Syncomr for promoting plant growth under drought stress and application thereof

Abstract

The present application belongs to the technical field of microbial growth promotion, and particularly relates to a composite microbial inoculant SynComR for promoting plant growth under drought stress and application thereof, both of which have plant growth promotion related functions, including the abilities of producing indole acetic acid, producing iron carrier, and dissolving inorganic phosphorus and decomposing organic phosphorus, etc., can improve the nutrient absorption and utilization efficiency of plants, promote root development and plant growth; meanwhile, the strains in the composite microbial inoculant SynComR have good drought tolerance, can still maintain high growth activity under drought stress conditions, and can enhance the adaptability of alfalfa to adverse environments through the synergistic effect among the strains. The above functions jointly act to promote the growth of alfalfa, improve the plant biomass, and enhance the drought resistance of alfalfa, and have good agricultural application value.

Description

Technical Field

[0001] This invention belongs to the field of microbial growth promotion technology, specifically relating to a compound microbial agent SynComR that promotes plant growth under drought stress and its application. Background Technology

[0002] With global climate change and the development of intensive agricultural production, soil salinization and drought have become increasingly prominent problems, posing significant challenges to forage production and sustainable agricultural development. Salt stress leads to ion imbalance, osmotic stress, and oxidative damage in plants, while drought stress affects water absorption and metabolic activities. Both inhibit plant growth and development, reducing crop yield and quality. Therefore, developing green and efficient biotechnological measures to improve crop salt and drought tolerance has become an important direction in modern agricultural research.

[0003] Alfalfa is an important legume forage crop widely cultivated worldwide, characterized by high yield, rich nutritional value, and strong nitrogen-fixing capacity, playing a vital role in livestock production and ecological restoration. However, alfalfa production is often affected by adverse environmental factors such as saline-alkali soil and insufficient water, leading to decreased seed germination rates, stunted plant growth, and reduced biomass, thus limiting its production potential. Traditional improvement measures mainly rely on stress-tolerant variety breeding, irrigation management, and the application of chemical regulators, but these methods generally suffer from high costs, limited applicability, or insufficient ecological benefits. Therefore, developing safe, environmentally friendly, and sustainable bio-regulation technologies has significant application value.

[0004] In recent years, research has revealed that plant endophytic bacteria, as beneficial microorganisms that colonize plant tissues long-term, form stable symbiotic relationships with their host plants. Seed endophytic bacteria, in particular, can be dispersed to the next generation of plants via seeds and preferentially colonize during seed germination and seedling establishment, significantly influencing early plant growth and development. Compared to exogenous microorganisms from soil or the rhizosphere, seed endophytic bacteria typically exhibit better host adaptability and colonization stability, continuously participating in plant nutrient acquisition, growth regulation, and microbial community construction. Therefore, endophytic bacteria resources derived from alfalfa seeds are considered an important resource for developing alfalfa-specific growth-promoting microbial agents.

[0005] Most existing microbial inoculants are constructed using single strains, but these often suffer from limited functionality, insufficient environmental adaptability, and unstable application effects under complex environmental conditions. With the development of plant microbiome research, it has become increasingly clear that the synergistic effects between multiple functional strains are crucial for plant health and environmental adaptation. Synthetic communities (SynCom) constructed based on different functional strains can fully leverage the functional complementarity and synergistic effects among strains, demonstrating superior application potential compared to single-strain in promoting plant growth and enhancing plant stress resistance.

[0006] However, current microbial growth-promoting agents developed for alfalfa production are mainly single-strain or general-purpose agents, with a lack of compound microbial agents based on alfalfa seed endophytic bacteria resources. Existing products still have limitations in terms of host adaptability, colonization stability, and long-term growth-promoting effects. With the development of plant microbiome research, constructing synthetic microbial communities using seed endophytic bacteria with clear sources and complementary functions has become an important direction for developing novel microbial agents. Therefore, developing a compound microbial growth-promoting agent composed of beneficial endophytic bacteria from alfalfa seeds and applying it to promote alfalfa growth and increase biomass production is of great significance for promoting green and efficient alfalfa production and the development and utilization of microbial resources. Summary of the Invention

[0007] To address the problems existing in the prior art, the primary objective of this invention is to provide a Streptomyces strain with potassium-solubilizing ability (…). Streptomyces G35 was deposited at the China Center for Type Culture Collection on June 10, 2026, with accession number CCTCC M 20261269.

[0008] The second objective of this invention is to provide a *Arthrobacter* ( Arthrobacter G43 was deposited at the China Center for Type Culture Collection on June 10, 2026, with accession number CCTCC M 20261270.

[0009] A third objective of this invention is to provide a *Arthrobacterium* ( Pseudarthrobacter L6 was deposited at the China Center for Type Culture Collection on June 10, 2026, with accession number CCTCC M 20261271.

[0010] The fourth objective of this invention is to provide a *Masses* strain (…). Massilia Y1 was deposited at the China Center for Type Culture Collection on June 10, 2026, with accession number CCTCC M 20261272.

[0011] The fifth objective of this invention is to provide a *Cocheria* genus ( KocuriaZ100 was deposited at the China Center for Type Culture Collection on June 10, 2026, with accession number CCTCC M 20261273.

[0012] The sixth objective of this invention is to provide a compound microbial agent, SynComR, that promotes plant growth under drought stress, wherein the compound microbial agent includes Streptomyces (… Streptomyces G35, Arthrobacter ( Arthrobacter G43, Arthroblastus ( Pseudarthrobacter L6, Masseilles ( Massilia Y1 and Cochlea ( Kocuria One or more of Z100 were deposited at the China Center for Type Culture Collection on June 10, 2026, with accession numbers CCTCC M20261269, CCTCC M 20261270, CCTCC M 20261271, CCTCC M 20261272, and CCTCC M 20261273, respectively.

[0013] The seventh objective of this invention is to provide the application of the aforementioned Streptomyces G35, the aforementioned Arthrobacter G43, the aforementioned Arthrobacterium L6, the aforementioned Masseuse Y1, the aforementioned Coxella Z100, or the aforementioned compound microbial agent SynComR in promoting plant growth under drought stress.

[0014] The eighth objective of this invention is to provide the application of the aforementioned Streptomyces G35, the aforementioned Arthrobacter G43, the aforementioned Arthrobacterium L6, the aforementioned Masseuse Y1, the aforementioned Coxella Z100, or the aforementioned compound microbial agent SynComR in formulations for improving the absorption of phosphorus / iron by plant roots.

[0015] The ninth objective of this invention is to provide the application of the aforementioned Streptomyces G35, the aforementioned Arthrobacter G43, the aforementioned Arthrobacterium L6, the aforementioned Masseuse Y1, the aforementioned Coxella Z100, or the aforementioned compound microbial agent SynComR in increasing the content of available potassium in soil or promoting the absorption of potassium by plants.

[0016] The tenth objective of this invention is to provide the application of the aforementioned Streptomyces G35, the aforementioned Arthrobacter G43, the aforementioned Arthrophagus L6, the aforementioned Masseuse Y1, the aforementioned Coxella Z100, or the aforementioned compound microbial agent SynComR in the preparation of microbial agents for remediating soil / water bodies contaminated with organophosphorus pesticides or for degrading organophosphorus pesticides.

[0017] By adopting the above technical solution, the present invention has the following beneficial effects: (1) The compound microbial agent for alfalfa of the present invention has a significant promoting effect on alfalfa growth, can significantly increase alfalfa dry weight and plant height, and at the same time improve alfalfa’s salt tolerance and drought resistance.

[0018] (2) The compound microbial agent SynComR constructed in this invention is composed of Streptomyces ( Streptomyces G35, Arthrobacter ( Arthrobacter G43, Arthroblastus ( Pseudarthrobacter L6, Masseilles ( Massilia Y1 and Cochlea ( Kocuria The SynComR strains, composed of Z100, all possess plant growth-promoting functions, including the production of indoleacetic acid (IAA), the production of siderophores, and the ability to dissolve inorganic phosphorus and decompose organic phosphorus. These abilities enhance the plant's nutrient absorption and utilization efficiency, promoting root development and plant growth. Simultaneously, the strains in SynComR exhibit good drought tolerance, maintaining high growth activity even under drought stress conditions. Furthermore, synergistic effects among the strains enhance alfalfa's adaptability to adverse environments. These combined functions promote alfalfa growth, increase plant biomass, and strengthen its drought resistance, demonstrating significant agricultural application value. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other embodiments can be obtained based on these drawings without creative effort.

[0020] Figure 1 Colony morphology Figure 2 Plant growth capacity under drought treatment Note: In the horizontal axis, W represents normal watering treatment, D represents drought treatment; CK is the sterilized culture medium control group, S is the compound microbial agent treatment group, and H is the Hogland nutrient solution control group; the vertical axis represents plant height (cm), fresh weight (g), dry weight (g), and leaf area (cm²), respectively. Figure 3 Comparison of growth of potted alfalfa under different treatments Note: DW is the drought + sterilized culture medium control group, DS is the drought + compound microbial agent treatment group, DH is the drought + Hogland nutrient solution control group; CK-W is the normal watering + sterilized culture medium control group, CK-S is the normal watering + compound microbial agent treatment group, CK-H is the normal watering + Hogland nutrient solution control group. Figure 4 Comparison of alfalfa plant growth under different treatments Note: Scale bar 5 cm; Treatment group number is the same as in Figure 3. Figure 5Physiological index analysis Detailed Implementation Embodiments of this embodiment will now be described in more detail with reference to the accompanying drawings. While some embodiments of this embodiment are shown in the drawings, it should be understood that this embodiment can be implemented in various forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided to provide a more thorough and complete understanding of this embodiment. It should be understood that the accompanying drawings and embodiments are for illustrative purposes only and are not intended to limit the scope of protection of this embodiment.

[0021] It should be noted that the methods described in the following embodiments are all conventional methods and can be obtained from the literature; the materials and consumables mentioned can be obtained through commercial channels.

[0022] In this invention, the culture medium formulation used is as follows: The LB medium formula is: 10g peptone, 5g yeast extract, 10g NaCl, 15g agar, and 1000mL distilled water.

[0023] The TSB medium formula is as follows: 17.0 g tryptone, 3.0 g soybean peptone, 5.0 g sodium chloride, 2.5 g dipotassium hydrogen phosphate, 2.5 g glucose, 15 g agar, and 1000 mL distilled water.

[0024] The R2A medium formula is as follows: 0.5 g yeast extract, 0.5 g peptone, 0.5 g casein hydrolysate, 0.5 g glucose, 0.5 g soluble starch, 0.3 g potassium dihydrogen phosphate, 0.024 g anhydrous magnesium sulfate, 0.3 g sodium pyruvate, 15 g agar, and 1000 mL distilled water.

[0025] The cellulose Congo red culture medium formula is as follows: 1 g sodium nitrate, 1.2 g disodium hydrogen phosphate, 0.9 g potassium dihydrogen phosphate, 0.5 g potassium chloride, 0.5 g magnesium sulfate heptahydrate, 0.5 g yeast extract, 0.5 g casein acid hydrolysate, 5 g microcrystalline cellulose, 0.2 g Congo red, 15 g agar, and 1000 mL distilled water.

[0026] The formula for the Monkina culture medium is as follows: 10 g glucose, 0.5 g ammonium sulfate, 0.3 g sodium chloride, 0.3 g magnesium sulfate heptahydrate, 0.03 g manganese sulfate, 0.3 g potassium chloride, 0.03 g ferrous sulfate heptahydrate, 2 g lecithin, 0.5 g yeast extract, 15 g agar, and 1000 mL distilled water.

[0027] The NBRIP medium formula is as follows: 10 g glucose, 0.1 g ammonium sulfate, 5 g magnesium chloride, 0.2 g potassium chloride, 0.25 g magnesium sulfate heptahydrate, 5 g calcium phosphate, 15 g agar, and 1000 mL distilled water.

[0028] The CAS detection medium formula is as follows: 0.0605 g of Chromium Azurite S (CAS), 0.0729 g of hexadecyltrimethylammonium bromide (HDTMA), 0.002645 g of ferric chloride hexahydrate, 0.29525 g of sodium dihydrogen phosphate dihydrate, 0.12135 g of disodium hydrogen phosphate dodecahydrate, 0.125 g of ammonium chloride, 0.0375 g of potassium dihydrogen phosphate, 0.0625 g of sodium chloride, 15 g of agar, and 1000 mL of distilled water.

[0029] Aleksandrov potassium-solubilizing / silicate bacteria culture medium: sucrose 5.0 g, MgSO4·7H2O 0.5 g, Na2HPO4 2.0 g, CaCO3 0.1 g, potassium feldspar powder 1.0 g, FeCl3·6H2O 0.005 g, distilled water to a final volume of 1000 mL, pH 7.0–7.5, agar 15 g / L.

[0030] Example 1: Isolation and Screening of Functional Strains 1. Rhizosphere soil sampling Using Gannong No. 3 alfalfa as the experimental material, rhizosphere soil samples were collected after the potted water control experiment. The loose soil on the surface of the plant roots was gently shaken off, and the rhizosphere soil tightly attached to the root surface was collected as the material for bacterial isolation.

[0031] 2. Strains Isolation and Purification Weigh a rhizosphere soil sample, add it to sterile water, shake thoroughly to mix, prepare a soil suspension, and then use 10... -3 ~10 -6 Serial dilutions were performed. Each serial dilution was plated onto TSA, R2A, and LB broth plates and incubated at 28–30 °C for 48 h. After colonies of different morphologies had grown on the plates, representative single colonies were selected based on colony color, morphology, edge characteristics, and surface features. These colonies were then continuously purified using the streak plating method to obtain pure cultures. A total of 276 culturable strains were isolated.

[0032] 3. Strain identification and screening of dominant strains Genomic DNA of the strain was extracted and purified, and PCR amplification was performed using the 16S rRNA gene as a molecular marker. The amplified products were sequenced by Sanger sequencing, and BLAST sequence alignment was performed using the NCBI database to determine the taxonomic position of the strain.

[0033] Simultaneously, based on the high-throughput sequencing results of 16S rRNA from alfalfa rhizosphere microorganisms, the dominant bacterial genera enriched under drought treatment were analyzed. The 16S rRNA identification results of the isolated strains were matched with the dominant bacterial genera obtained from high-throughput sequencing for screening, resulting in 20 strains from 6 dominant genera, including *Boswellia*. Bosea ) (1 strain: R60), Marseille species ( Massilia ) (4 strains: Y1, Y31, Y32, Y61), Arthrobacter ( Arthrobacter (5 strains: G43, R48, R8, R16, Y26), *Pseudomonas* spp. ( Pseudarthrobacter (4 strains: T31, L6, T1, L30), Coxella spp. ( Kocuria (4 strains: R23, R12, Z100, R10) and Streptomyces ( Streptomyces ) (2 strains: G35, G39).

[0034] Example 2: Screening of Functional Strains The plant growth-promoting and drought-resistant functions of 20 dominant strains were evaluated, including indoleacetic acid (IAA) production capacity, ACC deaminase activity, polyethylene glycol (PEG) drought resistance, organic and inorganic phosphorus solubility, potassium solubilization capacity, and biofilm formation capacity. Each indicator was tested in triplicate.

[0035] 1. Determination of indoleacetic acid (IAA) production capacity Single colonies were picked and inoculated into 10 mL of LB medium containing 100 mg / L tryptophan, and cultured at 30 ℃ and 180 r / min for 24 h. A blank LB medium containing 100 mg / L tryptophan was used as a negative control. After centrifugation at 10000 r·min⁻¹ for 10 min, 100 μL of the supernatant was transferred to a 96-well plate, mixed with an equal volume of Salkowski colorimetric solution, and incubated in the dark for 30 min before measuring the OD. 530 Using IAA standard solution as a positive control, the actual IAA yield was obtained by comparing it with the standard curve.

[0036] Table 1. Indoleacetic acid production capacity

[0037] Note: Different letters indicate significant differences between groups.

[0038] As shown in Table 1, after comparing the OD value (0.13±0.89) with that of the control (CK), and then inserting it into the standard curve, only 9 bacterial strains were found to have the ability to produce IAA, with strains R23, T1, G43, and Z100 exhibiting the highest IAA production capacity.

[0039] 2. Experimental protocol for determining the ACC deaminase capacity of microorganisms (SM medium method): (1) Preparation of culture medium The SM basal medium formula is as follows: 6.0 g dipotassium hydrogen phosphate, 3.0 g potassium dihydrogen phosphate, 0.2 g magnesium sulfate heptahydrate, 5.0 g glucose, 1000 mL distilled water, and pH adjusted to 7.0. SMA medium is the test group, consisting of 0.5 g / L ACC added to the SM basal medium. This component must be sterilized by filtration through a 0.22 μm filter before addition. SMN medium is the positive control group, consisting of 0.5 g / L (NH4)2SO4 (ammonium sulfate) added to the SM basal medium.

[0040] (2) Operating steps The tested bacterial strain was inoculated into SMN liquid medium for activation induction. After 20 h of shaking culture, the bacterial cells were collected by centrifugation at 4 °C, and the supernatant was discarded. The bacterial cells were washed with nitrogen-free SM medium and centrifuged again, repeated twice to completely remove residual nitrogen sources. The washed bacterial cells were resuspended in SM medium and inoculated into SMA liquid medium at a 5% inoculation rate. The medium was then incubated with shaking at 28 °C and 150 rpm for 48 h. After the incubation period, the absorbance (A) at 600 nm was measured using an ELISA reader, with uninoculated SM medium as a zero-control.

[0041] Table 2 ACC deaminase activity

[0042] Note: Different letters indicate significant differences between groups.

[0043] Table 2 shows that, after comparing the OD values ​​with the control (CK), only 14 bacterial strains exhibited ACC deaminase production activity. Among them, strains L6, R48, T1, and G43 showed the highest ACC deaminase production capacity. ACC deaminase effectively reduces the accumulation of ethylene precursors in alfalfa under drought stress by catalyzing the hydrolysis of 1-aminocyclopropane-1-carboxylic acid (ACC), thereby inhibiting stress-induced excessive ethylene production. This mechanism can alleviate the inhibitory effect of ethylene on root growth, promote alfalfa root development and water absorption capacity, and thus significantly enhance the plant's drought resistance.

[0044] 3. PEG tolerance test: TSB medium containing 15% (w / v) polyethylene glycol 6000 (PEG-6000) and conventional TSB medium without PEG-6000 were prepared to simulate drought stress conditions. The concentration of the endophytic bacterial suspension was adjusted to OD. 600 = 0.2, 3 μL was inoculated onto the surface of the two solid culture media mentioned above. Each strain was replicated three times under each treatment condition. After inoculation, the cultures were inverted at 28 ℃ for 3–5 days. The colony diameter (D) under stress conditions was measured and compared with the colony diameter (d) under normal control conditions. The colony size reduction ratio (SI = D / d) was used as the evaluation index to characterize the strain's tolerance to drought stress. The larger this ratio, the weaker the strain's tolerance to the stress condition.

[0045] Table 3. Drought Resistance of Polyethylene Glycol (PEG)

[0046] Note: When p < 0.001, it is represented by ***; when p < 0.01, it is represented by **; when p < 0.05, it is represented by *; when p ≥ 0.05, it is extremely insignificant and is represented by ns.

[0047] Table 3 shows that 15 bacterial strains exhibited significantly reduced colony size under 15% PEG-6000, 4 strains showed no significant change, and 1 strain showed a significant increase in colony size. Specifically, R16 showed an increase in colony size under simulated stress, while G35S, R10, G39, and R23 showed no significant change, and the remaining strains showed a decrease. Polyethylene glycol (PEG), as a high-molecular-weight permeabilizer, can simulate low water potential stress under arid soil conditions. The good growth ability of the target strains in high-concentration PEG medium directly reflects the stability of their cell membrane system to osmotic stress. This strong osmotic adaptability ensures that the strains can maintain normal metabolic activity and population density under water-deficient conditions in the plant rhizosphere, continuously exerting their growth-promoting function, thereby providing stable stress resistance support for the host plant.

[0048] 4. Determination of phosphorus solubility The phosphorus-solubilizing ability of the strains was determined using Monkina medium (organic phosphorus) and NBRIP medium (inorganic phosphorus). Endophytic bacterial cultures were adjusted to OD=0.2 and inoculated onto the two media, with 3 μL inoculated per treatment. Each strain was replicated three times on each medium. The cultures were incubated upside down at 28°C for 3–5 days. The diameter of the phosphorus-solubilizing zone (D) and the colony diameter (d) were measured. The phosphorus-solubilizing ability of the strains was represented by the solubilization index (SI=D / d); a higher ratio indicated a stronger phosphorus-solubilizing ability.

[0049] Table 4 Inorganic phosphorus solubility

[0050] Note: Different letters indicate significant differences between groups.

[0051] Table 4 shows that only 8 bacterial strains exhibited a clear zone on NBRIP medium. Y32 and Y1 showed the strongest ability to dissolve inorganic phosphorus. The phosphorus-solubilizing zones formed by the target strains on NBRIP inorganic phosphorus medium directly demonstrate their ability to secrete organic acids or phosphatases to dissolve insoluble phosphates. These strains can convert inorganic phosphorus, which is difficult for plants to utilize, into absorbable soluble phosphorus. This process increases the supply of available phosphorus in the rhizosphere.

[0052] Table 5 Organophosphorus solubility

[0053] Note: Different letters indicate significant differences between groups.

[0054] Table 5 shows that only 15 bacterial strains exhibited a clear zone on Monkina medium. Among them, Y1, R23, L30, and R48 showed the strongest organic phosphorus solubility. The phosphorus-solubilizing zone and its solubility index formed by the target strains on NBRIP inorganic phosphorus medium directly reflect their ability to dissolve insoluble inorganic phosphates. A higher phosphorus-solubilizing index indicates a higher efficiency in converting inorganic phosphorus that is difficult for plants to utilize into soluble phosphorus. This efficient phosphorus-solubilizing ability helps increase the supply of available phosphorus in the rhizosphere, improves the phosphorus nutrition status of plants under drought stress, and thus indirectly supports the normal physiological metabolism of plants under adverse conditions, ultimately enhancing the drought resistance of plants.

[0055] 5. Determination of potassium-soluble capacity: The potassium-solubilizing ability of the strains was determined using Alexandrov medium. Endophytic bacterial cultures were adjusted to OD=0.2 and inoculated onto the two aforementioned media, with 3 μL inoculated per treatment. Each strain was replicated three times on each medium. The cultures were incubated upside down at 28°C for 3–5 days. The diameter of the clear zone (D) and the colony diameter (d) were measured. The potassium-solubilizing ability of the strains was represented by the solubilization index (SI=D / d); a higher ratio indicated a stronger potassium-solubilizing ability.

[0056] Table 6 Potassium solubilization capacity

[0057] Note: Different letters indicate significant differences between groups.

[0058] Table 6 shows that only nine bacterial strains exhibited a distinct clear zone on Alexandrov medium. Among them, Z100, T31, Y26, and G35 demonstrated the strongest potassium-solubilizing ability. The clear zones formed by the target strains on Alexandrov medium and their potassium-solubilizing index directly reflect their ability to decompose silicate minerals and release soluble potassium. A higher potassium-solubilizing index indicates a higher efficiency in converting insoluble potassium in the soil into a form that can be absorbed by plants. This efficient potassium-solubilizing ability helps increase the supply of available potassium in the rhizosphere, improves the potassium nutrition status of plants under drought stress, and thus indirectly supports the normal physiological metabolism of plants under adverse conditions, ultimately enhancing the drought resistance of plants.

[0059] 6. Biofilm formation ability Dilute the bacterial suspension of the test strain to OD using LB medium. 600 =0.2, take 150 μL into a 96-well plate and incubate at 28℃ for 48 h. Discard the supernatant and wash unbound cells with 150 μL of sterile water (or PBS). After air-drying for 45 min, add 150 μL of 0.1% (w / v, 0.1 g in 100 mL) crystal violet solution and let stand for 20 min for staining. Then wash 3 times with sterile water and air-dry, add 200 μL of 95% ethanol or 33% glacial acetic acid, and let stand for 20 min to dissolve the crystal violet. Measure the absorbance at 590 nm, using blank culture medium as a control, and repeat 5 times for each sample. Set up the culture medium without SynCom seeding as a negative control, and twice its OD value is the cutoff value (Dc). Based on the comparison between the D value and the threshold value Dc, we can divide the results into three categories: (1) When the D value is greater than 2 times Dc, it indicates that the bacterial community has a strong biofilm formation ability; (2) When the D value is between Dc and 2 times Dc, it indicates that the bacterial community has a relatively weak biofilm formation ability; (3) When the D value is less than or equal to Dc, it is considered that the bacterial community does not form a biofilm or has a very weak formation ability.

[0060] Table 7 Biofilm formation capacity

[0061] Note: Different letters indicate significant differences between groups.

[0062] Table 7 shows that, after comparing the OD values ​​with the control (CK), only 7 bacterial strains exhibited strong biofilm production capabilities. Among them, G35 and G39 showed the strongest biofilm production. Biofilms, as an important barrier structure for bacterial colonization in the rhizosphere, protect the bacteria from direct damage caused by environmental stresses such as drought and hyperosmolarity, while also helping the strains form stable and persistent microcolon communities on the plant root surface. Strong biofilm formation capability means that the strains can maintain a high rhizosphere survival rate and population density under water-scarce conditions, thus continuously exerting their growth-promoting function and ultimately enhancing the plant's drought resistance.

[0063] Based on the above results, a screening process was conducted considering both phylogenetic representativeness and functional complementarity, building upon the dominant genera in drought response. First, six dominant genera closely related to drought response and capable of stable isolation and culture were identified based on 16S sequencing results. Within each genera, a comprehensive evaluation was performed using multiple indicators, including IAA production, ACC deaminase activity, PEG tolerance, organic and inorganic phosphorus solubility, potassium solubilization, and biofilm formation. To avoid assigning excessive weight to any single strain, representative strains were selected and mixed in equal proportions to construct a synthetic microbial community (SynCom). Five strains exhibiting excellent growth-promoting and drought-resistant properties were screened, namely *Streptomyces* (…). Streptomyces G35, Arthrobacter spp. Arthrobacter G43, *Arthrobacterium* ( Pseudarthrobacter L6, Masséria genus ( Massilia Y1 and Cochlea spp. Kocuria Z100.

[0064] Among them, Streptomyces ( Streptomyces G35 was deposited at the China Center for Type Culture Collection on June 10, 2026, with accession number CCTCC M 20261269. The deposit address is Wuhan University, Wuhan, China. The contact number is 027-68754052.

[0065] Arthrobacter spp. ( Arthrobacter G43 was deposited at the China Center for Type Culture Collection on June 10, 2026, with accession number CCTCC M 20261270. The deposit address is Wuhan University, Wuhan, China. The contact number is 027-68754052.

[0066] genus *Arthrobacter* ( Pseudarthrobacter L6 was deposited at the China Center for Type Culture Collection on June 10, 2026, with accession number CCTCC M 20261271. The deposit address is Wuhan University, Wuhan, China. The contact number is 027-68754052.

[0067] genus *Massezia* MassiliaY1 was deposited at the China Center for Type Culture Collection on June 10, 2026, with accession number CCTCC M 20261272. The depository address is Wuhan University, Wuhan, China. The contact number is 027-68754052.

[0068] genus Cochlea Kocuria Z100 was deposited at the China Center for Type Culture Collection on June 10, 2026, with accession number CCTCC M 20261273. The deposit address is Wuhan University, Wuhan, China. The contact number is 027-68754052.

[0069] In the following embodiments, the inventors prepared a compound bacterial agent from the above five strains and conducted preliminary experiments. They found that the performance of the prepared compound bacterial agent was significantly superior to that of any single strain. This superior performance included the ability to produce indoleacetic acid, polyethylene glycol, ACC dehydrogenase activity, potassium solubilization ability, inorganic phosphorus dissolution, organic phosphorus decomposition, and biofilm formation. The compound bacterial agent was also used in plant growth-promoting experiments, and the results showed a significant growth-promoting effect. To save space, some experiments have been omitted; the specific experiments are as follows: Example 3: Cultivation of Functional Strains The Streptomyces species obtained from screening in Example 2 ( Streptomyces G35, Arthrobacter spp. Arthrobacter G43, *Arthrobacterium* ( Pseudarthrobacter L6, Masséria genus ( Massilia Y1 and Cochlea spp. Kocuria Z100 was inoculated onto 1 / 2 TSB solid medium and incubated at 28 ℃ for 24–48 h. Single colonies were then picked for later use.

[0070] Single colonies of each strain were picked and inoculated into 200 mL sterile Erlenmeyer flasks containing 50 mL of 1 / 2 TSB liquid medium. The flasks were then shaken and cultured at 28 °C and 180 rpm for 48 h to obtain the fermentation broth of each strain.

[0071] After cultivation, the OD of the bacterial culture was determined by aseptic spectrophotometry. 600 The values ​​were adjusted to OD values ​​using sterile 1 / 2 TSB liquid medium. 600 =0.2, so that each strain has the same cell concentration, providing a basic bacterial solution for the subsequent preparation of compound bacterial agents.

[0072] The obtained bacterial solution has good activity and is free from contamination by other microorganisms, and can be used to prepare compound bacterial agents.

[0073] Example 4: Preparation of a liquid compound microbial agent for promoting growth and resisting drought in alfalfa Take the Streptomyces genus obtained in Example 2 ( StreptomycesG35, Arthrobacter spp. Arthrobacter G43, *Arthrobacterium* ( Pseudarthrobacter L6, Masséria genus ( Massilia Y1 and Cochlea spp. Kocuria Z100 was mixed in equal volumes under aseptic conditions to prepare alfalfa growth-promoting and drought-resistant compound bacterial solution.

[0074] The above-mentioned bacteria were tested to be of the same OD value, and then gently mixed. The volume ratio of each strain was 1:1:1:1:1. This mixture can be directly used for soaking alfalfa seeds, root irrigation, and potted or field application. The strains in the compound bacterial agent are derived from the dominant rhizosphere flora of alfalfa and have good functional complementarity and rhizosphere colonization ability.

[0075] Example 5: Verification of the growth-promoting and drought-resistant effects of synthetic microbial liquid inoculants 1. Method Using Gannong No. 3 alfalfa as the experimental material, a pot experiment was conducted to evaluate the growth-promoting and drought-resistant effects of the compound microbial agent of this invention. Streptomyces (… Streptomyces G35, Arthrobacter spp. Arthrobacter G43, *Arthrobacterium* ( Pseudarthrobacter L6, Masséria genus ( Massilia Y1 and Cochlea spp. Kocuria Z100 was inoculated into TSB liquid medium and cultured with shaking at 28 ℃ and 180 rpm for 48 h. After the culture was completed, the bacterial solutions were adjusted to the same cell concentration (OD600=0.2) and mixed in equal volumes to prepare a compound bacterial solution.

[0076] Before sowing, alfalfa seeds were soaked in a compound bacterial solution for inoculation. After sowing, 20 mL of the compound bacterial solution was applied to each pot, and after two weeks of cultivation, another 20 mL of the compound bacterial solution was applied. Subsequently, drought stress treatment was initiated, and plant growth indicators were measured after two weeks of cultivation. The control group was maintained at 75% field capacity during the experiment, while the drought group was maintained at 35% field capacity after two weeks of cultivation, and this was continued for two weeks.

[0077] The experiment set up two water conditions: watering treatment and drought treatment. Each water condition had three treatments: (1) adding an equal volume of sterile culture medium (CK); (2) adding the compound bacterial solution of the present invention (S); and (3) adding Hogland nutrient solution (H). A total of six treatments were set up. Each treatment adopted the same culture management measures and added an equal volume of Hogland nutrient solution (H) as a nutrient supplement control.

[0078] After the experiment, the plant height, aboveground fresh weight, aboveground dry weight, and leaf area of ​​each treatment alfalfa plant were measured to evaluate the growth-promoting and drought-resistant effects of the compound microbial agent of the present invention.

[0079] 2. Results Comparison of growth phenotypes of alfalfa under different treatments is as follows: Figure 2 As shown; the comparison results of potted alfalfa under different treatments are as follows. Figure 3 As shown; the results of the comparison of whole alfalfa plants under different treatments are as follows. Figure 4 As shown, under normal irrigation conditions, alfalfa treated with the compound microbial agent (CK-B) of this invention significantly outperformed the sterilized culture medium control (CK-W) and the Hoagland nutrient solution control (CK-H) in all growth indicators. Specifically, the average plant height reached 24.82 cm, an increase of approximately 38% compared to CK-W (17.95 cm); the average aboveground fresh weight was 2.148 g, an increase of approximately 83% compared to CK-W (1.173 g); and the average aboveground dry weight was 0.335 g, an increase of approximately 98% compared to CK-W (0.169 g). Furthermore, leaf area and root fresh weight also showed significant advantages, indicating that the compound microbial agent of this invention has a significant growth-promoting and yield-increasing effect under normal water conditions. Under drought stress conditions, alfalfa treated with the compound microbial agent (DB) of this invention also showed a significant drought resistance advantage. Compared with the drought control group (DW), the average plant height of the DB treatment group was 14.70 cm, an increase of approximately 39% compared with DW (10.57 cm); the average fresh weight of the aboveground parts was 0.661 g, an increase of approximately 41% compared with DW (0.468 g); and the average dry weight of the aboveground parts was 0.143 g, an increase of approximately 54% compared with DW (0.093 g). Meanwhile, the leaf area and root-to-shoot ratio of the DB treatment group were also superior to those of the DW and DH treatments, indicating that the compound microbial agent of this invention can effectively alleviate the inhibitory effect of drought stress on alfalfa growth and significantly enhance the drought resistance of the plants.

[0080] Example 6: Effects of compound microbial agents on the physiological characteristics of alfalfa under drought stress To further verify the effects of the compound microbial agent of this invention on the physiological characteristics of alfalfa under drought stress, functional leaves of plants under each treatment were collected, and the following physiological indicators were measured: 1. Relative water content of leaves (RWC) After weighing the fresh weight (FW) of the leaves, immerse the leaves in deionized water at 4℃ for 24 h. Remove the leaves, wipe off the surface moisture, and weigh the saturated fresh weight (TW). Then, place the leaves at 105℃ for 30 min to fix the green color, and then dry them at 80℃ to constant weight, weighing the dry weight (DW). Calculate the relative moisture content of the leaves using the formula RWC (%) = (FW - DW) / (TW - DW) × 100%.

[0081] 2. Malondialdehyde (MDA) content The MDA content was determined using the thiobarbituric acid (TBA) colorimetric method. 0.5 g of leaf sample was weighed, added to 5 mL of 10% trichloroacetic acid (TCA) solution, and homogenized. The mixture was centrifuged at 4 ℃ and 4000 rpm for 10 min. 2 mL of the supernatant was collected, and 2 mL of 0.6% TBA solution was added. The mixture was reacted in a boiling water bath for 15 min, then rapidly cooled. The absorbance values ​​at 450 nm, 532 nm, and 600 nm were measured, and the MDA content was calculated using the corresponding formula.

[0082] 3. Peroxidase (POD) activity The guaiacol method was used for determination. 0.5 g of leaf sample was weighed, and an appropriate amount of phosphate buffer (pH 7.0) was added. The sample was ground in an ice bath and centrifuged at 12000 rpm for 20 min at 4 ℃. The supernatant was used as the crude enzyme solution. The reaction system contained 50 mM phosphate buffer, 20 mM guaiacol, 40 mM H2O2, and an appropriate amount of crude enzyme solution. The absorbance change at 470 nm was measured, and one unit of enzyme activity (U) was defined as a change in absorbance of 0.01 μL per minute.

[0083] 4. Superoxide dismutase (SOD) activity The enzyme activity was determined using the nitroblue tetrazolium (NBT) photoreduction method. Crude enzyme extraction was performed using the same method as the POD method. The reaction system contained 50 mM phosphate buffer (pH 7.8), 130 mM methionine, 750 μM NBT, 100 μM EDTA-Na2, 20 μM riboflavin, and an appropriate amount of crude enzyme solution. The reaction was carried out under 4000 lx illumination for 20 min, and the absorbance at 560 nm was measured. One unit (U) of enzyme activity was defined as the inhibition of 50% of NBT photoreduction.

[0084] 5. Results The results are as follows Figure 5As shown, under normal watering conditions, the average relative water content of alfalfa leaves treated with the compound microbial agent of this invention (CK-B) reached 91.87%, significantly higher than the 83.60% of the sterilized culture medium control (CK-W), indicating that the compound microbial agent helps maintain good plant moisture status. Simultaneously, the average MDA content of the CK-B treatment was approximately 53.85 nmol / g, lower than the 81.33 nmol / g of CK-W, indicating that the compound microbial agent can effectively reduce the degree of cell membrane lipid peroxidation and alleviate membrane damage. Under drought stress conditions, the average relative water content of alfalfa leaves treated with the compound microbial agent of this invention (DB) was 76.19%, higher than the 74.67% of the drought control group (DW), indicating that the compound microbial agent helps mitigate drought-induced water loss. The average MDA content of the DB treatment was approximately 95.90 nmol / g, lower than the 116.09 nmol / g of DW, indicating that the compound microbial agent can effectively inhibit the accumulation of membrane lipid peroxidation products under drought stress and alleviate cell membrane damage. In addition, the DB treatment maintained high POD and SOD activities, and the antioxidant enzyme system responded more actively, which was beneficial for clearing drought-induced reactive oxygen species and further enhancing the drought resistance of the plants.

[0085] In summary, the compound microbial agent of this invention can significantly increase the biomass and relative leaf water content of alfalfa under normal irrigation conditions, and reduce the level of membrane lipid peroxidation. Under drought stress, it can effectively alleviate drought damage by maintaining high antioxidant enzyme activity, stabilizing cell membrane structure, and maintaining leaf moisture status, thus exhibiting both growth-promoting and drought-resistant effects. Compared with the corresponding control treatment, the compound microbial solution treatment of this invention can significantly promote alfalfa growth, increase plant height, fresh weight, dry weight, and leaf area. Under drought stress, the compound microbial solution treatment can effectively alleviate the inhibitory effect of drought on alfalfa growth, promote plant biomass accumulation, and improve plant drought resistance. It can effectively promote alfalfa growth and improve its growth performance under drought stress, demonstrating good application value.

[0086] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A Streptomyces species with potassium-solubilizing ability ( Streptomyces G35, characterized in that, It was deposited at the China Center for Type Culture Collection on June 10, 2026, with accession number CCTCC M 20261269.

2. A type of arthrobacterium ( Arthrobacter G43, characterized in that, It was deposited at the China Center for Type Culture Collection on June 10, 2026, with accession number CCTCC M 20261270.

3. A type of arthrobacterium ( Pseudarthrobacter L6 was deposited at the China Center for Type Culture Collection on June 10, 2026, with accession number CCTCC M 20261271.

4. A type of muscarinic bacteria ( Massilia Y1, characterized in that, It was deposited at the China Center for Type Culture Collection on June 10, 2026, with accession number CCTCC M 20261272.

5. A species of Coxella ( Kocuria Z100, characterized in that, It was deposited at the China Center for Type Culture Collection on June 10, 2026, with accession number CCTCC M 20261273.

6. A compound microbial agent, SynComR, for promoting plant growth under drought stress, characterized in that... The compound microbial agent includes Streptomyces ( Streptomyces G35, Arthrobacter ( Arthrobacter G43, Arthroblastus ( Pseudarthrobacter L6, Masseilles ( Massilia Y1 and Cochlea ( Kocuria One or more of Z100 were deposited at the China Center for Type Culture Collection on June 10, 2026, with accession numbers CCTCC M20261269, CCTCC M 20261270, CCTCC M 20261271, CCTCC M 20261272, and CCTCC M 20261273, respectively.

7. The application of Streptomyces G35 as described in claim 1, Arthrobacter G43 as described in claim 2, Bacillus pseudoarthroblastus L6 as described in claim 3, Massey's bacterium Y1 as described in claim 4, Coxella Z100 as described in claim 5, or the compound microbial agent SynComR as described in claim 6 in promoting plant growth under drought stress.

8. The application of Streptomyces G35 as described in claim 1, Arthrobacter G43 as described in claim 2, Bacillus pseudoarthroblastus L6 as described in claim 3, Massey's bacterium Y1 as described in claim 4, Coxella Z100 as described in claim 5, or the compound microbial agent SynComR as described in claim 6 in formulations for improving the absorption of phosphorus / iron by plant roots.

9. The application of Streptomyces G35 as described in claim 1, Arthrobacter G43 as described in claim 2, Bacillus pseudoarthroblastus L6 as described in claim 3, Massey's bacterium Y1 as described in claim 4, Coxella Z100 as described in claim 5, or the compound microbial agent SynComR as described in claim 6 in increasing the content of available potassium in soil or promoting the absorption of potassium by plants.

10. The application of Streptomyces G35 as described in claim 1, Arthrobacter G43 as described in claim 2, Bacillus pseudoarthroblastus L6 as described in claim 3, Massey's bacterium Y1 as described in claim 4, Coxella Z100 as described in claim 5, or the compound microbial agent SynComR as described in claim 6 in the preparation of microbial agents for remediating soil / water bodies contaminated with organophosphorus pesticides or for degrading organophosphorus pesticides.

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