A strain of Collimonas sp. D3 from a bamboo forest and its application

Abstract

This invention discloses a strain of *Collimonas sp. D3* from bamboo forests and its applications. *Collimonas sp. D3* is deposited at the China General Microbiological Culture Collection Center (CGMCC) on March 12, 2025, with accession number CGMCC NO:33803. This *Collimonas sp. D3* strain is easy to cultivate and grows rapidly; it possesses the ability to fix nitrogen, solubilize inorganic and organic phosphorus; it also has the function of producing proteases; it can be used as a soil remediation agent to promote plant growth; and when inoculated into the rhizosphere of bamboo seedlings, it can enhance bamboo height and biomass.

Description

Technical Field

[0001] This invention relates to a strain of Collimonas sp. D3 from moso bamboo forests and its applications, belonging to the fields of microbial technology and ecological restoration technology. Background Technology

[0002] While chemical fertilizers can improve plant yield and quality, excessive fertilization can reduce soil water retention capacity and fertility, decrease the number of microorganisms, and ultimately lead to soil nutrient imbalance. Microbial fertilizers are one of the main ways to replace chemical fertilizers and achieve environmentally friendly goals. These fertilizers use the life activities of microorganisms as their core element, leveraging these activities to help target crops obtain beneficial benefits. As a low-cost and environmentally friendly new type of fertilizer, microbial fertilizers can effectively improve soil, enhance fertility, increase crop yields, and reduce crop losses caused by pathogens, thus occupying an important position in the national agricultural green development strategy.

[0003] my country ranks among the world's top countries in terms of bamboo resources area, variety, and reserves. As of 2021, my country's bamboo forest area was 7.5627 million hectares. 2 This accounts for 3.31% of the forest area, of which bamboo forests cover 5.2776 million hectares. 2 Bamboo forests account for 69.78% of the total bamboo forest area. The bamboo forest ecosystem contains abundant microbial resources. Exploring these beneficial microbial resources is of great significance for solving environmental problems in bamboo forest management and promoting the green and healthy development of resources.

[0004] This invention isolates a strain of Collimonas sp. D3 from moso bamboo forests. It has the functions of nitrogen fixation, inorganic phosphorus solubilization, organic phosphorus solubilization, and protease production. It can be used as a soil remediation agent and to promote plant growth. Summary of the Invention

[0005] The first objective of this invention is to provide a strain of Collimonas sp. D3, a bacterium found in bamboo forests.

[0006] A second objective of this invention is to provide the application of a strain of *Collimonas sp. D3* from moso bamboo forests for soil remediation and to promote plant growth.

[0007] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0008] A strain of Collimonas sp. D3 from bamboo forest has been deposited at the China General Microbiological Culture Collection Center (CGMCC) on March 12, 2025, with accession number CGMCC NO:33803.

[0009]

[0010] The strain Collimonas sp. D3 of the bamboo forest strain of the present invention was obtained by the inventor through screening.

[0011] The aforementioned Collimonas sp. D3, a bacterium found in bamboo forests, can be used as a soil remediation agent or in the preparation of soil remediation agents. It promotes plant growth.

[0012] The aforementioned Collimonas sp. D3, a bacterium found in moso bamboo forests, can be used for nitrogen fixation, inorganic phosphorus decomposition, and / or organic phosphorus decomposition.

[0013] The aforementioned *Collimonas sp.* D3 strain from moso bamboo forests is used to increase the content of organic carbon, total nitrogen, hydrolyzable nitrogen, total phosphorus, and available phosphorus in the soil. When using it, the concentration should be 1×10⁻⁶. 9 Collimonas sp. D3 (cfu / ml) of *Bambusa pilosa* in bamboo forests was sprayed onto the soil at a rate of 25–30 L / mu (approximately 0.067 hectares). A second spraying was applied after a 7-day interval, for a total of two applications. Bamboo forest soil is preferred.

[0014] The aforementioned Collimonas sp. D3 from the moso bamboo forest can be used to produce proteases.

[0015] The aforementioned Collimonas sp. D3 fungus used in moso bamboo forests can be used to increase the height and biomass of moso bamboo. When using it, the concentration should be 1×10⁻⁶. 8 Collimonas sp. D3 (cfu / ml) was inoculated into the rhizosphere of three-leaf stage bamboo seedlings in moso bamboo forests at a dosage of 8–10 ml per seedling. After 120 days, compared to a control of equal volume of sterile physiological saline, the inoculated seedlings showed increased plant height and biomass of strain D3 by 25.34%–30.20% and 22.82%–34.23%, respectively.

[0016] The *Collimonas sp.* D3 strain from the bamboo forest of this invention exhibits colony diameters of 0.5–0.8 mm on TSB agar plates. The colonies are round, with neat edges, opaque, milky white on the front, smooth, moist, raised, soft in texture, and easily picked up. When cultured in TSB broth (17.0 g tryptone, 3.0 g soybean peptone, 5.0 g sodium chloride, 2.5 g dipotassium hydrogen phosphate, 2.5 g glucose, the remainder being water, pH 7.0 ± 0.5, sterilized at 115°C for 20 minutes), the strain density can reach 1 × 10⁻⁶. 9 cfu / ml.

[0017] Any techniques not mentioned in this invention are based on existing technologies.

[0018] The present invention relates to Collimonas sp. D3, a bacterium found in moso bamboo forests, which has the following beneficial effects: 1. It is easy to cultivate and grows rapidly; 2. This strain has the ability to fix nitrogen, solubilize inorganic phosphorus and organic phosphorus; 3. This bacterium has the function of producing proteases; 4. It can be used as a soil remediation agent to increase the content of organic carbon, total nitrogen, hydrolyzable nitrogen, total phosphorus and available phosphorus in the soil, and promote plant growth; 5. When inoculated into the rhizosphere of moso bamboo seedlings, it can be used to improve the height and biomass of moso bamboo. Attached Figure Description

[0019] Figure 1 This is the phylogenetic tree of Collimonas sp D3, a bacterium found in bamboo forests, according to the present invention.

[0020] Figure 2 This invention illustrates the growth of Collimonas sp. D3, a bacterium from the moso bamboo forest, on an organophosphate agar plate.

[0021] Figure 3 This invention describes the growth of Collimonas sp. D3, a bacterium from the moso bamboo forest, on an inorganic phosphorus medium plate.

[0022] Figure 4 This invention illustrates the growth of Collimonas sp. D3 from moso bamboo forest on Assumption nitrogen-fixing medium plates.

[0023] Figure 5 This invention describes the growth of Collimonas sp. D3 from moso bamboo forest on a skim milk powder culture medium plate. Detailed Implementation

[0024] To better understand the present invention, the following embodiments further illustrate the content of the present invention, but the content of the present invention is not limited to the following embodiments.

[0025] Example 1

[0026] Obtaining Collimonas sp. D3:

[0027] 1. Enrichment Culture: Take 10g of soil from a bamboo forest in Anji, Zhejiang Province, and prepare a suspension using 90mL of sterile physiological saline (0.9% by mass). Transfer 10mL of the suspension to 90mL of TSB liquid culture medium (tryptone 17.0g / L, soybean peptone 3.0g / L, sodium chloride 5.0g / L, dipotassium hydrogen phosphate 2.5g / L, glucose 2.5g / L, the remainder being water, pH 7.0±0.5, sterilized at 115℃ for 20 minutes), and incubate at 30℃ with shaking at 180rpm for 24h to obtain the enriched culture medium.

[0028] 2. Preparation of diluent: Take several sterile test tubes and add 9 mL of diluent (sterile physiological saline, 0.9% concentration) to each tube. Add 1 mL of enrichment culture medium to the first test tube, mix well, and you will get 10... -1 Diluent; replace with a new sterile pipette tip, take 1 mL of 10 -1 Transfer the diluent to the next test tube and mix well to obtain 10 -2 Diluent; and so on, to obtain 10 -3 10 -4 10 -5 10 -6 10 -7 10 -8 10 -9 Diluent. That is, this step yielded a dilution of 10... -1 10 -2 10 -3 10 -4 10 -5 10 -6 10 -7 10 -8 10 -9 There are a total of nine concentration gradients.

[0029] 3. Cultivation: Select 10 -7 10 -8 10 -9 Three dilution gradients were each 200 μL and spread onto organophosphate agar plates (10.0 g / L glucose, 0.5 g / L ammonium sulfate, 0.3 g / L sodium chloride, 0.3 g / L magnesium sulfate, 0.03 g / L manganese sulfate, 0.3 g / L potassium sulfate, 0.03 g / L ferrous sulfate, 5.0 g / L lecithin, 15.0 g / L agar, pH 7.0-7.5, autoclaved at 115°C for 30 min). After air-drying, the plates were inverted and incubated in a constant temperature incubator.

[0030] 4. Isolation and purification of the strain: Incubate on organophosphate agar plates at 30°C for 24 h, select single colonies with the largest phosphate-solubilizing zones, and streak them continuously on the surface of TSB solid medium (tryptone 17.0 g / L, soybean peptone 3.0 g / L, sodium chloride 5.0 g / L, dipotassium hydrogen phosphate 2.5 g / L, glucose 2.5 g / L, agar 15 g / L, the remainder being water, pH 7.0±0.5, sterilized at 115°C for 20 minutes), and incubate at 30°C for 24 h. Repeat this process 6 times to obtain pure strain D3.

[0031] 5. Glycerol preservation method: Select strain D3 obtained in step 4 and culture it in TSB liquid medium at 30℃ with shaking at 180 r / min for 24 h to obtain strain culture solution. Add 0.8 ml of strain culture solution and 0.2 ml of glycerol to a sterile strain preservation tube, mix thoroughly, and store in a -80℃ refrigerator.

[0032] 6. Strain identification

[0033] (1) Morphological characteristics:

[0034] In TSB liquid medium, it presents as a uniform turbidity with a small amount of precipitate, which disperses easily with gentle shaking. On TSB solid agar plates, the colony diameter is 0.5–0.8 mm. The colonies are round, with neat edges, opaque, milky white on the front, smooth, moist, raised, soft in texture, and easy to pick up.

[0035] (2) 16S rRNA sequence analysis:

[0036] Further 16S rRNA sequence analysis and alignment were performed on the isolated and screened strain D3. A pair of primers was designed based on the conserved sequence of bacterial 16S rDNA.

[0037] Upstream primer (27F): 5'-A AGAGTTTGATCMTGGCTCAG-3'

[0038] Downstream primer (1492R): 5'-GGTTACCTTGTTACGACTT-3'

[0039] PCR amplification was performed using the screened strains as templates. Reaction conditions: 95℃ pre-denaturation for 5 min, 94℃ denaturation for 50 s, 57℃ annealing for 30 s, 72℃ extension for 1 min 30 s, for 30 cycles. The target fragment was cloned and sequenced according to standard methods (Sambrook, et al., 2001). The sequencing sequence of strain D8 is as follows:

[0040] Collimonas sp D3

[0041] TCTGGTAAAACCCGCTCCCATGGTGTGACGGGCGGTGTGTACAAGACCCGGGAACG

[0042] TATTCACCGCGACATGCTGATCCGCGATTACTAGCGATTCCAACTTCATGTAGTCGAG

[0043] TTGCAGACTACAATCCGGACTACGATACACTTTCTGGGATTAGCTCCCCCTCGCGGG

[0044] TTGGCGGCCCTCTGTATGTACCATTGTATGACGTGTGAAGCCCTACCCATAAGGGCCA

[0045] TGAGGACTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCTCATTAGA

[0046] GTGCCCTTTCGTAGCAACTAATGACAAGGGTTGCGCTCGTTGCGGGACTTAACCCAA

[0047] CATCTCACGACACGAGCTGACGACAGCCATGCAGCACCTGTGTTACAGCTTTCTTTC

[0048] GAACACTCCCAAATCTCTTCGGGATTCTGTACATGTCAAGGGTAGGTAAGGTTTTTC

[0049] GCGTTGCATCGAATTAATCCACATCATCCACCGCTTGTGCGGGTCCCCGTCAATTCCT

[0050] TTGAGTTTTAATCTTGCGACCGTACTCCCCAGGCGGTCTACTTCACGCGTTAGCTGC

[0051] GTTACCAAGTCAATTAAGACCCGACAACTAGTAGACATCGTTTAGGGCGTGGACTAC

[0052] CAGGGTATCTAATCCTGTTTGCTCCCCACGCTTTCGTGCATGAGCGTCAGTGTTATCC

[0053] CAGGGGGCTGCCTTCGCCATCGGTATTCCTCCACATCTCTACGCATTTCACTGCTACA

[0054] CGTGGAATTCTACCCCCCTCTGACACACTCTAGCCGTGCAGTCACAAATGCCATTCC

[0055] CAGGTTAAGCCCGGGGATTTCACACCTGGCTTACACAACCGCCTGCGCACGCTTTAC

[0056] GCCCAGTAATTCCGATTAACGCTTGCACCCTACGTATTACCGCGGCTGCTGGCACGTA

[0057] GTTAGCCGGTGCTTATTCTTCAGGTACCGTCATTAGCAAGAGATATTAGCCCTCACCG

[0058] TTTCTTCCCTGACAAAAGAGCTTTACAACCCGAAGGCCTTCTTCACTCACGCGGCAT

[0059] TGCTGGATCAGGGTTGCCCCCATTGTCCAAAATTCCCCACTGCTGCCTCCCGTAGGA

[0060] GTCTGGGCCGTGTCTCAGTCCCAGTGTGGCTGGTCGTCCTCTCAGACCAGCTACTGA

[0061] TCGATGCCTTGGTGAGCCTTTACCTCACCAACTAGCTAATCAGATATCGGCCGCTCTA

[0062] TGAGCATGAGGTCTTGCGAGCCCCACTTTCATCCGTAGATCGTAT

[0063] The sequence determination results of the strain were compared with the 16S rRNA sequences registered in GenBank (accession numbers: NZ_JBANDC010000043.1, NZ_CP013235.1, NZ_CP013232.1, and NZ_CP013236.1, etc.), and the gene homology reached over 98.70%. Based on Bergey's Mannual of Determinative Bacteriology (Holt, JG, Gibbons, NE, 1994) and the Handbook of Systematic Identification of Common Bacteria (Dong Xiuzhu and Cai Miaoying et al., 2001), strain D3 was identified as *Syntropha sacchariformis* through morphological characteristics and 16S rRNA sequence analysis. A phylogenetic tree between strain D3 and the reference strain was constructed using the neighbor-joining method, as shown below. Figure 1 As shown.

[0064] Example 2

[0065] Determination of the organic phosphorus solubilizing ability of strain D3:

[0066] After activating the bacterial strain preserved on test tube slant, it was inoculated onto TSB liquid medium for further activation. After 48 hours of culture, this was used as a seed culture. 10 μL of the seed culture was then inoculated onto organophosphate agar plates (10.0 g / L glucose, 0.5 g / L ammonium sulfate, 0.3 g / L sodium chloride, 0.3 g / L magnesium sulfate, 0.03 g / L manganese sulfate, 0.3 g / L potassium sulfate, 0.03 g / L ferrous sulfate, 5.0 g / L lecithin, 15.0 g / L agar, pH 7.0-7.5, autoclaved at 115°C for 30 min). Two inoculation points were used per plate, and the process was repeated three times. Figure 2 As shown, after being cultured at 30℃ for 24 hours, a transparent zone was observed around the strain, indicating that the strain has the ability to solubilize organophosphates.

[0067] Example 3

[0068] Determination of inorganic phosphorus solubilization capacity of strain D3:

[0069] After activating the bacterial strain preserved on test tube slant, it was inoculated onto TSB liquid medium for further activation. After 24 hours of incubation, this was used as a seed culture. 10 μL of the seed culture was inoculated onto inorganic phosphorus medium plates (10.0 g / L glucose, 0.5 g / L ammonium sulfate, 0.3 g / L sodium chloride, 0.3 g / L magnesium sulfate, 0.03 g / L manganese sulfate, 0.3 g / L potassium sulfate, 0.03 g / L ferrous sulfate, 5.0 g / L calcium phosphate, 15.0 g / L agar, pH 7.0-7.5, autoclaved at 115°C for 30 min). Two inoculation points were used per plate, and the process was repeated three times. Figure 3 As shown, after incubation at 30℃ for 72 hours, a transparent zone was observed around the strain, indicating that the strain has the ability to dissolve organophosphates.

[0070] Example 4

[0071] Nitrogen fixation capacity determination of strain D3:

[0072] After activating the bacterial strain preserved in test tube slant culture, it was inoculated onto TSB liquid medium (17.0 g / L tryptone, 3.0 g / L soybean peptone, 5.0 g / L sodium chloride, 2.5 g / L dipotassium hydrogen phosphate, 2.5 g / L glucose, the remainder being water, pH 7.0±0.5, sterilized at 115℃ for 20 minutes) for activation. After culturing for 48 hours, this was used as seed culture. 10 μL of the seed culture was inoculated onto Assab nitrogen fixation medium plates (0.2 g / L potassium dihydrogen phosphate, 0.2 g / L sodium chloride, 0.2 g / L magnesium sulfate, 5.0 g / L calcium carbonate, 0.1 g / L calcium sulfate, 10 g / L mannitol, 15.0 g / L agar, pH 7.0-7.5, autoclaved at 115℃ for 30 minutes), with 2 inoculation points per plate, and repeated three times. Figure 4As shown, after being cultured at a constant temperature of 30℃ for 96 hours, the bacteria showed good growth, indicating that the strain has the ability to fix nitrogen.

[0073] Example 5

[0074] Determination of protease production capacity of strain D3:

[0075] After activating the bacterial strain preserved on test tube slant agar, it was inoculated onto TSB liquid medium for further activation and cultured for 24 hours to obtain the seed culture. 10 μL of the seed culture was then inoculated onto skim milk powder agar plates (15 g / L skim milk powder, 16 g / L agar powder, 1000 mL water, pH 7.0–7.2, sterilized at 115°C for 15 min), with two inoculation points per plate, and repeated three times. Figure 5 As shown, after incubation at 30℃ for 48 hours, a clear zone was observed around the strain, indicating that the strain can produce protease.

[0076] Example 6

[0077] Effects of strain D3 on the growth of moso bamboo seedlings:

[0078] After activating the bacterial strain preserved on test tube slant culture, it was inoculated onto TSB liquid medium for further activation and cultured for 24 hours to obtain the seed culture. 5 ml of the seed culture was inoculated into 1000 ml of TSB liquid medium and cultured at 30°C for 24 hours. After fermentation, the bacterial culture was centrifuged at 8000 rpm for 10 min at 4°C, the supernatant was discarded, and the culture was resuspended in sterile physiological saline. This resuspending process was repeated three times. The collected bacterial cells were adjusted to a concentration of 1×10⁻⁶ cells / mL with sterile physiological saline. 9 The above-mentioned bacterial agent, at a concentration of cfu / ml, was inoculated at a concentration of 10 ml into the rhizosphere of three-leaf stage moso bamboo seedlings, with an equal volume of sterile physiological saline as a control. The potted plants were grown under natural light and under uniform management. After 120 days, compared to the control, the plant height and biomass of the inoculated strain D3 increased by 25.34%–30.20% and 22.82%–34.23%, respectively.

[0079] Example 7

[0080] The effects of strain D3 on bamboo forest soil:

[0081] The bacterial strain preserved on test tube slant agar plates was activated on TSB solid plates. A loopful of a single colony was inoculated into 10 mL of TSB liquid medium and incubated at 30°C with shaking at 180 rpm for 12 h to obtain the inoculum. The strain was then inoculated into fresh LB liquid medium at a 1% (v / v) inoculation rate and incubated at 30°C with shaking at 180 rpm for 24 h to obtain the seed culture. 5 mL of the seed culture was inoculated into 500 mL of TSB liquid medium (10 g / L peptone, 10 g / L NaCl, 5 g / L yeast extract, the remainder being water, pH 7.0, sterilized at 121°C for 20 min) and incubated at 30°C for 24 h to obtain the fermentation broth with a bacterial count of 1 × 10⁻⁶. 8 CFU / ml. The fermentation broth was diluted 10 times with tap water and sprayed onto the bamboo forest at a rate of 30 L / mu. A second spraying was performed after 7 days, for a total of two sprayings. A control group was used without bacterial broth spraying. After 90 days, tests showed that, compared to the control, spraying with strain D3 significantly increased the content of organic carbon, total nitrogen, hydrolyzable nitrogen, total phosphorus, and available phosphorus in the soil (Table 1).

[0082] Table 1. Effects of strain D3 on the physicochemical properties of bamboo forest soil.

[0083] D3 control group Organic carbon (g / kg) 30.37±1.51a 16.10±0.67b Total nitrogen (g / kg) 2.08±0.09a 1.44±0.03b Hydrolyzable nitrogen (mg / kg) 231.32±2.96a 146.51±21.40b Total phosphorus (g / kg) 1.83±0.05a 1.54±0.08b Available phosphorus (mg / kg) 743.03±6.49a 477.26±8.84b

[0084] The above description, in conjunction with specific preferred embodiments, provides a further detailed explanation of the present invention. It should not be construed that the specific implementation of the present invention is limited to these descriptions. For those skilled in the art, several simple deductions can be made without departing from the concept of the present invention, and all such deductions should be considered within the scope of protection of the present invention.

Claims

1. A strain of Phaeoseptatia burghensis Collimonas sp D3, characterized in that: Deposited with the China General Microbiological Culture Collection Center on March 12, 2025, and assigned accession number CGMCC NO: 33803.

2. A bacterium of the species Arcanobacterium arundinaceus according to claim 1. Collimonas sp Use of D3, characterized in that: Use as a soil remediation agent, or in the preparation of a soil remediation agent.

3. Use according to claim 2, characterized in that: For nitrogen fixation, inorganic phosphorus solubilization, and / or organic phosphorus solubilization.

4. Use according to claim 2, characterized in that: For increasing the content of organic carbon, total nitrogen, hydrolyzable nitrogen, total phosphorus, and available phosphorus in soil.

5. Use according to claim 4, characterized in that: The concentration of 1 × 10 9 cfu / ml of B. phyllosphaerae Collimonas sp D3, according to 25~30 L / acre sprayed to the bamboo forest, and sprayed again after 7 days, a total of 2 times.

6. Use according to claim 2, characterized in that: For producing protease.

7. Use according to claim 2, characterized in that: For increasing the height and biomass of Phyllostachys edulis.

8. Use according to claim 7, characterized in that: The concentration of 1 × 10 9 cfu / ml of P. bambusicola Collimonas sp D3, inoculated to the rhizosphere of the three-leaf stage of Phyllostachys edulis seedling, with the dosage of 8~10 ml per Phyllostachys edulis.

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