Method for relieving saline-alkali stress and continuous cropping obstacles of cotton by using combination of phyllobacterium and aged cotton stalk charcoal

Abstract

The invention provides a method for relieving saline-alkali stress and continuous cropping obstacles of cotton by combined application of phyllobacterium and aged cotton stalk charcoal, which comprises the following steps of: aiming at soil with saline-alkali stress and continuous cropping obstacles of cotton; the phyllobacterium sp. P2 CCTCC No. M2025416 which is screened from rhizosphere soil of the Salmatopsis hastate and has saline-alkali resistance, drought resistance, growth promotion and disease prevention is combined with the aged cotton stalk charcoal for combined application, and a comprehensive evaluation result shows that the combined application of the phyllobacterium sp. P2 and the aged cotton stalk charcoal has the best effect of relieving saline-alkali stress and continuous cropping obstacles of the cotton compared with single application, so that the growth of the cotton can be promoted, and the yield of the cotton is increased. The cotton rhizosphere soil salinity is reduced, and the nutrient content is increased; the quantity of pathogenic bacteria fusarium oxysporum of cotton fusarium wilt in rhizosphere soil is reduced; meanwhile, the growth of microorganisms utilizing phenolic acid autotoxic substances is promoted, and the content of the microorganisms in the soil is reduced, so that the growth of continuous cropping cotton in the moderate saline-alkali soil and the micro-ecological environment of rhizosphere soil are improved.

Description

Technical Field

[0001] The present invention relates to the technical field of cotton soil improvement, and in particular to a method for improving cotton soil subjected to saline-alkali stress and continuous cropping obstacles by jointly applying Leaf Bacterium bacteria and aged cotton stalk charcoal, thereby promoting cotton growth and improving the rhizosphere soil microecological environment. Background Art

[0002] Xinjiang, located in the inland region, is characterized by drought, low rainfall, and water shortages. A large proportion of arable land is salinized. Xinjiang produces over 90% of my country's total cotton output, with a high percentage of cotton cultivation, widespread planting, and long-term continuous cropping. The presence of extensive salinized cotton fields in Xinjiang makes it difficult for cotton seedlings to emerge and maintain, restricting their growth and impacting both yield and profitability. Furthermore, long-term continuous cropping leads to nutrient imbalances in the rhizosphere soil, soil compaction, and reduced air and water permeability. This inhibits soil microbial activity, exacerbating pests and diseases, and increasing the accumulation of soil-borne pathogens such as Verticillium wilt and Fusarium wilt. Furthermore, the accumulation of phenolic autotoxic substances in the rhizosphere significantly inhibits cotton growth, leading to a decline in cotton fiber quality indicators such as fiber length and strength, severely impacting yield and economic profitability. Therefore, salinization and continuous cropping have become major constraints affecting Xinjiang's cotton industry. These factors interact through complex physiological and ecological mechanisms, severely limiting cotton yield and quality.

[0003] Currently, soil improvement in cotton fields under saline-alkali stress and continuous cropping problems can be achieved through breeding salt-tolerant and disease-resistant varieties, rational crop rotation, increased application of organic fertilizers and biofertilizers, green biological pest control, and effective irrigation and salt removal. These measures can improve the rhizosphere soil microecological environment and promote cotton growth. Microbial fertilizers are a promising biocontrol measure. Screening for indigenous microbial strains that are well adapted to the saline-alkali and arid environment of Xinjiang, promote crop growth, improve the soil microecological environment, inhibit the growth and reproduction of pathogens, degrade phenolic acid autotoxicants, and can be used for biofertilizer production is crucial for effectively alleviating saline-alkali stress and continuous cropping problems in cotton. Biochar, a carbon-rich material produced by the pyrolysis of biomass under anoxic conditions, has high porosity and specific surface area, providing a favorable habitat for microorganisms. It can also improve soil structure, increase soil nutrient content, adsorb autotoxicants, promote the growth of beneficial microorganisms, and inhibit pathogens, making it an excellent carrier for microbial agents. Compared to fresh biochar, aged biochar has more developed pores, enhanced polarity, and improved nutrient release capacity, making it more conducive to microbial colonization. Aged biochar, through its structure-function synergistic optimization, demonstrates more efficient and lasting environmental benefits than fresh biochar in soil improvement, pollution remediation, and carbon sequestration. It is particularly suitable for complex contaminated sites and long-term agricultural management. Future efforts will require precise control of the aging process to further enhance its cost-effectiveness and environmental safety.

[0004] Phyllobacterium belongs to the family Phyllobacteraceae of the order Rhizobiales and is of great significance in the field of agricultural microbial applications. It has the ability to fix nitrogen, produce growth hormones (such as auxin, cytokinin, etc.) and polysaccharides, and has a degradation effect on some organic pollutants. It can be used for soil improvement and bioremediation in agricultural production. There are currently no case reports on the combined application of Phyllobacterium bacteria and aged cotton stalk charcoal in preventing and treating cotton salinity stress and continuous cropping obstacles. Therefore, this application combines Phyllobacterium with aged cotton stalk charcoal, and the synergistic complementarity between the two can improve the growth of cotton, improve the rhizosphere soil environment, reduce the salt content in the soil, inhibit the growth of pathogens, degrade phenolic acid self-toxic substances, and help enhance the salinity and continuous cropping improvement effects of cotton. Summary of the Invention

[0005] In response to the above-mentioned problems arising in the cotton production process under saline-alkali stress and continuous cropping obstacles, the present invention provides a method for promoting cotton growth, improving the microecological environment of cotton rhizosphere soil, and alleviating saline-alkali stress and continuous cropping obstacles by jointly applying Phyllobacterium sp. P2 CCTCC No. M2025416 and aged cotton stalk charcoal, thereby alleviating saline-alkali stress and continuous cropping obstacles, thereby achieving sustainable development of cotton.

[0006] To achieve the above object, the present invention provides the following technical solutions:

[0007] A Phyllobacterium sp. P2 CCTCC No. M2025416 bacterium was isolated and purified from the rhizosphere soil of the halophyte Halophyte. It exhibits salt-alkali and drought tolerance, growth promotion, and disease prevention properties. This strain has been deposited with the China Center for Type Culture Collection (CCTCC), an international depository for microorganisms under the Budapest Treaty, at Wuhan University, 16 Luojiashan Road, Wuchang District, Wuhan, Hubei Province, China, 430072, on March 10, 2025, under the CCTCC No. M2025416. Its 16S rRNA genome sequence is shown in SEQ ID NO: 1.

[0008] Furthermore, the present application also provides a bacterial agent of Phyllobacterium sp. P2, which is obtained by fermenting the strain according to claim 1.

[0009] The Phyllobacterium sp. P2 bacterial agent is a liquid bacterial agent.

[0010] Furthermore, the present application also provides a method for preparing a Phyllobacterium sp. P2 inoculum, comprising the following steps: inoculating the Phyllobacterium sp. P2 in a NA solid culture medium containing 2% NaCl for activation, and then transferring it to a NB liquid culture medium containing 2% NaCl, wherein 50 mL of the liquid culture medium is placed in a 500 mL conical flask, and cultured at 30°C and 180 rpm for 18 hours to obtain a seed solution; inoculating the seed solution into a NB liquid fermentation medium containing 2% NaCl at a ratio of 1:100, and culturing at 30°C and 200 rpm for 24 hours, wherein the maximum concentration of the inoculum can reach 5.5×10 9 cfu / ml.

[0011] Furthermore, the present application also provides a use of a Phyllobacterium sp. P2 bacterial agent in improving saline-alkali stress in cotton.

[0012] Furthermore, the present application also provides an application of a Phyllobacterium sp. P2 bacterial agent in overcoming the obstacles of continuous cotton cropping.

[0013] Furthermore, the present application also provides a method for improving cotton growth under saline-alkali stress and continuous cropping disorders. In the method, cotton seeds of uniform size are selected, washed with sterile water, and then soaked in a 1% Phyllobacterium sp. P2 bacterial agent overnight; 2% aged cotton stalk charcoal is applied as base fertilizer during the planting process.

[0014] Preferably, the aged cotton stalk charcoal is prepared by the following steps: collecting cotton stalks without obvious pests and diseases and with less impurities, crushing them and placing them in a carbonization furnace for carbonization at a carbonization temperature of 400-450°C for 1-1.5 hours. After the carbonization is completed, cooling them to 25°C in a cooler, sieving them through a 0.25mm sieve and grinding them to obtain fresh cotton stalk biochar powder. The carbonized cotton stalk charcoal is placed in a natural environment and aged for two years to obtain aged cotton stalk charcoal.

[0015] Furthermore, the present application also provides application of the above-mentioned method for improving cotton growth under saline-alkali stress and continuous cropping obstacles in cotton planting.

[0016] Based on the above technical solution, the present invention mainly has the following beneficial effects:

[0017] The present invention discloses a superior salt-tolerant, growth-promoting bacterium, Phyllobacterium sp. P2 CCTCC No. M2025416, isolated from the rhizosphere soil of the halophyte Halotrichia sinensis. This bacterium exhibits salt-alkali and drought tolerance, growth-promoting properties, and disease prevention. When applied in combination with aged cotton stalk charcoal to continuously cropped cotton soils plagued by soil-borne diseases and severe salinization, the bacterium promotes cotton growth, reduces rhizosphere soil salinity, and increases nutrient content. It also reduces the number of Fusarium spp., the pathogen responsible for cotton wilt, in the soil. Furthermore, it promotes the growth of microorganisms that utilize phenolic acid autotoxicants and reduces their content, thereby improving the growth of continuously cropped cotton and the rhizosphere soil microecological environment in moderately saline-alkali soils, achieving sustainable cotton development. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Shown are the colony morphology and bacterial morphology of Phyllobacterium sp.P2 CCTCC No.M2025416.

[0019] Figure 2 Shown is the phylogenetic tree of Phyllobacterium sp. P2 CCTCC No. M2025416 strain constructed based on 16S rRNA.

[0020] Figure 3 Shown is the effect of combined application of Phyllobacterium sp.P2 CCTCC No.M2025416 strain and aged cotton stalk charcoal on the relative abundance of Fusarium oxysporum in cotton soil.

[0021] Figure 4 Shown is the utilization effect of phenolic acid autotoxic substances by cotton rhizosphere soil microorganisms when Phyllobacterium sp.P2 CCTCC No.M2025416 strain is applied in combination with aged cotton stalk charcoal.

[0022] Figure 5 Shown is the degradation effect of phenolic acid autotoxic substances in cotton rhizosphere soil by the combined application of Phyllobacterium sp.P2 CCTCC No.M2025416 strain and aged cotton stalk charcoal. DETAILED DESCRIPTION

[0023] The following will be combined with the contents of the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. However, the present invention is not limited to the following embodiments. All raw and auxiliary materials used in the present invention, as well as the selected bacterial culture methods, are conventional reagents, methods, and equipment in the art. The percentages mentioned in the present invention are all weight percentages unless otherwise specified.

[0024] The 2% NaCl NA (nutrient agar) culture medium (g / L) comprises: 10.0% peptone, 3.0% beef powder, 20.0% NaCl, 15.0-20.0% agar, and a pH of 7.3±0.1.

[0025] 2% NaCl NB (nutrient broth) liquid fermentation medium (g / L): peptone 10.0, beef powder 3.0, NaCl 20.0, pH 7.2±0.2.

[0026] Example 1: Isolation and screening of Phyllobacterium sp.P2 CCTCC No.M2025416

[0027] Soil samples were collected from the rhizosphere of the Xinjiang halophyte Halophyte. Microorganisms were isolated and purified using the gradient dilution method. The strains were stored on a slant of NA medium containing 2% NaCl for later use. 10 g of soil sample was weighed and placed in 100 mL of sterile water. Sterilized glass beads were added and shaken at 150 rpm and 30°C for 30 minutes to fully disperse the sample. 100 μL of the supernatant was aseptically aspirated and added to 900 μL of sterile water. The supernatant was diluted with sterile water to make 10 -2 -10 -5 Concentration dilution. Take 100 μL of each concentration dilution, spread it on a NA solid culture medium plate containing 2% NaCl using a conventional coating method, and culture it in a 30°C constant temperature incubator for 72 hours. After colonies grow on the plate, pick a single colony on the plate for purification and culture until there are no other colonies. After salt tolerance determination and screening of the growth-promoting properties of the selective culture medium, a strain numbered P2 was selected and transferred to a slant containing 2% NaClNA culture medium for storage. Example 2: Identification of Phyllobacterium sp.P2 CCTCCNo.M2025416 strain

[0028] (1) Colony and bacterial morphology and physiological and biochemical characteristics

[0029] The results of the growth condition study showed that the P2 strain was a Gram-negative bacterium, aerobic, motile, and rod-shaped (attached Figure 1When the colonies grow for 5 days in NA medium containing 2% NaCl, they are round, white, smooth, moist, translucent, raised, soft, and have mucus. When picked with an inoculation loop, a distinct sticky feeling can be felt (see attached). Figure 1 The strain grows best at 28°C-34°C, pH 6.0, and 2%-5% NaCl. Physiological and biochemical testing revealed that strain P2 tested positive for oxidase, catalase, and catalase. It is a chemoheterotrophic bacterium that utilizes various sugars or organic acid salts as carbon sources and does not hydrolyze starch, pectin, or cellulose. Growth-promoting screening tests demonstrated that the strain has growth-promoting properties, including polysaccharide production, nitrogen fixation, siderophore production, and ACC deaminase production, with an IAA (indole-3-acetic acid) yield of 77.857 mg / L.

[0030] (2) In the GN III assay, the available carbon sources were: D-mannitol, aminoacetyl-L-proline, D-galacturonic acid, melibiose, D-fructose, D-arabinol, L-alanine, L-galacturonolactone, D-trehalose, D-galactose, inositol, L-aspartic acid, D-glucuronic acid, N-acetyl-D-glucosamine, D-fructose, D-glucose-6-phosphate, L-glutamic acid, glucuronamide, α-ketoglutaric acid, acetoacetic acid, L-fructose, D-fructose-6-phosphate, L-histamine, mucic acid, D-malic acid, D-turanose, N-acetyl-D-galactosamine, L-rhamnose, D-aspartic acid, L-pyroglutamic acid, L-malic acid, acetic acid, inosine, L-serine, saccharic acid, bromo-succinic acid, and formic acid.

[0031] (3) Molecular identification of strain P2

[0032] Extraction of PCR template DNA: The purified bacteria were inoculated into 2% NaCl NA solid medium and cultured at 30°C for 2 days. The bacteria were collected and the total genomic DNA was extracted using a DNA extraction kit.

[0033] PCR amplification: Specific primers were used: 227F: 5'-AGAGTTTGATCCTGGCT CAG-3', 1492R: 5'-GGTTACCTTGTTACGACTT-3'; the total volume of the PCR reaction system was 25 μL, and the PCR amplification conditions were: 94°C for 5 min; 94°C for 45 s, 56°C for 45 s, and 72°C for 45 s, 30 cycles; 72°C for 10 min.

[0034] Sequencing: The PCR amplification product was detected by electrophoresis and purified before sequencing to obtain a 1373 bp 16S rRNA sequence, as shown in SEQ ID NO: 1 provided in the attached document. The obtained sequence was compared and analyzed using the common NCBI website. A homology search of the sequenced DNA sequence with the GeneBank database revealed that P2 had the highest homology with the 16S rRNA sequence of Phyllobacterium ifriqiyense (Lepidoptera: Tunisiensis), reaching 99.93%. A phylogenetic tree of the bacterium was constructed using MEGA software, as shown in the attached document. Figure 2 .

[0035] Strain P2 was taxonomically identified and named Phyllobacterium sp. through analysis of colony morphology, bacterial morphology, physiological and biochemical characteristics, and 16S rRNA gene sequence. This strain has been deposited with the China Center for Type Culture Collection (CCTCC), a Budapest Treaty International Depository for Microorganisms, at Wuhan University, 16 Luojiashan Road, Wuchang District, Wuhan, Hubei 430072, China. The deposit date is March 10, 2025, and the deposit number is CCTCC No. M2025416.

[0036] Example 3: Preparation of Phyllobacterium sp.P2 CCTCC No.M2025416 bacteria

[0037] After activation, the genus P2 was inoculated on NA solid medium containing 2% NaCl and transferred to NB liquid medium containing 2% NaCl (50 mL in a 500 mL Erlenmeyer flask) and cultured at 30°C and 180 rpm for 18 hours to obtain seed liquid. The seed liquid was inoculated into NB liquid fermentation medium containing 2% NaCl at a ratio of 1:100 and cultured at 30°C and 200 rpm for 24 hours. The highest concentration of the inoculum reached 5.5×10 9 cfu / ml, and the microbial agent is obtained. In actual use, the fermentation agent can be diluted a certain multiple before use according to needs.

[0038] Example 4: Effect of Phyllobacterium sp. P2 CCTCC No. M2025416 on Salt Tolerance and Growth Promotion of Cotton

[0039] Cotton seeds of uniform size were selected, washed with sterile water, and then soaked overnight in the bacterial suspension prepared in Example 3, diluted to 1%. Sterile water was used as a control. Ten cotton seeds, treated with the bacterial suspension and sterile water (CK), were placed in culture dishes containing 0, 5, 7.5, 10, and 15 g / kg NaCl concentrations for a hydroponic culture experiment. The seeds were incubated at 30°C and watered regularly to maintain a constant NaCl concentration. After two weeks of cultivation, various growth indicators of the cotton plants in the experimental and control groups were measured, including seedling emergence rate, root length, plant height, and fresh weight. The cotton growth measurement results, shown in Table 1, show that, compared to the CK treatment, the P2 seed soaking treatment maintained a significantly better growth-promoting effect on cotton seedlings with increasing salt concentration. In particular, at a NaCl concentration of 15 g / kg, the P2 seed soaking treatment significantly increased seedling emergence rate, root length, plant height, and fresh weight by 50%, 170%, 650%, and 180%, respectively, compared to the CK treatment. This indicates that Leaf Bacterium can improve the salt tolerance of cotton and promote its growth.

[0040] Table 1: Effects of seed soaking with Phyllobacterium P2 strain on cotton growth under different salt stress concentrations

[0041]

[0042] Example 5: Preparation of aged cotton stalk biochar

[0043] Cotton stalks free of obvious pests and diseases and low in impurities are collected, crushed, and then carbonized in a carbonization furnace. The carbonization temperature is 400-450°C for 1-1.5 hours. After carbonization, the biochar is cooled to 25°C in a cooler, sieved through a 0.25mm mesh, and ground to obtain fresh cotton stalk biochar powder. The carbonized cotton stalk charcoal is then placed in a natural environment and aged for two years to obtain aged cotton stalk charcoal.

[0044] Example 6: Combined application of Phyllobacterium sp. P2 CCTCC No. M2025416 and aged cotton stalk charcoal

[0045] The soil used in this application was collected from a moderately saline-alkali soil (8g / kg) from a long-term cotton cropping operation at the Paotai Soil Improvement Experimental Station, 121st Regiment, 8th Agricultural Division, Xinjiang Production and Construction Corps, Shihezi Reclamation Area, Xinjiang. The topsoil, litter, film residue, gravel, and weeds were removed, and the 0-20cm topsoil layer was excavated. The soil was thoroughly mixed and sieved for use in potted plant experiments.

[0046] Select cotton seeds of the same size, wash them with sterile water, and soak them in the bacterial suspension diluted to 1% prepared in Example 3 overnight. The control treatment is sterile water. The moderate saline-alkali soil prepared in the early stage is placed in a plastic flower pot with a diameter of 20 cm and a height of 15 cm at 3.0 kg per pot for standby use. The experiment set up 4 potted treatments, namely, the sowing treatment of cotton seeds soaked in sterile water (CK), the treatment of applying 2% aged cotton stalk charcoal as base fertilizer and soaking cotton seeds in sterile water (B), the soaking treatment of leaves with a suspension of bacteria (P2), and the treatment of soaking leaves with 2% aged cotton stalk charcoal (B+P2). Ten cotton seeds with different treatments are sown in a flower pot at a sowing depth of about 3 cm, and grown in an outdoor natural environment for 50 days. Example 7: Effect of the combination of leaves with bacteria Phyllobacterium sp.P2 CCTCC No.M2025416 and aged cotton stalk charcoal on cotton growth under saline-alkali stress and continuous cropping obstacles.

[0047] Various growth indicators of the cotton plants grown in the potted test under different treatments in Example 6, including seedling emergence rate, root length, plant height, fresh weight, and leaf chlorophyll content, were measured. The cotton growth measurement results, shown in Table 2, indicate that the combined application of the Phyllobacterium P2 strain and aged cotton stalk charcoal (B+P2) significantly increased the seedling emergence rate by 66.67% compared to the CK treatment and the aged cotton stalk charcoal treatment alone (B), respectively. However, the combination did not increase the seedling emergence rate compared to the Phyllobacterium treatment alone (P2). Compared with the CK treatment, the plant height of the treatments with aged cotton stalk charcoal alone, Leafbacillus P2 alone, and Leafbacillus P2 combined with aged cotton stalk charcoal increased significantly by 18.25%, 27.75%, and 36.00%, respectively; the root length increased significantly by 43.79%, 9.26%, and 44.92%, respectively; the fresh weight increased significantly by 28.26%, 36.87%, and 29.65%, respectively; and the chlorophyll content increased significantly by 67.57%, 87.75%, and 97.12%, respectively. These results indicate that the combined application of Leafbacillus P2 and aged cotton stalk charcoal can increase the plant height, root length, and chlorophyll content of cotton compared with the single application treatment.

[0048] Table 2: Effects of combined application of Phyllobacterium P2 and aged cotton stalk charcoal on cotton growth under salt stress

[0049]

[0050] Example 8: Effects of Phyllobacterium sp. P2 CCTCC No. M2025416 combined with aged cotton stalk charcoal on the microecology of cotton rhizosphere soil under saline-alkali stress and continuous cropping disorder

[0051] 1. Effect of combining Phyllobacterium sp.P2 CCTCC No.M2025416 with aged cotton stalk charcoal in reducing soil salinity and increasing nutrient content in continuous cotton cropping

[0052] The pH, total salt, and nutrient content of the rhizosphere soil of cotton plants grown in a potted experiment using a combination of Leaf Bacillus P2 and aged cotton stalk charcoal were measured. The results of the cotton physical and chemical property measurements, shown in Table 3, indicate that compared to the CK treatment, application of aged cotton stalk charcoal alone and Leaf Bacillus P2 alone did not reduce salt content, while the combined application of Leaf Bacillus P2 and aged cotton stalk charcoal reduced total salt content by 7.50%. Application of aged cotton stalk charcoal alone and Leaf Bacillus P2 combined with aged cotton stalk charcoal significantly increased available nitrogen and available potassium by 8.71% and 91.29%, and by 189.03% and 234.84%, respectively. Application of aged cotton stalk charcoal alone, alone, or in combination with aged cotton stalk charcoal significantly increased available phosphorus by 112.75%, 2.68%, and 150.34%, respectively; organic matter by 97.09%, 2.75%, and 143.94%, respectively; and total nitrogen by 47.50%, 5.00%, and 92.50%, respectively. This indicates that the combined application of Phyllobacterium sp. P2CCTCC No. M2025416 with aged cotton stalk charcoal significantly increased soil nutrient content and reduced total salt content compared to the single application treatment.

[0053] Table 3: Effects of combined application of Phyllobacterium P2 and aged cotton stalk charcoal on the physical and chemical properties of rhizosphere soil in continuously cropped cotton under salt stress

[0054]

[0055] 2. Effect of combining Phyllobacterium sp. P2 CCTCC No. M2025416 with aged cotton stalk charcoal on reducing the relative abundance of cotton pathogenic fungi

[0056] Fusarium wilt caused by Fusarium oxysporum is one of the main soil-borne diseases of cotton. Long-term continuous cropping leads to the accumulation of pathogens in the soil. Based on the results of high-throughput sequencing of cotton rhizosphere soil fungi, see Appendix Figure 3As shown in the results, the relative abundance of Fusarium in the soils treated with aged cotton stalk charcoal alone, Leaf Bacterium P2 alone, and Leaf Bacterium P2 combined with aged cotton stalk charcoal significantly decreased by 34.79%, 18.97%, and 36.83%, respectively, compared with the CK treatment. The combined application of Leaf Bacterium sp. P2 CCTCC No. M2025416 with aged cotton stalk charcoal showed a positive effect in inhibiting the decrease in the relative abundance of Fusarium oxysporum and preventing and controlling cotton wilt.

[0057] 3. The combined application of Phyllobacterium sp. P2 CCTCC No. M2025416 strain and aged cotton stalk charcoal increases the utilization of phenolic acids by cotton rhizosphere soil microorganisms

[0058] The utilization of carbon sources by microorganisms in the rhizosphere soil of the four cotton treatments was determined using the Biolog microplate technique. The optical density values ​​of the utilization of phenolic acids (2-hydroxybenzoic acid and 4-hydroxybenzoic acid) after 120 h can be seen. Figure 4 As shown in the results, treatments CK and P2 did not utilize phenolic acids, while treatments B and B+P2 utilized phenolic acids, with optical density values ​​increasing by 3.00% and 45.10%, respectively. In particular, the ability of cotton rhizosphere soil microorganisms to metabolize phenolic acid autotoxic substances was significantly enhanced after the combined application of Phyllobacterium sp. P2 CCTCC No. M2025416 with aged cotton stalk charcoal. This suggests that the combined application of Phyllobacterium sp. P2 CCTCC No. M2025416 with aged cotton stalk charcoal can promote the growth of microorganisms that utilize phenolic acid as a carbon source, thereby reducing the accumulation of autotoxic substances in the soil and ultimately making the soil ecological environment of continuously cropped cotton healthier and more stable.

[0059] 4. Effect of combining Phyllobacterium sp. P2 CCTCC No. M2025416 with aged cotton stalk charcoal to reduce phenolic autotoxic substances in cotton continuous cropping soil

[0060] Based on LC-MS technology, non-targeted determination of metabolites in the soil was performed. Detection revealed that the phenolic acid autotoxicants in the long-term cotton continuous cropping soil metabolites used in the present invention mainly included p-hydroxycinnamic acid, 4-hydroxycoumarin, and p-hydroxyphenylpropionic acid. Compared with the CK treatment, the phenolic acid autotoxic substances in the soil treated with the combined application of Phyllobacterium sp. P2 and aged cotton stalk charcoal were significantly reduced by 55.11%; the application of aged cotton stalk charcoal alone showed a downward trend, decreasing by 1.56%, but the difference was not significant; while the treatment with the application of Phyllobacterium sp. P2 alone increased by 9.47%, which also did not reach a significant difference. This indicates that the combined application of Phyllobacterium sp. P2 CCTCC No. M2025416 and aged cotton stalk charcoal promoted the growth of microorganisms with phenolic acid degrading functions in the soil, thereby reducing the autotoxic substances in the cotton continuous cropping soil, which is consistent with the results of the Biolog assay. Example 9: Evaluation of the effect of combining Phyllobacterium sp. P2 CCTCC No. M2025416 with aged cotton stalk charcoal on alleviating saline-alkali stress and continuous cropping problems in cotton

[0061] Based on the data from the above examples, this application used the TOPSIS method to analyze data on 17 metrics, including growth indicators (seedling emergence rate, root length, plant height, fresh weight, and chlorophyll content) corresponding to different cotton potted plant treatments; soil physicochemical properties (pH, total salt, available nitrogen, available phosphorus, available potassium, organic matter, and total nitrogen); soil microbial utilization of phenolic acids; the relative abundance of Fusarium oxysporum in the soil; and soil phenolic acid autotoxicants (p-hydroxycinnamic acid, 4-hydroxycoumarin, and p-hydroxyphenylpropionic acid). The four treatments were ranked according to their comprehensive proximity to each other, providing a comprehensive evaluation of cotton growth and rhizosphere soil microecology. The results show that the proximity index (Ci) of the cotton treatments, from best to worst, was B+P2 treatment > B treatment > P2 treatment > CK treatment. The B+P2 treatment had the best overall performance, with a statistical value of 0.942; followed by B treatment and P2 treatment, with statistical values ​​of 0.427 and 0.219, respectively; and the CK treatment had the lowest statistical value, 0.092. It can be seen that the use of Phyllobacterium sp.P2 CCTCC No.M2025416 combined with aged cotton stalk charcoal can better promote cotton growth under saline-alkali stress and continuous cropping obstacles, and create a healthier cotton rhizosphere soil microecological environment.

[0062] Table 4: Evaluation of the application effect of combined application of Phyllobacterium P2 and aged cotton stalk charcoal

[0063] deal with <![CDATA[Distance from the optimal value (D i + )]]> <![CDATA[Distance from the worst value (D i - )]]> <![CDATA[Statistic C i > Ranking CK 1.589 0.161 0.092 4 B 1.177 0.877 0.427 2 P2 1.550 0.435 0.219 3 B+P2 0.100 1.613 0.942 1

[0064] The above embodiments are only for illustrating the technical concept and features of the present invention in a specific scenario. Its purpose is to enable people who need this technology to understand the content of the present invention and implement it. It does not limit the scope of protection of the present invention. Any equivalent changes or modifications made according to the spirit of the present invention should be included in the scope of protection of the present invention.

Claims

1. A bacterium of the genus Leafbacterium Phyllobacterium sp. P2, characterized in that The strain has been deposited in the China Center for Type Culture Collection (CCTCC) with the strain deposit number CCTCC No. M2025416 and the deposit date: March 10, 2025. Its 16S rRNA genome sequence is shown in SEQ ID NO:

1.

2. A Leaf Bacterium Phyllobacterium sp. P2 bacterial agent, characterized in that, The bacterial agent is obtained by fermentation of the strain according to claim 1.

3. A Leaf Bacterium bacterium as claimed in claim 2 Phyllobacterium sp. P2 bacterial agent is a liquid bacterial agent.

4. A Leaf Bacterium Phyllobacterium sp. P2 bacterial agent preparation method, characterized in that, The steps include: Phyllobacterium After activation, sp. P2 was inoculated on NA solid medium containing 2% NaCl and then transferred to NB liquid medium containing 2% NaCl. 50 mL of the liquid medium was placed in a 500 mL Erlenmeyer flask and cultured at 30°C and 180 rpm for 18 h to obtain seed solution. The seed solution was inoculated into NB liquid fermentation medium containing 2% NaCl at a ratio of 1:100 and cultured at 30°C and 200 rpm for 24 h. The highest concentration of the inoculum reached 5.5×10 9 cfu / ml.

5. A Leaf Bacterium Phyllobacterium Application of sp. P2 bacterial agent in improving saline-alkali stress in cotton.

6. A Leaf Bacterium Phyllobacterium Application of sp. P2 fungicide in overcoming the obstacles of continuous cropping of cotton.

7. A method for improving cotton growth under saline-alkali stress and continuous cropping obstacles, characterized in that: In the method, cotton seeds of uniform size are selected, washed with sterile water, and then soaked overnight in the bacterial agent provided in claim 2 diluted to 1%; and 2% aged cotton stalk charcoal is applied as base fertilizer during the planting process.

8. The method for improving cotton growth under saline-alkali stress and continuous cropping obstacles according to claim 7, characterized in that: The aged cotton stalk charcoal is prepared by the following steps: collecting cotton stalks without obvious pests and diseases and with few impurities, crushing them and placing them in a carbonization furnace for carbonization at a carbonization temperature of 400-450°C for 1-1.5 hours. After the carbonization is completed, cooling them to 25°C in a cooler, sieving them through a 0.25mm sieve and grinding them to obtain fresh cotton stalk biochar powder. The carbonized cotton stalk charcoal is placed in a natural environment and aged for two years to obtain aged cotton stalk charcoal.

9. Use of the method for improving cotton growth under saline-alkali stress and continuous cropping obstacles as claimed in claim 7 in cotton planting.

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