Composite microbial agent and application thereof in promoting plant growth

Abstract

The invention discloses a composite microbial agent and application thereof in promoting plant growth. The invention discloses a compound microbial agent, which is characterized in that the compound microbial agent comprises Paenibacillus gelatinolyticus PG04 and Lysinibacillus capsici LC43, the compound microbial agent is preserved in the China Center for Type Culture Collection (CCTCC), and the preservation numbers of the compound microbial agent are respectively CCTCC M 2026304 and CCTCC M 2026305. The two strains have excellent salt tolerance. The two components complement each other in function and have synergistic interaction. Pot experiments show that wheat growth can be remarkably promoted by applying the complex microbial inoculant. The microbial agent disclosed by the invention is eco-friendly and controllable in cost, and has a wide application prospect in agricultural utilization.

Description

Technical Field

[0001] This invention relates to the fields of microbial technology and sustainable agriculture. Specifically, this invention provides a compound microbial inoculant and its application in promoting plant growth. Background Technology

[0002] Saline-alkali land is a widely distributed type of low-yield soil on Earth, characterized by high concentrations of soluble salts (such as...). , , , Salineage (including salinization) severely restricts crop growth and yield. Statistics show that approximately 25% of irrigated land globally is affected by salinization, resulting in about 15% of land losing its productivity, with crop yield reductions in some areas reaching as high as 70%-100%. my country has a large area of ​​saline-alkali land, and developing and utilizing saline-alkali land resources is of significant strategic importance for ensuring food security and promoting sustainable agricultural development.

[0003] Wheat (Triticum aestivum L.), as one of the world's major food crops, is quite sensitive to salt stress. Wheat grown in saline-alkali land often exhibits stunted growth, reduced tillering, yellowing and wilting of leaves, ultimately leading to a significant reduction in yield.

[0004] Traditional methods for improving saline-alkali land mainly include physical methods (such as topsoil and salt leaching), chemical methods (such as applying soil conditioners like gypsum and humic acid), and engineering drainage. However, these methods often involve large-scale engineering projects, high costs, slow results, and are prone to causing secondary salinization. Furthermore, long-term use of chemical conditioners may disrupt soil microbial diversity and ecological balance.

[0005] In recent years, utilizing salt-tolerant microorganisms to improve saline-alkali land and promote crop growth has become a research hotspot. Currently, various microbial strains with salt-tolerant growth-promoting properties have been reported both domestically and internationally, such as *Pseudomonas*, *Bacillus*, and *Kushneria*. However, existing research largely focuses on the application of single strains, and their adaptability to complex saline-alkali environments and the stability of their growth-promoting effects are often limited, making it difficult to truly realize their industrial value.

[0006] Therefore, developing compound microbial agents that can effectively promote wheat growth under salt stress, have multiple growth-promoting functions, and are ecologically safe is of great practical significance for the efficient utilization of saline-alkali land agriculture and the improvement of wheat production capacity. Summary of the Invention

[0007] The present invention aims to at least partially solve one of the technical problems existing in the prior art. To this end, the present invention provides a compound microbial agent and its application in promoting plant growth.

[0008] According to one aspect of the present invention, a composite microbial agent is provided, the composite microbial agent comprising Paenibacillus gelatinilyticus and Lysinibacillus capsica.

[0009] Preferably, the *Paenibacillus gelatinilyticus* strain PG04 is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC M 2026304; and the *Lysinibacillus capsica* strain LC43 is deposited at the same collection with accession number CCTCC M 2026305.

[0010] Preferably, the bacterial agent is a liquid bacterial agent or a solid bacterial agent, containing live cells of the strain PG04 and the strain LC43 and / or their metabolites.

[0011] Preferably, in the bacterial agent, the effective viable count of strains PG04 and LC43 is maintained at 4 × 10⁻⁶. 8 Approximately CFU / mL.

[0012] According to another aspect of the present invention, a strain of Paenibacillus gelatinilyticus PG04 is provided, which is deposited at the China Center for Type Culture Collection with accession number CCTCC M2026304.

[0013] According to another aspect of the present invention, a strain of *Lysinibacillus capsica*, LC43, is provided, which is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC M 2026305.

[0014] According to another aspect of the present invention, the application of the above-mentioned compound microbial inoculant, strain PG04, or strain LC43 in promoting plant growth under salt stress or without salt stress is provided.

[0015] Preferably, the plant is wheat (Triticum aestivum L.).

[0016] Preferably, the application includes increasing at least one of the following: plant biomass, plant height, root length, chlorophyll content, proline content, catalase activity, peroxidase activity, or superoxide dismutase activity.

[0017] Preferably, the compound microbial agent, or the strain PG04, or the strain LC43 is applied to the roots of wheat plants or to the soil where wheat is planted, wherein salt stress refers to a concentration in the soil that inhibits the normal growth of wheat.

[0018] Compared with the prior art, the present invention has the following beneficial effects.

[0019] The *Paenibacillus gelatinilyticus* PG04 and *Lysinibacillus capsica* LC43 used in this invention both exhibit excellent salt stress tolerance and can grow normally in culture media containing 2% to 7% NaCl. Some strains can tolerate salt concentrations as high as 10% to 15%. This high osmotic pressure adaptability enables them to efficiently colonize, survive stably, and continuously promote growth in saline-alkali soils.

[0020] From a functional perspective, the two strains possess different growth-promoting functional profiles and can form a synergistic and complementary mechanism of action in the plant rhizosphere. When combined, they can achieve comprehensive improvement of the plant rhizosphere environment through functional complementarity. This multi-target synergistic mechanism exhibits a more comprehensive and stable growth-promoting effect than a single strain.

[0021] Pot experiments showed that the application of the compound microbial agent described in this invention significantly alleviated the growth inhibition of wheat caused by salt stress. Compared with the salt-stressed control group, the wheat treated with the compound microbial agent showed a 33.24% increase in plant height, a 63.28% increase in chlorophyll content, and a 60.13% increase in total biomass (P < 0.05). In contrast, wheat inoculated with single strains PG04 and LC43, respectively, showed increases in plant height of 23.68% and 25.83%, chlorophyll content of 21.38% and 50.57%, and biomass of 30.17% and 36.71%, respectively. Therefore, the growth-promoting effect of the compound microbial agent is significantly better than that of a single strain, exhibiting a clear synergistic effect.

[0022] Regarding physiological stress resistance, the compound microbial agent described in this invention can enhance the plant's physiological adaptability to salt stress by inducing its own defense system. Specifically, it promotes the accumulation of proline in wheat leaves to maintain cell turgor pressure and alleviate osmotic stress; at the same time, it significantly increases the activities of superoxide dismutase, peroxidase, and catalase, effectively scavenging reactive oxygen species induced by salt stress and reducing membrane lipid peroxidation damage; in addition, it can increase chlorophyll content, maintain photosynthetic efficiency, ensure the accumulation of photosynthetic products, and enable wheat to maintain normal growth and development under salt stress conditions.

[0023] Furthermore, the microbial agent described in this invention is a pure microbial preparation, which will not cause secondary pollution to the soil after application, nor will it damage the soil microbial community structure, thus conforming to the concepts of green agriculture and sustainable development. The preparation process of this microbial agent is simple, and it can be produced on a large scale through conventional liquid fermentation at low cost. Its application methods are flexible, and it can be combined with existing agronomic practices such as root irrigation and drip irrigation, making it suitable for promotion and application in saline-alkali wheat-growing areas. It is of great value for increasing wheat yields in saline-alkali areas, promoting the resource utilization of saline-alkali land, and ensuring food security.

[0024] In summary, this invention provides a unique, complementary, and synergistic compound microbial agent that can effectively promote wheat growth under salt stress and improve its physiological resistance. It also has the advantages of multiple improvement effects, eco-friendliness, and controllable cost, and has broad application prospects in saline-alkali land agricultural utilization, degraded land ecological restoration, and sustainable agricultural development. Attached Figure Description

[0025] The accompanying drawings, which are included to provide a further understanding of the invention and form part of this invention, illustrate exemplary embodiments of the invention and are used to explain the invention, but do not constitute an undue limitation of the invention. In the drawings:

[0026] Figure 1 These are photographs of the colony morphology of Paenibacillus gelatinilyticus PG04(a) and Lysinibacillus capsica LC43(b) provided in the embodiments of the present invention.

[0027] Figure 2 This is a neighbor-joining phylogenetic tree constructed based on the homology of the 16S rRNA gene sequence of strain Paenibacillus gelatinilyticus PG04, according to an embodiment of the present invention.

[0028] Figure 3 This is a neighbor-joining phylogenetic tree constructed based on the homology of the 16S rRNA gene sequence of strain Lysinibacilluscapsica LC43, according to an embodiment of the present invention.

[0029] Figure 4 This is a circular map of the whole genome of Paenibacillus gelatinilyticus PG04 provided according to an embodiment of the present invention.

[0030] Figure 5This is a circular map of the whole genome of Lysinibacillus capsica LC43 provided in an embodiment of the present invention.

[0031] Figure 6 This is a graph showing the results of salinity tolerance testing of Paenibacillus gelatinilyticus PG04 and Lysinibacillus capsica LC43 at different NaCl concentrations, according to embodiments of the present invention.

[0032] Figure 7 These are phenotypic photographs of the inhibitory effect of salt stress on wheat growth according to embodiments of the present invention.

[0033] Figure 8 This is a graph showing the promoting effect of applying Paenibacillus gelatinilyticus PG04 single bacteria, Lysinibacillus capsica LC43 single bacteria, and a compound of the two bacteria on wheat growth under salt stress conditions, as provided in the embodiments of the present invention. In the graph, a is plant height, b is chlorophyll content, c is whole plant dry and fresh weight, and d is sprout dry and fresh weight. If the characters above the bars in the graph do not contain the same letter, it indicates a significant difference (P<0.05).

[0034] Figure 9 These are photographs showing the effects of applying Paenibacillus gelatinilyticus PG04, Lysinibacillus capsica LC43, and a compound inoculum CPDS to wheat under salt stress conditions, as provided in embodiments of the present invention.

[0035] Figure 10 This is a graph showing the effect of applying compound microbial agent CPDS under salt-free conditions on wheat growth according to an embodiment of the present invention. In the graph, a is the total weight of the whole plant, b is the dry fresh weight of the buds, c is the dry fresh weight of the roots, d is the plant height, and e is the root length. If the characters above the bars in the graph do not contain the same letters, it indicates that there is a significant difference (P<0.05).

[0036] Figure 11 These are comparative photographs showing the effect of applying compound microbial agent CPDS under salt-free conditions on the growth phenotype promotion of wheat, according to embodiments of the present invention (left is the control group CK, right is the treatment group CPDS).

[0037] Figure 12These are another set of comparative photos showing the effect of applying compound microbial agent CPDS under salt-free conditions on wheat growth, as provided in the embodiments of the present invention (leftmost and second from the left are control group CK; rightmost and second from the right are treatment group CPDS).

[0038] Figure 13 This is a graph showing the effects of applying Paenibacillus gelatinilyticus PG04, Lysinibacillus capsica LC43, and a combination thereof on wheat metabolites and stress resistance indicators under salt stress conditions, as provided in embodiments of the present invention. In the graph, a represents catalase (CAT) activity, b represents superoxide dismutase (SOD) activity, c represents peroxidase (POD) activity, and d represents proline content. If the characters above the bars in the graph do not contain the same letter, it indicates a significant difference (P<0.05). Detailed Implementation

[0039] While the invention can be embodied in many different forms, what is disclosed herein are specific illustrative embodiments that demonstrate the principles of the invention. It should be emphasized that the invention is not limited to the specific embodiments illustrated herein. Furthermore, any section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter.

[0040] The following examples are provided to help those skilled in the art better understand the present invention. It should be noted that the following examples are not intended to limit the scope of protection claimed by the present invention, but are merely illustrative. Unless otherwise specified, the raw materials, reagents, or devices mentioned in the following examples are commercially available or obtained through known existing methods.

[0041] Microbial materials:

[0042] Paenibacillus gelatinilyticus PG04 and Lysinibacillus capsica LC43 were deposited on January 30, 2026, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Bayi Road, Wuchang District, Wuhan, Hubei Province, China, with accession numbers CCTCC M 2026304 and CCTCC M 2026305, respectively.

[0043] Example 1: Screening and identification of *Paenibacillus gelatinilyticus* PG04 and *Lysinibacillus capsica* LC43 strains.

[0044] 1.1 Screening of strains

[0045] Paenibacillus gelatinilyticus PG04 and Lysinibacillus capsica LC43 were collected from salt-tolerant plants Suaeda salsa and Limonium bicolor in the Yuncheng Salt Lake Nature Reserve, Shanxi Province, respectively. Appropriate amounts of the collected plant samples were rinsed with tap water and placed in 50 ml centrifuge tubes. In a clean bench, sterile water, 300 ml of 75% ethanol, sterile water, 5% NaClO, sterile water, and sterile water were added sequentially to six beakers, and sterile water was added to a wash bottle. The five-step disinfection method was followed: rinsing with tap water three times (already completed), rinsing with sterile water, immersion in ethanol for 1 minute, rinsing with sterile deionized water, and disinfection with 5% sodium hypochlorite for 2 minutes, followed by rinsing 3-4 times. After rinsing, the samples were placed on filter paper for 2 minutes to allow surface moisture to evaporate.

[0046] Fill four 15 ml centrifuge tubes with 9 ml of sterile water. Chop the drained plant material into small pieces and grind it in a mortar. If the juice is insufficient during grinding, add PBS solution in small amounts several times, grinding until a paste-like consistency is achieved, ensuring that 1 ml of the paste is available for aspiration. Transfer 1 ml of the paste to a centrifuge tube containing 9 ml of sterile water; this is 10 ml of the paste. -1 The endophytic bacterial culture at different dilutions can be serially diluted to obtain 10. -3 ~10 -5 Diluent. Take 200 μL of a dilution of 10. -3 10 -4 and 10 -5 Plant tissue homogenate suspensions were spread onto enrichment medium plates, with three replicates for each concentration. The plates were incubated at 30°C for 48 to 72 hours, and the growth of the strains was observed. Single colonies were then transferred to identical solid medium plates and streaked to obtain pure cultures of single colonies. The enrichment medium formulation was as follows: yeast extract 0.5 g; peptone 0.5 g; casein hydrolysate 0.5 g; glucose 0.5 g; soluble starch 0.5 g; potassium dihydrogen phosphate 0.3 g; anhydrous magnesium sulfate 0.024 g; sodium pyruvate 0.3 g; agar 15 g; distilled water 1000 ml; pH 7.2±0.2.

[0047] 1.2 Identification of strains

[0048] The selected and cultured strains were placed under an optical microscope to observe their morphology. (Photos are shown below.) Figure 1 As shown, after 48 hours of cultivation, single colonies of Paenibacillus gelatinilyticus PG04(a) were white, round, and had distinctly white edges. Single colonies of Lysinibacillus capsica LC43 were pale yellow, round, and had a raised and smooth surface.

[0049] Bacterial genomic DNA extraction kits were used to extract bacterial DNA, and PCR amplification was performed using universal primers for the 16S rDNA gene to identify the strain species. The primer sequences for PCR amplification are as follows:

[0050] 27F: 5'-AGAGTTTGATCCTGGCTCAG-3'

[0051] 1492R:5'-TACGGYTACCTTGTTAYGACTT-3'

[0052] Using genomic DNA from the sample as a template, PCR was performed with the primers listed above. The system is shown in Table 1.

[0053] Table 1 PCR Reaction System

[0054]

[0055] The sequence reaction conditions were: ① 94℃ for 5 min; ② 94℃ for 40 s; ③ 56.5℃ for 40 s; ④ 72℃ for 1 min and 30 s; ⑤ ②~④ were repeated 32 times; ⑥ 72℃ for 10 min; ⑦ 16℃ for 10 min.

[0056] The target bands of the PCR amplification products of Paenibacillus gelatinilyticus PG04 and Lysinibacillus capsica LC43 were 1494 bp and 1479 bp, respectively.

[0057] The nucleotide sequence of the 16S rRNA gene fragment of the gelatinilyticus strain Paenibacillus gelatinilyticus PG04 is SEQ ID NO: 1:

[0058]

[0059] The nucleotide sequence of the 16S rRNA gene fragment of *Lysinibacillus capsica* LC43 is SEQ ID NO: 2.

[0060]

[0061] Based on Ezbiocloud sequence comparison, the 16S rRNA sequences with the highest sequence similarity in the Ezbiocloud library were selected as references. Phylogenetic trees between the strain and the reference strains were constructed using the Mega11 software with the nearest neighbor method. The phylogenetic tree construction results for Paenibacillus gelatinilyticus PG04 and Lysinibacillus capsica LC43 are shown below. Figure 2 , 3 As shown.

[0062] The whole genome of *Paenibacillus gelatinilyticus* PG04 was sequenced using the Illumina second-generation high-throughput sequencing platform. The genome size was 4,452,451 bp, with a GC content of 41.6%. The predicted number of coding genes was 3,932; 25 rRNAs; 76 tRNAs; 7 gene islands with a total length of 144,073 bp; and 1 prephage structure with a length of 39,704 bp. The whole genome of *Lysinibacillus capsica* LC43 was sequenced, showing a genome size of 4,750,950 bp and a GC content of 37.43%. The predicted number of coding genes was 4,808; 34 rRNAs; 108 tRNAs; 12 gene islands with a total length of 258,663 bp; and 1 prephage structure with a length of 61,754 bp. The circular genome maps of Paenibacillus gelatinilyticus PG04 and Lysinibacillus capsica LC43 are as follows: Figure 4 , 5 As shown.

[0063] Example 2: Evaluation of the growth-promoting performance of *Paenibacillus gelatinilyticus* PG04 and *Lysinibacillus capsica* LC43.

[0064] To systematically evaluate the plant growth-promoting potential of culturable plant endophytic bacteria, functional analyses were conducted on the strains in terms of auxin synthesis, enzyme activity, polysaccharide secretion, nutrient acquisition, and stress tolerance. The specific methods are as follows. The culture medium formulations used for various functional tests are shown in Table 3.

[0065] 1. Indoleacetic acid (IAA) production capacity

[0066] The purified strain was inoculated into King's B liquid medium, and the production of IAA was detected using the Salkowski colorimetric method. Strains that initially tested positive were cultured at 28 °C and 150 rpm for 3 days, then centrifuged (12,000 rpm, 5 min). The supernatant was mixed with an equal volume of Salkowski reagent, and the reaction was allowed to proceed for 30 min. The absorbance was then measured at 530 nm. The IAA content was calculated by comparing with a standard curve. Uninoculated King's B medium served as a negative control.

[0067] 2. Cellulase activity

[0068] The strain was streaked onto selection medium containing sodium carboxymethyl cellulose (CMC-Na) and cultured at 28 °C for 7 days. After culture, the culture was stained with 0.5% Congo red for 60 min, followed by destaining with 1 N NaCl for 30 min. The appearance of a clear hydrolysis zone around the colony indicated cellulase activity. The cellulose-degrading ability was assessed by measuring the ratio of the clear zone to the colony diameter.

[0069] 3. Extracellular polysaccharide (EPS) production capacity

[0070] The strain was inoculated into LB solid medium and cultured at 30 °C for 48 h. The production of EPS was determined by observing whether the colonies exhibited a mucous or viscous appearance.

[0071] 4. Nitrogen fixation capacity

[0072] Strains capable of producing EPS were inoculated onto Ashby nitrogen-free solid medium, and their growth was observed. Strains that could grow normally under nitrogen-free conditions were identified as having nitrogen-fixing ability.

[0073] 5. Phosphorus solubility

[0074] The phosphate-solubilizing ability of the strains was assessed using NBRIP and Menkina solid medium (containing insoluble tricalcium phosphate). After culturing the strains at 30 °C for 7 days, the phosphate solubility index (SI) was calculated by measuring the diameter of the transparent phosphate-solubilizing zone around the colonies. The formula is as follows:

[0075] SI = (diameter of lysing zone + diameter of colony) / diameter of colony.

[0076] 6. Sidephore generation capability

[0077] The strain was inoculated onto CAS (Chrome Azurol S) medium and cultured at 28 °C for 5 days. A change in medium color from blue to orange indicates that the strain produced siderophores; the size of the orange halo reflects the strength of siderophore production.

[0078] 7. Salt tolerance

[0079] After preparing the bacterial strain into a suspension, it was inoculated into R2A liquid medium containing different NaCl concentrations (2%, 3.5%, 5%, 7%, 10%, 15%) and cultured for one week at 30 °C and 150 rpm. After the culture was completed, the medium was shaken thoroughly, and the OD was measured at 600 nm. 600 The values ​​were used to assess the growth ability of the strain at different salt concentrations.

[0080] Table 3. Culture medium formulations used for functional testing

[0081]

[0082] Note: Trace element solution (g / L): Ferrous sulfate heptahydrate, 0.1 g; Manganese chloride tetrahydrate, 0.1 g; Zinc sulfate heptahydrate, 0.1 g

[0083] like Figure 6 As shown, both *Paenibacillus gelatinilyticus* PG04 and *Lysinibacillus capsica* LC43 can grow in salt solutions of different concentrations (Lysinibacillus capsica LC43 has slightly weaker salt tolerance). Their growth is inhibited to varying degrees with increasing NaCl concentration, but overall they exhibit strong salt tolerance. This indicates that these strains possess good osmotic regulation capabilities and cell membrane stability in high osmotic pressure environments. This stability allows them to survive and function continuously in soils with severe salt accumulation, giving them a competitive advantage in the saline soil microecology.

[0084] The results of the growth-promoting experiments are shown in Table 4. The results of this study indicate that *Paenibacillus gelatinilyticus* PG04 possesses multiple growth-promoting functions, including cellulase production, phosphate solubilization, extracellular polysaccharide (EPS) synthesis, and biological nitrogen fixation, while *Lysinibacillus capsica* LC43 possesses multiple growth-promoting functions, including siderophore production and cellulase production. This synergistic growth-promoting mechanism lays the physiological foundation for the strains' function in complex habitats: by continuously providing endogenous growth regulators and key nutrients to plant roots, these strains can not only effectively regulate the morphological development of plant roots but also significantly optimize rhizosphere nutrient absorption efficiency, thus demonstrating great application potential in promoting plant biomass accumulation.

[0085] Table 4 Results of the growth-promoting characteristic test

[0086]

[0087] Example 3: Effects of a complex of *Paenibacillus gelatinilyticus* PG04 and *Lysinibacillus capsica* LC43 on wheat growth and physiological characteristics under salt stress.

[0088] The effects of salt-tolerant growth-promoting bacterial strains on wheat growth under salt stress were tested using pot experiments. NaCl was selected... , and A series of salt solutions with total salt concentrations of 0 g / kg, 3 g / kg, 6 g / kg, 9 g / kg, 12 g / kg, and 15 g / kg were prepared according to a molar ratio of 1:1:1:1 and used for soil irrigation. The results showed that when the salt solution concentration reached 12 g / kg, wheat growth was significantly inhibited, specifically manifested as stunted growth, yellowing and wilting of leaves, etc. Figure 7 To evaluate the effects of selected bacterial isolates on plant growth under saline conditions, a pot experiment was conducted. Potting soil was used in the experiment, and NaCl was selected. , and A salt solution with a total salt concentration of 12 g / Kg was prepared by mixing the amounts of the substances in a 1:1:1:1 ratio. The solution was then used to thoroughly water the potting soil to prepare saline-alkali soil (150 g potting soil + 120 mL salt solution). The salt content of the soil was measured to be 5.33 g / Kg based on its wet weight.

[0089] 1. Wheat seedlings

[0090] Select plump, uniformly sized wheat seeds and immerse them in 75% (v / v) ethanol. Shake the seeds inverted position to ensure thorough contact with the ethanol, then let them stand for no more than 3 minutes. Remove the ethanol, add sterile water, shake thoroughly, and then remove the sterile water. Next, add a 1% sodium hypochlorite solution (available chloride ions), shake thoroughly, and let stand for no more than 8 minutes. Remove the sodium hypochlorite solution and rinse the seeds 4-5 times with sterile water. Place the seeds in sterile petri dishes lined with two layers of moist sterile filter paper and incubate at 28°C in the dark. Once the seeds show signs of sprouting, select seedlings of uniform growth and transplant them into seedling trays. The experiment used potting soil and added a salt mixture (NaCl, sodium chloride ... , and The culture medium is mixed with soil and vermiculite in a ratio of 3:1 (v / v), and 15 seeds are sown in each pot.

[0091] 2. Preparation and application of microbial agents

[0092] The experiment consisted of four treatment groups: CK (salt-treated and sterile), PG04 (salt-treated and sterile), LC43 (salt-treated and sterile), and CPDS (salt-treated and sterile) to verify the growth-promoting effect of the strain on wheat under salt stress.

[0093] The bacterial strain stored at 4°C was slowly thawed, inoculated into LB liquid medium, and cultured with shaking for 24 h. Then, 1% of the inoculum was added to freshly prepared LB liquid medium and cultured in a shaker at 28°C in the dark for 72 h. It was then stored at 4°C for later use. Before use, the culture was centrifuged, rinsed three times with sterile water, and diluted with sterile ultrapure water to control the colony count to within OD0.05. 600 An inoculum with a concentration of 0.5 was used to obtain a uniform total bacterial count. The aim was to maintain consistent initial bacterial concentrations in the three inoculation groups (PG04, LC43, and CPDS) at inoculation, ensuring a substantially similar number of viable bacteria per unit volume and guaranteeing the comparability and reproducibility of experimental results. CPDS was prepared by mixing PG04 and LC43 strains at a 1:1 volume ratio. Before mixing, the bacterial concentrations of both strains were adjusted to OD values. 600 =0.5, to ensure a consistent number of viable bacteria during mixing, thereby guaranteeing a relatively balanced inoculation amount of each strain in the compound microbial agent.

[0094] Specifically, after wheat seedlings have grown 3-4 pairs of true leaves (approximately 14 days) after being transplanted into seedling trays, the soil around the wheat roots is irrigated with 1 mL of a prepared bacterial suspension. The control group received irrigated with sterile water. Irrigation was performed every 3 days for a total of 2 times. In terms of application, the bacterial strain is prepared into a standardized bacterial suspension through liquid fermentation and then applied to the plant roots. The bacteria colonize the rhizosphere, forming a stable micro-ecological community, continuously releasing growth-promoting metabolites and interacting positively with the plant roots. This inoculation process can be combined with water-saving technologies such as drip irrigation to achieve precise, quantitative, and efficient application, making it suitable for large-scale application in farmland and saline-alkali land.

[0095] 4. Growth index measurement

[0096] Wheat growth indicators were measured: plant height and root length were measured using a ruler, and fresh and dry weights of the above-ground and underground parts were measured using an electronic balance. Chlorophyll content was measured using ultraviolet spectrophotometry. Each indicator was replicated in nine cases. The experimental data were analyzed and plotted using Excel and GraphPad Prism (10.1.2) software. A statistically significant difference was indicated if the characters above the bars in the graph did not contain the same letter (P<0.05). Inoculation with salt-tolerant growth-promoting bacteria to prepare a bacterial suspension could alleviate the growth inhibition of wheat caused by salt stress to some extent, as shown in the figure. Figure 8 and Figure 9 As shown in the figure. Compared with the blank control group that was subjected to salt stress but did not have bacterial suspension added, the plant height, chlorophyll content, and biomass of wheat treated with strain PG04 increased by 23.68%, 21.38%, and 30.17%, respectively (P < 0.05); the plant height, chlorophyll content, and biomass of wheat treated with strain LC43 increased by 25.83%, 50.57%, and 36.71%, respectively (P < 0.05); and the plant height, chlorophyll content, and biomass of wheat treated with strain CPDS increased by 33.24%, 63.28%, and 60.13%, respectively (P < 0.05).

[0097] The experiment consisted of two treatment groups: CK (salt-free and sterile) and CPDS (salt-free and bacterial) to verify the growth-promoting effect of the strain on wheat under no-stress conditions. Specifically, after wheat seedlings had grown 3-4 pairs of true leaves (approximately 14 days) after being transplanted into seedling trays, the soil around the wheat roots was irrigated with 1 mL of a prepared bacterial suspension. The control group received irrigated with sterile water. Irrigation was performed every 3 days for a total of 2 times. In terms of application, the strain was prepared into a standardized bacterial suspension through liquid fermentation and then applied to the plant roots. The bacteria colonized the rhizosphere, forming a stable micro-ecological community, continuously releasing growth-promoting metabolites and interacting positively with the plant roots. This inoculation process can be combined with water-saving technologies such as drip irrigation to achieve precise, quantitative, and efficient application, making it suitable for large-scale application in farmland and saline-alkali land. Figure 10 , 11 As shown in Figures 1 and 12, wheat treated with strain CPDS showed increases in plant height, root length, aboveground biomass, underground biomass, and total biomass (fresh weight) of 18.88%, 14.78%, 12.78%, 9.33%, and 11.99%, respectively. Aboveground biomass, underground biomass, and total biomass (dry weight) increased by 19.52%, 12.51%, and 16.13%, respectively (P < 0.05).

[0098] 5. Physiological index measurement

[0099] Wheat growth indicators were determined: Proline, catalase (CAT), peroxidase (POD), and superoxide dismutase (SOD) levels in wheat treated with *Paenibacillus gelatinilyticus* PG04, *Lysinibacillus capsica* LC43, and a compound microbial agent under salt stress were measured. Proline levels were determined using the ninhydrin colorimetric method. Antioxidant enzyme activities were determined according to the methods described in *Plant Physiology Experimental Techniques Tutorial*. Specifically, POD activity was determined using the guaiacol method, CAT activity was determined using UV spectrophotometry, and SOD activity was determined using a superoxide dismutase (SOD) activity assay kit (WST-8, colorimetric method). The experimental data were analyzed and plotted using Excel and GraphPad Prism (10.1.2) software. A statistically significant difference (P<0.05) was indicated if the characters above the bars in the graph did not contain the same letter. Figure 13 As shown, under salt stress, LC43, PG04, and their compound microbial agent CPDS can all affect wheat stress-related physiological indicators, but their modes of action differ. Compared with single-strain treatments, CPDS showed a significant advantage in CAT and SOD activities, indicating that the combination of LC43 and PG04 can more effectively activate the wheat's antioxidant defense system. Regarding POD activity, there was no significant difference between CPDS and LC43 treatments, both significantly higher than PG04 and CK, indicating that the compound treatment retained the promoting effect of LC43 on POD. Notably, LC43 significantly increased proline content, while the CPDS treatment had the lowest proline content, suggesting that the compound microbial agent does not simply superimpose the effects of single strains, but may instead promote a shift in wheat's salt tolerance strategy under salt stress from a proline accumulation-based response to one primarily focused on enhancing the antioxidant enzyme system. In summary, CPDS mainly alleviates salt stress by enhancing antioxidant capacity, demonstrating the complementary advantages of the compound microbial agent in stress resistance regulation.

[0100] The purpose of this invention is to provide a compound microbial agent, Paenibacillus gelatinilyticus PG04 and Lysinibacillus capsica LC43, and its application. The compound microbial agent is suitable for improving the growth of plants under salt stress, and has multiple improvement effects, strong adaptability, and eco-friendliness. It has broad application prospects for promoting the growth of plants in saline-alkali land, helping the ecological restoration of saline-alkali land, sustainable agricultural development, and the management of degraded land.

[0101] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A compound microbial agent, characterized in that, The compound microbial agent contains Paenibacillus gelatinilyticus and Lysinibacillus capsica.

2. The composite microbial agent according to claim 1, characterized in that, The *Paenibacillus gelatinilyticus* strain PG04 is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC M 2026304; the *Lysinibacillus capsica* strain LC43 is deposited at the same collection with accession number CCTCC M 2026305.

3. The composite microbial agent according to claim 2, characterized in that, The bacterial agent is a liquid or solid bacterial agent, containing live cells of strains PG04 and LC43 and / or their metabolites.

4. The composite microbial agent according to claim 3, characterized in that, In the bacterial agent, the effective viable count of strains PG04 and LC43 is maintained at 4 × 10⁻⁶. 8 Approximately CFU / mL.

5. A strain of *Paenibacillus gelatinilyticus*, PG04, characterized in that... It is deposited at the China Center for Type Culture Collection, with accession number CCTCC M 2026304.

6. A strain of *Lysinibacillus capsica*, LC43, characterized in that... It is deposited at the China Center for Type Culture Collection, with accession number CCTCC M 2026305.

7. The application of the compound microbial agent according to any one of claims 1-4, or the strain PG04 according to claim 5, or the strain LC43 according to claim 6 in promoting plant growth under salt stress or without salt stress.

8. The application according to claim 7, characterized in that, The plant in question is wheat (Triticum aestivum L.).

9. The application according to claim 8, characterized in that, The application includes increasing at least one of the following: plant biomass, plant height, root length, chlorophyll content, proline content, catalase activity, peroxidase activity, or superoxide dismutase activity.

10. The application according to claim 7, characterized in that, The compound microbial agent, or the strain PG04, or the strain LC43 is applied to the roots of wheat plants or to the soil where wheat is planted. Salt stress refers to a concentration in the soil that inhibits the normal growth of wheat.

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