown in Table 1. Draw the standard curve of IAA as shown in Figure 3 shown.

[0063] Table 1 Standard curve data

[0064]

[0065] The concentration of IAA produced by each group of strains was calculated according to the standard curve. The calculation formula is y = 3.8391x + 0.0888, where R 2 =0.9842, and the calculated results of the IAA concentration of each group of strains are shown in Table 2. Three replicates were set for each sample, and single-factor ANOVA analysis was performed using SPSS26 software to compare the significance of the mean differences.

[0066] Table 2 IAA concentration results of each group of strains

[0067]

[0068]

[0069] 4. Experimental results:

[0070] The results of different strains producing plant growth hormone indoleacetic acid are shown in Table 3, and the color development results of the strain culture supernatant with Salkowski reagent are shown in Figure 3 As shown. It can be seen that Acidovorax delftii htf4 can efficiently synthesize indoleacetic acid (yield of 75.74 mg / L) without process optimization, and has a strong ability to produce indoleacetic acid. It is worth noting that the color depth of the solution in the Salkowski color reaction is positively correlated with the IAA content, and the reaction solution of the htf4 strain shows the darkest color ( Figure 4), which is highly consistent with the quantitative detection results (Table 1). Based on its excellent IAA production ability, this strain can be used as an excellent strain for industrial production of IAA and can also be developed into a microbial agent with growth-promoting function.

[0071] Table 3 Results and identification information of different strains producing plant hormone indoleacetic acid

[0072]

[0073]

[0074] Different lowercase letters indicate significant differences among different bacterial solution treatment groups (P<0.05).

[0075] Example 4

[0076] Experiment on promoting the growth of pepper seedlings by Acidovorax delftii HTF4

[0077] 1. Preparation of experimental materials

[0078] Test soil was obtained from ordinary field soil from the Qingdao Experimental Base of the Tobacco Research Institute of the Chinese Academy of Agricultural Sciences in Jimo District, Qingdao. After natural air drying, the soil was filtered through a 0.25 mm pore size and then placed in standard 9 cm diameter pots at a rate of 110 g per pot.

[0079] 2. Experimental treatment

[0080] Pepper seedlings that have grown to two true leaves were selected and, after strict screening (selecting pepper seedlings of similar growth and size), two plants were planted in prepared pots. Each pot was watered with 30 mL of sterilized water to ensure normal acclimatization and initial growth of the seedlings.

[0081] 3. Bacterial Liquid Preparation

[0082] The preserved htf4 strain was streaked on NA solid medium and cultured in the dark at 28°C for 24 hours. A typical single colony was picked and streaked for a second time for 24 hours to ensure the activity of the strain. A typical colony was inoculated into NB liquid medium and cultured at 28°C and 180 rpm for 24 hours. After the culture was completed, the cells were collected by centrifugation at 8000 rpm for 5 minutes, resuspended in sterile water and adjusted to OD 600 = working concentration of 0.3.

[0083] 4. Processing Implementation

[0084] After planting, the seedlings were first watered with 30 mL of the prepared bacterial suspension. Three days later, 30 mL of sterile water was added. Six days after planting, a second watering with the same concentration and dosage as the initial treatment was performed. Thereafter, watering with sterile water was maintained every three days. The control group was watered with the same amount of sterile water throughout the entire process, and all other management measures remained the same as those for the treatment group.

[0085] 5. Index determination

[0086] Pepper seedlings treated 15 days later and the control group seedlings Figure 5 As shown, various growth indicators were measured 15 days after treatment, including counting the number of effective leaves and measuring plant height. The plants were then divided into aboveground and underground parts, dried at 65°C to constant weight, and the dry matter weight of each part was measured. The specific results are shown in Table 4.

[0087] Table 4 Results of various growth index measurements of pepper seedlings (15 days)

[0088] Number of effective leaves Plant height / cm Above ground dry weight / g Underground dry weight / g CK 4±1.17b 7.90±0.6b 0.08±0.006b 0.053±0.006b htf4 6±0.75a 10.67±1.03a 0.107±0.011a 0.081±0.004a

[0089] 6. Test results

[0090] Table 2 shows that, compared with the control group, treatment with the htf4 strain demonstrated significant growth-promoting effects: the number of effective leaves increased by 50%, plant height increased by 35.06%, aboveground dry weight increased by 33.75%, and underground dry weight increased by 52.83%. All indicators showed significant differences (p < 0.05).

[0091] Based on the above, it can be seen that the Acidovorax Delftii htf4 provided by the present invention can produce high amounts of indoleacetic acid, promote plant growth, and has important application value in agricultural production.

[0092] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creative work, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. A strain of Delftia acidovorans htf4, with a deposit number of CGMCC No. 34123.

2. A microbial agent, characterized in that: The active ingredient of the microbial agent includes the Acidovorax delftii htf4 according to claim 1.

3. The microbial agent according to claim 2, characterized in that The microbial agent further comprises agriculturally acceptable adjuvants.

4. The microbial agent according to claim 2, characterized in that The agriculturally acceptable auxiliary material includes one or more of an adsorption material, a liquid suspension matrix and an embedding material.

5. Use of the Acidovorax delftii htf4 according to claim 1 or the microbial agent according to any one of claims 2 to 4 in producing indoleacetic acid and / or promoting crop growth.

6. A method for producing indoleacetic acid, characterized in that: The following steps are involved: The Acidovorax delftii htf4 according to claim 1 is fermented and cultured using a culture medium containing tryptophan to obtain a fermentation liquid containing indoleacetic acid.

7. The method according to claim 6, characterized in that The concentration of tryptophan in the tryptophan-containing culture medium is 0.1-10 mM.

8. A method for promoting plant growth, characterized in that: The following steps are involved: The planted plants are treated with the Acidovorax delftii htf4 according to claim 1 or the microbial agent according to any one of claims 2 to 4.

9. The method according to claim 8, characterized in that The dosage of the microbial agent is 20-50 mL / plant, and the OD of the microbial agent is 20-50 mL / plant. 600 The value is 0.3~1.

0.

10. The method according to claim 8 or 9, characterized in that The plant is pepper.

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