Leclercia adecarboxylata strain bp-4 and application thereof

Abstract

The present application relates to the field of microbial technology, in particular to a non-decarboxylation leclercia BP-4 and its application. The non-decarboxylation leclercia (Leclercia adecarboxylata) BP-4 is preserved in China typical culture preservation center on June 24, 2026, and the preservation number is CCTCC NO: M20261419. The non-decarboxylation leclercia BP-4 provided in the present application is isolated from the rhizosphere soil of white and has high efficient phosphorus solubilization effect and excellent IAA production, iron carrier production and nitrogen fixation capacity, and also has potassium solubilization effect, and can significantly promote the growth of white. The present application provides high-quality strain resources and reliable technical support for the development of new microbial growth promoting agent and the construction of white green ecological cultivation system, and has good industrial application prospect.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a non-decarboxylated Leucobacterium BP-4 and its applications. Background Technology

[0002] Bletilla striata (Thunb.) Reichb.f. is a perennial medicinal plant belonging to the genus Bletilla in the family Orchidaceae. Its dried tuber is a traditional and widely used Chinese medicinal material, rich in active substances such as Bletilla polysaccharides, bibenzyl groups, and phenanthrene. It has the effects of astringing and stopping bleeding, reducing swelling and promoting tissue regeneration, and is widely used in the fields of medicine, cosmetics, and health food.

[0003] The raw materials for medicinal Bletilla striata mainly come from artificial cultivation. The karst region in southwestern my country is the main production area of ​​Bletilla striata. The total phosphorus content of the soil in this region is higher than that in non-karst regions. However, the available phosphorus in the soil is easily combined with calcium ions to form insoluble calcium phosphate salts, resulting in a lack of available phosphorus in the soil. At present, there are the following production shortcomings in field cultivation: (1) Bletilla striata is a shallow-rooted orchid with weak root absorption capacity and high dependence on available phosphorus in the soil. However, the phosphorus in the soil is mostly in inorganic / organic combined forms such as insoluble calcium phosphate, dicalcium phosphate, calcium phytate, and lecithin, which cannot be directly absorbed and utilized by the roots of Bletilla striata; (2) Applying phosphate fertilizer from external sources is still the main way to supplement phosphorus in Bletilla striata cultivation. However, the Ca in karst soil is high. 2+ Fe 3+ Al 3+ Plasma is easy to fix with phosphorus, resulting in low phosphorus fertilizer utilization. Excessive phosphorus fertilizer not only increases planting costs but also easily causes environmental pollution, which does not meet the requirements of green and standardized planting of Chinese medicinal materials. (3) The existing Bletilla striata growth-promoting microbial resources are relatively simple. At present, research and application are mostly focused on orchid mycorrhizal fungi such as sclerotium and rhizoctonia. Fungi have problems such as long propagation cycle, high industrial fermentation cost, and poor colonization stability in the field environment. (4) In view of the scarcity of Bletilla striata rhizosphere-adapted phosphorus-soluble growth-promoting bacteria resources, there is a lack of dedicated functional strains that can soluble phosphorus and directionally increase the yield of Bletilla striata.

[0004] Based on this, the present invention provides a non-decarboxylating Leucobacterium BP-4 isolated from the rhizosphere soil of Bletilla striata, which can efficiently dissolve inorganic insoluble phosphorus, has excellent IAA production, iron carrier production and nitrogen fixation capabilities, and also has potassium solubilizing effects, and can significantly promote the growth of Bletilla striata. It aims to solve the above-mentioned technical problems, provide high-quality strain resources for the development of new microbial growth-promoting agents, and provide technical support for the construction of an ecological green cultivation system for Bletilla striata. Summary of the Invention

[0005] The purpose of this invention is to provide a non-decarboxylated Leucobacterium BP-4 strain and its applications.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: The non-decarboxylating Leclercia BP-4 described in this invention is named Leclercia adecarboxylata BP-4 and was deposited at the China Center for Type Culture Collection on June 24, 2026, with accession number CCTCC NO: M20261419.

[0007] The application of the non-decarboxylated Leucobacterium BP-4 or its culture in phosphorus solubilization according to the present invention.

[0008] Preferably, the application of the non-decarboxylated Leuciscus BP-4 or its culture in phosphorus solubilization according to the present invention specifically involves: co-culturing the non-decarboxylated Leuciscus BP-4 bacterial solution with inorganic or organic phosphorus to dissolve insoluble phosphorus; wherein the co-culturing conditions are specifically as follows: the culture medium is NBRIP inorganic phosphorus liquid medium or Monkina organic phosphorus liquid medium, the phosphorus source of the culture medium is selected from any one of calcium phosphate, dicalcium phosphate, aluminum phosphate, ferric phosphate, lecithin, and calcium phytate, and the phosphorus source concentration is 2.5-12.5 g / L; the initial pH of the culture medium is 4.0-8.0; OD 600 The inoculation amount of non-decarboxylated Lechnerella BP-4 bacterial suspension with a value of 0.8 is 1%-5%.

[0009] Further preferably, in the application of the non-decarboxylated Leucella BP-4 or its culture in phosphorus solubilization, the co-culture conditions are specifically as follows: the culture medium is NBRIP inorganic phosphorus liquid medium, the phosphorus source is dicalcium phosphate, and the phosphorus source concentration is 5 g / L; the initial pH of the culture medium is 7.0; OD 600 The inoculum size of the non-decarboxylated Lechner's BP-4 bacterial suspension with a value of 0.8 was 2%.

[0010] The application of the non-decarboxylated Leucobacterium BP-4 or its culture in potassium solubilization according to the present invention.

[0011] The application of the non-decarboxylated Leucobacterium BP-4 or its culture in the production of IAA as described in this invention.

[0012] The application of the non-decarboxylated Leucobacterium BP-4 or its culture in siderogenic carriers as described in this invention.

[0013] The application of the non-decarboxylating Leucobacter BP-4 or its culture in nitrogen fixation as described in this invention.

[0014] The present invention relates to the application of the non-decarboxylated Leucobacterium BP-4 or its culture in promoting plant growth or in the preparation of microbial agents or bio-organic fertilizers that promote plant growth.

[0015] Preferably, in the application of the non-decarboxylated Leucobacterium BP-4 or its culture in promoting plant growth or in the preparation of microbial agents or bio-organic fertilizers that promote plant growth, the plant is Bletilla striata.

[0016] The beneficial effects of this invention are: 1. This invention, through qualitative and quantitative screening, screened a non-decarboxylating Leuciscus BP-4 strain with highly efficient phosphorus-solubilizing activity from the rhizosphere soil of Bletilla striata. A series of single-factor experiments determined the optimal phosphorus-solubilizing conditions for this strain to be: calcium hydrogen phosphate as the phosphorus source; phosphorus source concentration of 5 g / L; inoculum size of 2%; and initial pH of the culture medium of 7. Under these optimal phosphorus-solubilizing conditions, the phosphorus-solubilizing capacity of non-decarboxylating Leuciscus BP-4 can reach 671.0-681.4 mg / L.

[0017] 2. The non-decarboxylating Leucella BP-4 provided by this invention possesses excellent IAA production, nitrogen fixation, and siderophore production capabilities, while also exhibiting potassium solubilization. Quantitative results show that strain BP-4 secretes 83.73±4.87 μg / mL of IAA; its potassium solubilization is 52.60±0.80 μg / mL, significantly increased by 4.08% compared to the control; and its nitrogen fixation reaches 0.041±0.005 mg / mL, significantly increased by 371.4% compared to the control. Quantitative results of siderophore production show that the IAA content of the blank control is significantly higher than that of the control. 630 The value was 0.430, while the three replicates A of strain BP-4 630 The SU values ​​of the iron carriers were significantly reduced (0.293-0.257), and the SU values ​​of the iron carriers were all above 31%.

[0018] 3. Through tissue culture and pot experiments, it was verified that the non-decarboxylated Lucidis bacterium BP-4 provided by this invention can significantly promote the growth of Bletilla striata. In tissue culture, the inoculation treatment with non-decarboxylated Lucidis bacterium BP-4 showed a highly significant growth-promoting effect on both above-ground and overall growth indicators of Bletilla striata: plant height, total biomass, number of leaves, and leaf length increased by 77.58%, 71.95%, 87.39%, and 82.33%, respectively, compared with the control group. Pot experiments showed that in sterilized soil (SS), inoculation with BP-4 increased the total biomass, tuber fresh weight, and leaf length of Bletilla striata, with the tuber fresh weight increasing by 37.46%; in unsterilized soil (NS), after inoculation with BP-4, the plant height, leaf length, and tuber fresh weight of Bletilla striata all increased significantly compared with the uninoculated control group, by 8.67%, 12.28%, and 19.82%, respectively, and all growth indicators were the highest among all treatment groups.

[0019] Preservation Information: Strain name: LeclerciaadecarboxylataBP-4; Depository: China Center for Type Culture Collection; Address of collection: No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province; Deposit date: June 24, 2026; Accession number: CCTCCNO: M20261419. Attached Figure Description

[0020] Figure 1 The results show the quantitative screening of phosphate-solubilizing bacteria; Figure 2 The colony morphology of strain BP-4 and the results of Gram staining microscopy are shown. Figure 3 A phylogenetic tree of strain BP-4 constructed based on the 16S rRNA gene sequence; Figure 4 Qualitative results for IAA production, potassium solubilization, nitrogen fixation, and siderophore production of strain BP-4; Figure 5 Quantitative results of IAA production, potassium solubilization, and nitrogen fixation of strain BP-4; Figure 6 The results of phosphate solubilization optimization for strain BP-4; Figure 7 The phosphorus solubility of strain BP-4 under optimal phosphorus solubility conditions; Figure 8 The effect of strain BP-4 on the growth of Bletilla striata in IPMS; Figure 9 Flowchart of the potted plant experiment; Figure 10 The effect of strain BP-4 on the growth of Bletilla striata in a pot experiment. Detailed Implementation

[0021] The technical solution of the present invention will be described in detail below with reference to specific embodiments. The following embodiments are for explanation and illustration only, and do not constitute a limitation on the technical solution of the present invention.

[0022] Example 1

[0023] Leclercia adecarboxylata BP-4, a non-decarboxylating Leclercia, was deposited at the China Center for Type Culture Collection on June 24, 2026, with accession number CCTCC NO: M20261419.

[0024] Example 2

[0025] The non-decarboxylated Leucobacterium BP-4 from Example 1 was used for phosphorus solubilization, specifically as follows: Non-decarboxylating Leucella BP-4 was co-cultured with inorganic phosphorus to dissolve insoluble phosphorus. The co-culture conditions were: NBRIP inorganic phosphorus liquid medium, with dicalcium phosphate as the phosphorus source at a concentration of 5 g / L; initial pH of the medium was 7.0; OD... 600 The inoculum size of the non-decarboxylated Lechner's BP-4 bacterial suspension with a value of 0.8 was 2%.

[0026] Example 3

[0027] The non-decarboxylated Leucobacterium BP-4 from Example 1 was used for phosphorus solubilization, specifically as follows: Non-decarboxylating Leucella BP-4 was co-cultured with inorganic phosphorus to degrade insoluble phosphorus. The co-culture conditions were as follows: the culture medium was NBRIP inorganic phosphorus liquid medium, the phosphorus source was dicalcium phosphate at a concentration of 2.5 g / L, and the initial pH of the medium was 4.0; OD 600 The inoculum size of the non-decarboxylated Lechner's BP-4 bacterial suspension with a value of 0.8 was 1%.

[0028] Example 4

[0029] The non-decarboxylated Leucobacterium BP-4 from Example 1 was used for phosphorus solubilization, specifically as follows: Non-decarboxylating Leucella BP-4 was co-cultured with inorganic phosphorus to degrade insoluble phosphorus. The co-culture conditions were as follows: the culture medium was NBRIP inorganic phosphorus liquid medium, the phosphorus source was dicalcium phosphate at a concentration of 12.5 g / L, and the initial pH of the medium was 8.0; OD 600 The inoculum size of the non-decarboxylated Lechner's BP-4 bacterial suspension with a value of 0.8 was 5%.

[0030] Example 5

[0031] The non-decarboxylated Leucobacterium BP-4 from Example 1 was used for IAA production, specifically as follows: OD 600 BP-4 bacterial culture with a pH of 0.8 was inoculated at a rate of 1% into LB liquid medium supplemented with 100 mg / L L-tryptophan and cultured with shaking at 28°C and 150 r / min for 2 days to produce IAA.

[0032] Example 6

[0033] The non-decarboxylating Lekkelsen BP-4 from Example 1 was used for potassium solubilization, specifically as follows: OD 600 BP-4 bacterial culture with a pH of 0.8 was inoculated into potassium-solubilizing liquid medium at an inoculation rate of 1% and cultured at 28℃ and 180r / min for 3-5 days to solubilize potassium.

[0034] Example 7

[0035] The non-decarboxylated Lekkelsen BP-4 from Example 1 was used as a siderogenic carrier, specifically as follows: OD 600 BP-4 bacterial suspension with a pH of 0.8 was inoculated into MKB liquid medium at a 1% inoculation rate and cultured on a shaker at 28°C and 180 r / min for 2-3 days to produce siderophores.

[0036] Example 8

[0037] The non-decarboxylating Leucobacter BP-4 from Example 1 was used for nitrogen fixation, specifically as follows: OD 600 BP-4 bacterial culture with a value of 0.8 was inoculated into Ashby liquid medium at an inoculation rate of 1% and cultured at 28℃ and 180r / min for 4 days to fix nitrogen.

[0038] Example 9

[0039] The non-decarboxylated Leucobacterium BP-4 from Example 1 was used to promote the growth of Bletilla striata, specifically as follows: Strain strain BP-4 was inoculated into LB liquid medium and cultured at 28℃ and 150 r / min for 24 h with constant temperature shaking. The bacterial concentration was then adjusted to 1×10⁻⁶ with sterile water. 8 CFU / mL was used to obtain the fermentation broth of strain BP-4; 10 mL of the fermentation broth of strain BP-4 was inoculated around Bletilla striata, and inoculated once every 15 days for a total of 90 days to promote the growth of Bletilla striata.

[0040] To further verify the reliability of the present invention and select the optimal solution, the inventors conducted a series of experiments, as follows: 1. Isolation, screening and identification of bacterial strains 1.1 Isolation of strains The test material was collected from the rhizosphere soil of Bletilla striata planted in karst mountain soil 90 days after planting. After removing the top 2-3 cm of soil and weeds, the Bletilla striata was uprooted. Large clumps of soil attached to the roots were gently shaken off, leaving only about 1-2 mm of soil tightly attached to the roots. The soil was then rapidly transported to the laboratory at 4℃. The rhizosphere soil was gently brushed off with a soft brush, and the collected rhizosphere soil was evenly mixed and divided into 3 portions. These portions were then flash-frozen in liquid nitrogen and stored at -80℃.

[0041] Soil suspension: Add Bletilla striata rhizosphere soil and water to a 250 mL sterile Erlenmeyer flask at a ratio of 1:9 (m / v), shake at 28℃ for 30 min, let stand for 3 min, and take the supernatant as the soil suspension. Dilute sequentially to a concentration of 10. -1 -10 -6 The suspension was spread into Monkina organic / inorganic phosphorus medium and incubated at 28°C in the dark for 2-5 days. The growth of the colonies was then observed.

[0042] Isolation and purification of bacterial strains: Observe the growth of colonies in the soil suspension spread on the culture medium. Colonies with phosphate-solubilizing zones are picked out with a sterile toothpick and inoculated into new LB agar plates for culture. Repeat the inoculation until single colonies appear. Inoculate the isolated rhizosphere phosphate-solubilizing bacteria into LB agar plates and incubate at 28°C for 2-3 days before use.

[0043] 1.2 Screening of strains 1.2.1 Qualitative Screening Representative strains obtained from the initial plate screening were activated on solid culture medium, then inoculated into LB liquid medium and cultured at 28℃ and 180 rpm for 24 h to reach the logarithmic growth phase. Their OD values ​​were then determined. 600 Adjust to 0.8. Then, use a pipette to transfer 10 μL of bacterial culture onto a Monkina organophosphate medium plate, repeating the process three times. Incubate at 28°C for 7 days, measure the diameter of the phosphate-solubilizing zone D and the diameter of the bacterial strain d, and calculate the phosphate-solubilizing index SI (SI = D / d) of the phosphate-solubilizing strain.

[0044] The results are shown in Table 1. The results showed that the phosphorus solubility index (SI) of each phosphate-solubilizing bacterium ranged from 1.24 to 3.15. Among them, 12 strains had an SI value > 2.0, with BP-4 having the highest SI value, followed by BP-2, BP-5, BP-11, BP-13, BP-14, BP-15, BP-18, BP-21, BP-75, BP-19, and BP-34.

[0045] .

[0046] 1.2.2 Quantitative Screening The strains obtained from the initial plate screening (SI > 2.0) were inoculated into LB liquid medium and cultured at 28°C and 180 rpm for 24 h. Their OD values ​​were then analyzed. 600 Adjust the pH to 0.8. Add the prepared phosphate-solubilizing bacteria suspension to 50 mL of NBRIP liquid medium at a 2% inoculation rate and incubate. Use an equal volume of sterile LB liquid medium as a control. Perform triplicate incubation, incubating at 28℃ and 150 rpm for 7 days. After incubation, centrifuge at 10000 rpm and 4℃ for 10 min. Determine the available phosphorus content of the supernatant using the sodium bicarbonate extraction-molybdenum antimony spectrophotometric method.

[0047] The results are as follows Figure 1 As shown in the figure. Twelve bacterial strains with an SI value greater than 2.0 were activated and inoculated into NBRIP liquid medium for culture. Their available phosphorus content was measured, with soluble phosphorus ranging from 46.53 to 300.86 mg / L. Among them, BP-4 had the highest soluble phosphorus content (300.86 ± 1.78 mg / L), significantly higher than the other 11 strains (46.53–215.63 mg / L), which is consistent with the qualitative results.

[0048] 1.3 Identification of strain BP-4 1.3.1 Morphological identification of strain BP-4 After purification, strain BP-4 was streaked onto LB solid medium for observation. The results are as follows: Figure 2As shown in the figure. The results showed that BP-4 colonies were milky white, with a moist and smooth surface. Single colonies were slightly convex, translucent, and had neat edges. Gram staining showed that they were Gram-negative bacteria.

[0049] 1.3.2 Molecular biological identification of strain BP-4 DNA template preparation: Single colonies of BP-4 were picked and cultured in LB medium until the logarithmic growth phase. The bacterial cells were then collected, and bacterial DNA was extracted using a bacterial sample genomic DNA extraction kit (Beijing Lanjieke Technology Co., Ltd.). Using the extracted DNA as a template, amplification was performed using universal primers 27F and 1492R. Primer information is shown in Table 2. The PCR reaction system and amplification program are shown in Tables 3 and 4, respectively. The cycle number was 34, yielding the amplified products.

[0050] ; ; ; Electrophoretic detection of PCR amplification products: Electrophoresis conditions were as follows: 1% agarose gel (containing 0.2 g agarose and 20 mL TAE buffer), 1X TAE electrophoresis buffer, 360 V for 12 min. The PCR product was loaded in a volume of 1 μL, mixed with 1 μL loading buffer, and then spotted. Results were observed under UV light using a gel imaging system. The length of the amplified fragment was determined using a 10000 DNA Marker as the standard molecular weight reference. The amplified product band should be located at 1000-1100 bp of the standard.

[0051] PCR product purification and sequencing were performed by Sangon Biotech (Shanghai) Co., Ltd.

[0052] Phylogenetic tree construction: Log in to NCBI, perform BLAST homology comparison between the 16S rRNA sequence obtained from sequencing (as shown in SEQ ID NO.1) and sequences in the GenBank database, obtain the representative strain with the highest similarity, record its accession number and the most similar strain, and download the rDNA ITS sequences of similar strains. Use MEGA 11 to construct a phylogenetic tree (as shown in SEQ ID NO.1). Figure 3 As shown in Table 5, the strains most similar to BP-4 are listed below.

[0053] ; A 1500-1600 bp fragment was amplified from the strain's genomic DNA using primers 27F and 1492R. Sequencing was performed, and the results were aligned using BLAST in GenBank. The results showed that strain BP-4 had high sequence similarity to strains in the *Leclercia* genus. Therefore, the sequence of a reference strain of *Leclercia* was downloaded from GenBank for phylogenetic analysis. In the constructed phylogenetic tree, BP-4 clustered with multiple strains of *Leclercia adecarboxylata*, forming a terminal branch with 98% support. Base similarity comparisons showed that BP-4 shared over 99% sequence similarity with strains of the *Leclercia* genus. Therefore, based on both morphological and molecular biological characteristics, strain BP-4 was preliminarily identified as *Leclercia adecarboxylata*.

[0054] SEQ ID NO.1 (16S rRNA sequence of strain BP-4): CATGTATACAAGTGGTAAGCGCCCTCCCGAAGGTTAAGCTACCTACTTCTTTTGCAACCCACTCCCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGTAGCATTCTGATCTACGATTACTAGCGATTCCGACTTCATGGAGTCGAGTTGCAGACTCCAATCCGGACTACGACGCACTTTATGAGGTCCGCTTGCTCTCGCGAGGTCGCTTCTCTTTGTATGCGCCATTGTAGCACGTGTGTAGCCCTACTCGTAAGGGCCATGATGACTTGACGTCATCCCCACCTTCCTCCAGTTTATCACTGGCAGTCTCCTTTGAGTTCCCGGCCTAGCCGCTGGCAACAAAGGATAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATTTCACAACACGAGCTGACGACAGCCATGCAGCACCTGTCTCAGAGTTCCCGAAGGCACCAAAGCATCTCTGCTAAGTTCTCTGGATGTCAAGAGTAGGTAAGGTTCTTCGCGTTGCATCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCATTTGAGTTTTAACCTTGCGGCCGTACTCCCCAGGCGGTCGACTTAACGCGTTAGCTCCGGAAGCCACTCCTCAAGGGAACAACCTCCAAGTCGACATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTTGCTCCCCACGCTTTCGCACCTGAGCGTCAGTCTTTGTCCAGGGGGCCGCCTTCGCCACCGGTATTCCTCCAGATCTCTACGCATTTCACCGCTACACCTGGAATTCTACCCCCCTCTACAAGACTCTAGCCTGCCAGTTTCGAATGCAGTTCCCAGGTTGAGCCCGGGGATTTCACATCCGACTTGACAGACCGCCTGCGTGCGCTTTACGCCCAGTAATTCCGATTAACGCTTGCACCCTCCGTATTACCGCGGCTGCTGGCACGGAGTTAGCCGGTGCTTCTTCTGCGGGTAAC 2. Growth-promoting function of strain BP-4 2.1 Experimental Methods (1) Qualitative detection of IAA production, potassium solubilization, nitrogen fixation and siderophore production The strain was inoculated into LB liquid medium and cultured at 28℃ and 150 r / min until the logarithmic growth phase. Its OD value was then determined. 600 Dilute to 0.8.

[0055] The bacterial culture was inoculated at a 1% inoculum into LB liquid medium supplemented with 100 mg / L L-tryptophan and cultured with shaking at 28℃ and 150 rpm for 2 days. A blank control group (without inoculation) was set up, and each group was biologically replicated three times. The resulting fermentation broth was centrifuged at 12000 rpm for 10 min, and the supernatant was mixed with an equal volume of Salkowski's colorimetric solution and incubated in the dark for 30 min. 100 μL of the mixture was dropped onto a white ceramic plate to observe the color change, with a 50 μg / mL IAA standard solution used as a positive control.

[0056] Take 10 μL of bacterial suspension and inoculate it onto potassium-solubilizing solid medium, Ashby solid medium, and modified CAS iron-affinity detection agar medium, respectively. Each treatment is set up in triplicate and incubated at 28℃ in the dark for 3-5 days. Observe and record: the growth of the strain on potassium-solubilizing solid medium, whether a clear zone forms around the colony on Ashby medium, and whether an orange-yellow halo appears on modified CAS agar medium.

[0057] (2) Quantitative detection of IAA production, potassium solubilization, nitrogen fixation and siderophore production IAA content detection: Take the IAA detection solution in the qualitative test and read its absorbance at a wavelength of 530 nm using a UV spectrophotometer. Calculate the IAA content according to the standard curve.

[0058] Potassium solubilization assay: 10 mL of the potassium solubilization culture medium fermentation broth of the strain was placed in a digestion tube, 2 mL of 6% H2O2 was added, and the mixture was heated in a boiling water bath for 1 h. After cooling, the mixture was placed in a centrifuge and centrifuged at 8000 r / min for 10 min. The supernatant was transferred to a new centrifuge tube, and the potassium concentration in the supernatant was determined using a flame spectrophotometer.

[0059] Nitrogen fixation detection: 1% of the culture medium was inoculated into 50 mL of Ashby liquid medium and incubated at 28℃ and 180 r / min for 4 days. A blank control (no inoculation) was used, and each treatment was repeated three times. 15 mL of the liquid was transferred to a digestion tube and heated to 150℃ to evaporate the liquid. 2 g of a 10:1 mixture of K₂SO₄ and CuSO₄ powder and 5 mL of concentrated sulfuric acid were added sequentially to the digestion tube as a reagent blank control. The tube was placed in a digestion oven, and the temperature was gradually increased until the liquid turned a clear, pale blue-green color. The solution was then cooled to room temperature. The digested solution was transferred without loss to a distillation tube and nitrogen was distilled using a Kjeldahl nitrogen analyzer. The final distillate was titrated with 0.01 mol / L standard acid. Record the volume of standard acid used to change the sample solution from blue-green to just turning purple-red, denoted as V (mL); record the volume of standard acid used to change the reagent blank solution from blue-green to just turning purple-red, denoted as V0 (mL); calculate the nitrogen content in the fermentation broth using the following formula: N (mg / mL) = (V - V0) × 0.01 × 14.01 ÷ 15.

[0060] Siderophore content detection: Inoculate the bacterial strain into MKB liquid medium at a 1% inoculum rate and culture on a shaker at 28℃ and 180 r / min for 2-3 days. Centrifuge an appropriate amount of bacterial suspension at 10000 r / min for 10 min. Mix 3 mL of the supernatant with an equal volume of 1×CAS detection solution (10×CAS detection solution and water are mixed at a 1:10 ratio) and let stand in the dark for 30 min. Measure the absorbance (As) at 630 nm, using double-distilled water as a control to zero the sample. Separately, mix an equal volume of blank liquid medium with 1×CAS detection solution and use its absorbance as a reference value (Ar). Calculate the absorbance using the formula ([(Ar-As) / Ar]×100%).

[0061] 2.2 Experimental Results Qualitative results of IAA production, potassium solubilization, nitrogen fixation, and siderophore production of strain BP-4 are shown below. Figure 4 The results showed that BP-4 could grow normally on potassium-solubilizing solid medium, form a clear zone on Ashby solid medium, and produce an orange-yellow halo characteristic of heparin on modified CAS agar medium. IAA qualitative results showed that the positive control group and the BP-4 fermentation broth group exhibited a color reaction, while the blank control group showed no color reaction. Preliminary results indicate that strain BP-4 has the ability to produce IAA, solubilize potassium, fix nitrogen, and produce siderophores.

[0062] Quantitative results of IAA production, potassium solubilization, and nitrogen fixation of strain BP-4 are shown in the figure. Figure 5The results showed that strain BP-4 secreted 83.73±4.87 μg / mL of IAA; its potassium solubilization was 52.60±0.80 μg / mL, significantly higher than the control by 4.08%; and its nitrogen fixation reached 0.041±0.005 mg / mL, significantly higher than the control by 371.4%. Quantitative results of siderophore production (Table 6) showed that the blank control had A... 630 The value was 0.430, while the three replicates A of strain BP-4 630 The levels were significantly reduced (0.293-0.257), and the SU values ​​of siderophores produced were all above 31%. In summary, strain BP-4 has the ability to produce IAA, solubilize potassium, fix nitrogen, and produce siderophores.

[0063] .

[0064] 3. Optimization of phosphorus solubilization conditions for strain BP-4 3.1 Experimental Methods (1) Determination of the solubility of strain BP-4 for different phosphates Based on 0.5% (w / v) NBRIP inorganic phosphorus liquid medium, the phosphorus source Ca3(PO4)2 was replaced by equal amounts of CaHPO4, AlPO4, and FePO4; the phosphorus source in the Monkina organic phosphorus liquid medium was replaced by lecithin and calcium phytate at a concentration of 0.2% (w / v), and basal liquid media with different phosphorus sources were prepared. After activating strain BP-4, the OD of the bacterial culture was adjusted with sterile water. 600 The phosphorus concentration was adjusted to 0.8, and 2% of the inoculum was added to each culture medium (1:50). An equal volume of sterile water was used as a blank control, and the inoculation was repeated three times. The culture was carried out at 28℃ and 150 r / min for 7 days with constant temperature shaking. After the culture was completed, samples were taken and centrifuged at 10000 rpm and 4℃ for 10 min. The supernatant was collected to determine the effective phosphorus concentration.

[0065] (2) Determination of the solubility of strain BP-4 for different concentrations of phosphate A series of basal liquid culture media were prepared with phosphate concentration gradients of 2.5 g / L, 5.0 g / L, 7.5 g / L, 10.0 g / L, and 12.5 g / L. The OD of the bacterial culture was adjusted after BP-4 activation. 600 The phosphorus concentration was increased to 0.8, and the culture medium was inoculated at a rate of 2% (1:50). An equal volume of sterile water was used as a blank control, and the inoculation was repeated three times. The culture was carried out at 28℃ and 150 rpm for 7 days with constant temperature shaking. After the culture was completed, the sample was taken, centrifuged and the effective phosphorus concentration of the supernatant was determined according to the method in item "(1)" above.

[0066] (3) Effect of pH on the phosphorus solubility of strain BP-4 The initial pH of the culture medium was adjusted to 4.0, 5.0, 6.0, 7.0, and 8.0, respectively, to prepare basal culture media with different pH values. After activation of strain BP-4, the OD of the bacterial culture was adjusted. 600 The phosphorus concentration was increased to 0.8, and 2% of the culture medium (1:50) was inoculated. An equal volume of sterile water was used as a blank control, and the inoculum was repeated three times. The culture was carried out at 28℃ and 150 rpm for 7 days with constant temperature shaking. After the culture was completed, samples were taken, centrifuged and the effective phosphorus concentration was determined according to the method in section (1) above.

[0067] (4) Effect of inoculum size on the phosphorus solubility of strain BP-4 Prepare basic liquid culture medium, and adjust the OD of the bacterial culture after activating strain BP-4. 600 The phosphorus concentration was increased to 0.8, and the culture medium was inoculated at 1%, 2%, 3%, 4%, and 5% respectively, with an equal volume of sterile water as a blank control. The inoculum was repeated three times. The culture was kept at 28℃ and 150 rpm for 7 days. After the culture was completed, the samples were taken, centrifuged and the effective phosphorus concentration was determined according to the method in section (1) above.

[0068] 3.2 Experimental Results The results of the optimization of phosphate solubilization conditions for strain BP-4 are shown in the figure. Figure 6 The results showed that strain BP-4 had a significantly higher phosphorus solubility for dicalcium phosphate than calcium phosphate, ferric phosphate, aluminum phosphate, egg yolk lecithin, and calcium phytate. The phosphorus solubility peaked at a phosphorus source concentration of 5 g / L, and decreased with further increases in concentration. An inoculum size of 2% yielded the best phosphorus solubility, which significantly decreased with further increases in inoculum size. The highest phosphorus solubility was observed at pH 7.0, with no significant difference compared to pH 6 and pH 8. Based on these results, the optimal phosphorus solubility conditions for strain BP-4 are: dicalcium phosphate as the phosphorus source, a phosphorus source concentration of 5 g / L, an inoculum size of 2%, and an initial culture medium pH of 7.

[0069] 3.3 Verification of optimal phosphate-solubilizing conditions for strain BP-4 Based on the optimization results of the single-factor experiments above, the optimal phosphorus-solubilizing conditions for strain BP-4 were determined to be: dicalcium phosphate as the phosphorus source, a phosphorus source concentration of 5 g / L, an inoculum size of 2%, and an initial pH of 7 in the culture medium. Using this optimal combination of conditions, a basic NBRIP liquid medium was prepared, and the activated OD... 600 Non-decarboxylated Leucobacterium BP-4 bacterial culture with a pH adjusted to 0.8 was inoculated at the optimal inoculum size and cultured at 28℃ and 150 rpm for 7 days with constant temperature shaking. Then, it was centrifuged at 10000 rpm and 4℃ for 10 min, and the supernatant was collected to determine the effective phosphorus concentration.

[0070] The results are as follows Figure 7As shown in the figure. The results showed that under the conditions of calcium hydrogen phosphate as the phosphorus source, phosphorus source concentration of 5 g / L, inoculum amount of 2%, and initial pH of culture medium of 7, the phosphorus solubility of strain BP-7 could reach 671.0-681.4 mg / L.

[0071] 4. Growth-promoting effect of strain BP-4 on Bletilla striata 4.1 Tissue Culture The growth-promoting effect of rhizosphere phosphate-solubilizing bacteria on Bletilla striata was investigated by symbiotic culture of sterile Bletilla striata seedlings and rhizosphere phosphate-solubilizing bacteria.

[0072] Preparation of fermentation broth: 30 μL of BP-4 bacterial culture was added to 1 mL of LB liquid medium and incubated at 28℃ and 150 r / min with constant shaking for 24 h. An appropriate amount of bacterial culture was added to 50 mL of LB liquid medium and incubated at 28℃ and 150 r / min with constant shaking for 12 h, and the concentration was adjusted to 1×10⁻⁶. 8 The fermentation broth of the strain was obtained by dispersing CFU / mL and set aside for later use.

[0073] Disinfection of Bletilla striata tubers: Take one-year-old Bletilla striata tissue culture seedlings, cut off the leaves and roots with sterile scissors, wash away residual culture medium with sterile water, and blot dry with sterile filter paper. Select Bletilla striata tubers of uniform size, soak in 75% alcohol for 1 minute, rinse 2-3 times with sterile water, soak in 0.1% HgCl solution for 4 minutes, and rinse 3-4 times with sterile water.

[0074] In a clean bench, culture was conducted using double-layered filter paper. Sterilized Bletilla striata tubers were placed between the walls of the culture flasks and the filter paper for fixation. Each flask contained 50 mL of liquid culture medium (4.57 g / L MS powder of potassium dihydrogen phosphate desulfurization + 93.2 mg / L KCl + 20 g / L sucrose + 170 mg / L CaHPO4). After sterilization at 116℃ for 30 min and cooling, sterile LB (NB) and bacterial fermentation broth (HB) were added at a 1% inoculum rate. Five Bletilla striata tubers were inoculated into each flask, with 10 flasks per group. The flasks were placed in a light-controlled culture chamber at 23-25℃, with a light duration of 8-12 h per day and a light intensity of 2000-3000 Lux. The growth of the Bletilla striata tissue culture seedlings was observed regularly. After 45 days of culture, the growth of the Bletilla striata tissue culture seedlings was observed and measured, including plant height, biomass, leaf length, and number of leaves.

[0075] The results are as follows Figure 8 As shown in the results, the non-decarboxylated Leucobacterium BP-4 inoculation treatment showed a highly significant growth-promoting effect on the aboveground and overall growth indicators of Bletilla striata: the plant height, total biomass, number of leaves, and leaf length of Bletilla striata increased by 77.58%, 71.95%, 87.39%, and 82.33% respectively compared with the control.

[0076] 4.2 Potted Cultivation Preparation of fermentation broth: BP-4 strain was cultured in LB medium at 28°C with constant temperature and shaking for 24 h. The bacterial concentration was then adjusted to 1×10⁻⁶ with sterile water. 8 CFU / mL, store for later use.

[0077] Potted plant group setup: The soil substrate was prepared by mixing purchased nutrient soil and mountain soil at a ratio of 3:4 (v:v). Anhydrous dicalcium phosphate was mixed with the soil at a ratio of 200 mg / kg. The experimental soil was divided into two types: unsterilized soil (NS) and sterilized soil (SS). Twenty parallel samples were set up for each type of experimental soil, including 10 blank control group (NB) and 10 BP-4 inoculated treatment group (HB), resulting in 4 experimental treatments: SS-NB, SS-HB, NS-NB, and NS-HB, with 10 replicates for each treatment. The corresponding soil substrate was added to each pot, and seven one-year-old Bletilla striata seedlings with similar growth were selected and planted in each pot. Then, 10 mL of bacterial suspension was inoculated around each Bletilla striata seedling in the BP-4 treatment group, while an equal amount of sterile water was added to the CK control group. All potted plants were cultured uniformly for 90 days. The potted plant experiment flowchart is shown below. Figure 9 As shown. Treatment was performed every 15 days. Sterile water was used for weekly irrigation according to specific growth requirements. Destructive sampling was conducted on day 90. The roots and tubers of *Bletilla striata* were washed, and the plant height, biomass, tuber fresh weight, and leaf length of individual *Bletilla striata* plants in each group were measured.

[0078] The results are as follows Figure 10 As shown in the results, in sterilized soil (SS), inoculation with BP-4 did not significantly increase the height of Bletilla striata, but only slightly increased the total biomass, tuber fresh weight, and leaf length, with the tuber fresh weight increasing by 37.46%. In unsterilized soil (NS), after inoculation with BP-4, the height, leaf length, and tuber fresh weight of Bletilla striata were significantly higher than those of the uninoculated control group, increasing by 8.67%, 12.28%, and 19.82%, respectively, with all growth indicators being the highest among all treatment groups.

[0079] In summary, the non-decarboxylating phosphate-solubilizing bacterium BP-4 screened in this study can significantly promote the growth of Bletilla striata.

[0080] Although the present invention has been described in detail above with general descriptions, specific embodiments, and experiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A non-decarboxylating Leucobacterium BP-4 strain, characterized in that, The non-decarboxylating Leclercia BP-4 was named Leclerciaadecarboxylata BP-4 and was deposited at the China Center for Type Culture Collection on June 24, 2026, with accession number CCTCC NO: M20261419.

2. The application of the non-decarboxylated Leucobacterium BP-4 or its culture as described in claim 1 in phosphorus solubilization.

3. The application according to claim 2, characterized in that, The specific application involves co-culturing non-decarboxylated Leukes bacillus BP-4 bacterial culture with inorganic or organic phosphorus to dissolve insoluble phosphorus. The specific co-culturing conditions are as follows: the culture medium is NBRIP inorganic phosphorus liquid medium or Monkina organic phosphorus liquid medium; the phosphorus source is selected from any one of calcium phosphate, dicalcium phosphate, aluminum phosphate, ferric phosphate, lecithin, and calcium phytate; the phosphorus source concentration is 2.5-12.5 g / L; the initial pH of the culture medium is 4.0-8.0; OD... 600 The inoculation amount of non-decarboxylated Lechnerella BP-4 bacterial suspension with a value of 0.8 is 1%-5%.

4. The application according to claim 3, characterized in that, The specific conditions for co-culture were as follows: the culture medium was NBRIP inorganic phosphorus liquid medium, the phosphorus source was dicalcium phosphate, and the phosphorus source concentration was 5 g / L; the initial pH of the culture medium was 7.0; OD 600 The inoculum size of the non-decarboxylated Lechner's BP-4 bacterial suspension with a value of 0.8 was 2%.

5. The application of the non-decarboxylating Leucobacterium BP-4 or its culture as described in claim 1 in potassium solubilization.

6. The application of the non-decarboxylated Leucobacterium BP-4 or its culture as described in claim 1 in the production of IAA.

7. The application of the non-decarboxylated Leucobacterium BP-4 or its culture as described in claim 1 in siderogenic carriers.

8. The application of the non-decarboxylating Leucobacterium BP-4 or its culture as described in claim 1 in nitrogen fixation.

9. The application of the non-decarboxylating Leucobacterium BP-4 or its culture as described in claim 1 in promoting plant growth or in the preparation of microbial agents or bio-organic fertilizers that promote plant growth.

10. The application according to claim 9, characterized in that, The plant in question is Bletilla striata.

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