Klebsiella Michaeli MX-56 and application thereof

Abstract

The invention discloses a Klebsiella Michaeli MX-56 strain and application thereof, and belongs to the technical field of biological agriculture. The Klebsiella michelianensis MX-56 disclosed by the invention is preserved in the Guangdong Microbial Culture Collection Center, the preservation number is GDMCC No: 66361, the preservation time is May 19, 2025, and the preservation address is No. 100, Xianlie Middle Road, Guangzhou City. The invention also discloses an application of the Klebsiella michelianensis MX-56 in preparation of a divaricate saposhnikovia root growth promoting agent and a method for promoting growth of divaricate saposhnikovia root by using the Klebsiella michelianensis MX-56. A root irrigation method is adopted to irrigate MX-56 bacterial liquid. The divaricate saposhnikovia root endophytic bacterium Klebsiella michelianensis MX-56 disclosed by the invention has the growth promoting characteristics of fixing nitrogen, dissolving inorganic phosphorus, and secreting IAA (Indoleacetic Acid) and an iron-producing carrier; the strain can be stably colonized in the soil around the divaricate saposhnikovia roots; a good growth promoting effect is achieved on wind prevention.

Description

Technical Field

[0001] This invention belongs to the field of bio-agricultural technology, specifically relating to a strain of Klebsiella Michigani MX-56 and its applications. Background Technology

[0002] *Saposhnikovia divaricata* (Turcz.) Schisck., belonging to the genus *Saposhnikovia* in the family Apiaceae, is used medicinally for its unbolted, dried roots. It is a commonly used and widely distributed traditional Chinese medicine with significant effects such as relieving exterior syndromes, dispelling wind, eliminating dampness, and stopping spasms. Wild *Saposhnikovia divaricata* resources are mainly distributed in Northeast, Northwest, and North my country, with a concentrated distribution in the central and western regions of Jilin Province. However, with the continuous growth of market demand, over-harvesting has led to a sharp decline in wild *Saposhnikovia divaricata* resources, posing a serious challenge to its sustainable utilization. Against this backdrop, artificial cultivation of *Saposhnikovia divaricata* has become the main source of market supply.

[0003] The key problem currently facing the cultivation of Saposhnikovia divaricata lies in the widespread phenomenon of indiscriminate fertilization. This not only fails to effectively increase yield but also leads to a decline in the quality of medicinal materials, hindering the healthy development of the industry. Seeking green and efficient cultivation techniques has become an urgent need. Microbial inoculants, as biological agents developed based on beneficial microorganisms, have become a research hotspot in the field of medicinal plant cultivation due to their significant advantages such as being green, environmentally friendly, safe, reliable, and sustainable. Some commercially available microbial inoculants have already demonstrated multiple effects in agricultural production, including promoting plant growth, increasing yield, improving quality, improving soil, and enhancing plant disease resistance. However, existing microbial inoculants still have significant limitations: firstly, the diversity of their microbial composition is generally insufficient; secondly, their main strains are mostly derived from the plant rhizosphere and soil environment. In practical applications, these microorganisms are easily affected by external environmental conditions (such as soil type and climate) and differences in host plants, leading to unstable and unreliable growth-promoting effects.

[0004] In contrast, plant endophytic bacteria refer to a class of bacteria that colonize the interior of healthy plant tissues (such as roots, stems, leaves, and seeds) in part or all of their life cycle without causing obvious diseases. Compared to rhizosphere and soil microorganisms, endophytic bacteria, because they colonize within the plant and are directly protected by the host tissue, occupy a more stable and dominant ecological niche, and theoretically can produce a more direct and effective growth-promoting effect on the host plant. Their growth-promoting mechanisms mainly include two aspects: (1) direct effects: such as secreting plant growth hormones (such as IAA), biological nitrogen fixation, dissolving insoluble phosphorus, and dissociating potassium minerals to promote plant growth; (2) indirect effects: such as inhibiting the growth of pathogens, inducing systemic resistance in plants, and secreting extracellular polysaccharides to promote the binding of roots with soil to indirectly promote plant growth.

[0005] Although research on endophytic bacteria in Saposhnikovia divaricata has a certain foundation, current technologies mainly focus on their antibacterial activity and antibacterial substances. Systematic research on the growth-promoting characteristics of Saposhnikovia divaricata endophytic bacteria (such as their ability to produce hormones, fix nitrogen, solubilize phosphorus, and solubilize potassium), especially the screening of functional endophytic bacteria with significant growth-promoting effects and their application in Saposhnikovia divaricata cultivation to solve yield and quality problems, is currently lacking. Therefore, screening endophytic bacterial resources with efficient and stable growth-promoting effects on the Saposhnikovia divaricata host and developing specialized microbial preparations suitable for high-yield and high-quality Saposhnikovia divaricata cultivation to compensate for the shortcomings of existing technologies has become a key technical problem urgently needing to be solved in this field. Summary of the Invention

[0006] One of the objectives of this invention is to provide an endophytic bacterium for Saposhnikovia divaricata, Klebsiella michiganensis MX-56, which possesses growth-promoting properties including nitrogen fixation, inorganic phosphorus dissolution, IAA secretion, and siderophore production; it can stably colonize in the rhizosphere soil of Saposhnikovia divaricata; and it has a good growth-promoting effect on Saposhnikovia divaricata.

[0007] A second objective of this invention is to provide the application of Klebsiella michiganensis MX-56 in the preparation of a windbreak growth promoter.

[0008] A third objective of this invention is to provide a wind-resistant growth promoter comprising Klebsiella michiganensis MX-56.

[0009] The fourth objective of this invention is to provide a method for promoting the growth of *Klebsiella michiganensis* MX-56.

[0010] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0011] The first aspect of this invention discloses a strain of Klebsiella michiganensis MX-56, deposited at the Guangdong Microbial Culture Collection Center, with accession number GDMCC No:66361, deposited on May 19, 2025, at No. 100 Xianlie Middle Road, Guangzhou.

[0012] The second aspect of this invention discloses the application of Klebsiella michiganensis MX-56 in the preparation of a windbreak growth promoter.

[0013] In some embodiments of the present invention, the windbreak growth promoter has at least one of the following functions: nitrogen fixation, dissolution of inorganic phosphorus, secretion of IAA, and iron carrier.

[0014] The third aspect of the present invention discloses a windproof growth promoter comprising the above-mentioned Klebsiella michiganensis MX-56 and a carrier.

[0015] The fourth aspect of the present invention discloses a method for promoting the growth of Saposhnikovia divaricata using the above-mentioned Klebsiella michiganensis MX-56, which employs the root irrigation method to irrigate the roots with MX-56 bacterial solution.

[0016] In some embodiments of the present invention, the bacterial concentration is 0.5 × 10⁻⁶. 9 ~10×10 9 CFU / mL.

[0017] In some embodiments of the present invention, the bacterial concentration is 1×10⁻⁶. 9 CFU / mL.

[0018] In some embodiments of the present invention, the bacterial solution is irrigated by root irrigation after planting and seedling establishment.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] The endophytic bacterium MX-56 provided by this invention exhibits significant and multiple beneficial effects in promoting growth. Through rigorous functional screening, this strain was confirmed to possess four key growth-promoting properties for *Saposhnikovia divaricata*: nitrogen fixation, inorganic phosphorus dissolution, indoleacetic acid (IAA) secretion, and siderophore production. This combination of multifunctionalities gives it a unique advantage in promoting nutrient absorption and growth of *Saposhnikovia divaricata*. Systematic morphological, physiological, and biochemical analyses, along with 16S rRNA gene sequencing, identified strain MX-56 as *Klebsiella michiganensis*, laying a reliable taxonomic foundation for its subsequent applications.

[0021] Most importantly, this invention thoroughly verified the colonization ability of strain MX-56 in the soil environment and its actual growth-promoting effect on Saposhnikovia divaricata. Colonization dynamics studies using the rifampicin-resistant labeling method showed that this strain exhibited a trend of initial decline, then increase, and then decline again in the rhizosphere soil of Saposhnikovia divaricata, ultimately achieving stable colonization. This provides a crucial guarantee for its continued function in the rhizosphere. Pot experiments further confirmed its excellent growth-promoting effect; treatment with strain MX-56 significantly improved key morphological indicators and overall biomass of Saposhnikovia divaricata. Particularly noteworthy is its outstanding promoting effect on root development, manifested in a significant increase in root length, diameter, and weight, which provides a foundation for Saposhnikovia divaricata to build a more robust absorption system and enhance its stress resistance.

[0022] The most significant application value of this invention lies in the fact that the total content of cimicifuga glycoside and 5-O-methylvisamidol glycoside in Saposhnikovia divaricata plants treated with strain MX-56 was significantly increased by 30.23% compared to the control group, successfully meeting the quality standards stipulated in the Pharmacopoeia of the People's Republic of China. This achievement not only directly demonstrates the enormous potential of this strain in improving the quality of Saposhnikovia divaricata, especially the effective components of medicinal Saposhnikovia divaricata, but also provides valuable high-quality strain resources and solid scientific theoretical support for the development of novel and efficient microbial fertilizers. Therefore, this invention provides an environmentally friendly and highly effective technical path for achieving green, high-yield, and high-quality cultivation of Saposhnikovia divaricata, especially as a traditional Chinese medicinal herb. Attached Figure Description

[0023] Appendix Figure 1 An illustration of the nitrogen-fixing effect of the windproof endophytic bacteria MX-56;

[0024] Appendix Figure 2 The image shows the effect of the endophytic bacteria MX-56 in dissolving inorganic phosphorus.

[0025] Appendix Figure 3 Qualitative staining effect of IAA production by the endophytic bacteria MX-56 of *Saposhnikovia divaricata*;

[0026] Appendix Figure 4 An illustration illustrating the effect of siderophore production by the windproof endophytic bacteria MX-56;

[0027] Appendix Figure 5 The images show the morphology and Gram staining of the endophytic bacteria strain MX-56 from Saposhnikovia divaricata. The left image shows the morphology of the strain, and the right image shows the Gram staining.

[0028] Appendix Figure 6 Phylogenetic tree of strain MX-56 constructed based on the 16S rRNA gene sequence; attached. Figure 7 This is a diagram illustrating the growth morphology of the windbreak plant. Detailed Implementation

[0029] The specific embodiments of the present invention will be further described in detail below with reference to the examples. The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention. The present invention covers all possible alternatives, improvements, and equivalents within the scope of the claims. Specific techniques or conditions not specified in the following examples are conventional techniques or conditions, or techniques or conditions described in the literature in the art, or according to the product manual.

[0030] The wind-proof sample described in this embodiment of the invention: 2-year-old healthy wind-proof plants were collected from the Medicinal Botanical Garden of Jilin Agricultural University (43°48′23″N, 125°24′57″). During collection, the plants were dug out completely along the root direction of the wind-proof plants with a shovel. Plants with no surface damage were selected, and after removing the attached soil, they were placed in sterile bags and kept at low temperature and brought back to the laboratory for further experimental treatment.

[0031] The culture medium and reagents described in this embodiment of the invention are as follows:

[0032] Beef extract peptone (NA) medium: 3g beef extract, 10g peptone, 5g NaCl, 18g agar, 1000mL distilled water, pH 7.0-7.2, sterilized at 121℃ for 30min. NB liquid medium is NA medium without agar. Assumption medium: 10g mannitol, 5.0g CaCO3, 0.2g NaCl, 0.2g MgSO4·7H2O, 0.2g KH2PO4, 0.1g CaSO4, 18g agar, 1000mL distilled water, pH 6.9-7.1, sterilized at 121℃ for 30min. Pikovasky's medium (PKO medium): 10g glucose, 5.0g Ca3(PO4)2, 5.0g yeast extract, 5.0g (NH4)2SO4, 0.2g NaCl, 0.2g KCl, 0.1g MgSO4·7H2O, 0.025g bromophenol blue, 0.002g FeSO4·7H2O, 18g agar, 1000mL distilled water, pH 7.0-7.2, sterilized at 121℃ for 30min. CAS medium: Chromium azurite S (CAS) 60.5 mg, hexadecyltrimethylammonium bromide (HDTMA) 72.9 mg, FeCl3·6H2O 2.645 mg, NaH2PO4·2H2O 295.25 mg, Na2HPO4·12H2O 1213.5 mg, NH4Cl 125 mg, KH2PO4 37.5 mg, NaCl 62.5 mg, agar 18 g, distilled water 1000 mL, pH 6.7-6.9, sterilized at 121℃ for 30 min. MKB medium: 5g butyrate hydrolyzed protein, 15mL glycerol, 2.5g MgSO4·7H2O, 2.5g K2HPO4·7H2O, pH 7.2-7.4, sterilized at 121℃ for 30min; Salkowski colorimetric solution: 320mL 0.5mol / L FeCl3, 600mL 98% H2SO4, 1000mL distilled water.

[0033] Bacterial micro-biochemical identification tubes, Qingdao Haibo Biotechnology Co., Ltd.; 98% rifampicin; 10% NaClO solution; 75% ethanol.

[0034] Example 1

[0035] This embodiment discloses the isolation and purification of Klebsiella michiganensis MX-56 of the present invention.

[0036] After rinsing the roots of 2-year-old Saposhnikovia divaricata plants under running water to remove surface soil, the roots were washed several times in sterile water under sterile conditions. They were then soaked in 75% ethanol for 3 minutes, rinsed with sterile water, and then soaked in a NaClO solution with an effective chlorine content of 3% for 5 minutes. Next, they were washed with 75% ethanol for 1 minute (to remove residual NaClO solution from the sample surface), and then washed with sterile water 5 times. Finally, the surface moisture of the Saposhnikovia divaricata root tissue was blotted dry with sterile filter paper.

[0037] To check the thoroughness of root surface disinfection, 200 μL of the final sterile water rinse solution was spread onto NA medium after disinfection. The roots of *Saposhnikovia divaricata* were then rolled once on the NA medium, and the culture dishes were incubated under the same isolation conditions. Endophytic bacteria were isolated from the disinfected *Saposhnikovia divaricata* root tissue using the dilution plating method, and the disinfected *Saposhnikovia divaricata* roots were divided into uniformly sized tissue blocks.

[0038] Take 1g of tissue block and place it in a sterile mortar. Add 9mL of sterile water and grind thoroughly. Take 0.1mL of the tissue fluid and add it to a centrifuge tube containing 0.9mL of sterile water. Shake well. This yields 10... -2 The diluent was diluted sequentially to 10 using this method. -3 ~10 -5 Diluted solutions of different concentrations were taken and spread 200 μL onto NA solid medium. Each treatment was repeated 3 times. The petri dishes were inverted and incubated in the dark at 32°C. The bacterial strains were repeatedly purified using the streak plating method. When the morphology and color of the strains were consistent, the glycerol tubes were frozen at -80°C.

[0039] In this embodiment, plant tissue isolation and gradient dilution plating methods were used to isolate endophytic bacteria from the roots of Saposhnikovia divaricata on NA medium. No colonies grew on the control culture dish, proving that the isolated bacteria were all Saposhnikovia divaricata endophytic bacteria. The strains were streaked and purified, and a total of 202 endophytic bacteria strains were obtained, including the strain MX-56 described in this invention.

[0040] Example 2

[0041] This embodiment discloses the investigation of the nitrogen fixation, inorganic phosphorus dissolution, IAA secretion, and siderophore production characteristics of strain MX-56.

[0042] 1. Qualitative and quantitative determination of nitrogen fixation capacity of strains

[0043] 1.1 Qualitative Analysis: Endophytic bacteria MX-56 were inoculated onto Ashby nitrogen-free medium, and their growth on the medium was used to determine their nitrogen-fixing activity. Strain MX-56 was subcultured 3–8 times consecutively on solid Ashby nitrogen-free medium. Subculturing 3 times with good growth indicated a "+" nitrogen fixation ability, meaning the strain had strong nitrogen fixation capacity; subculturing 5 times with good growth indicated a "++" nitrogen fixation ability, meaning the strain had strong nitrogen fixation capacity; subculturing 8 times with good growth indicated a "+++" nitrogen fixation ability, meaning the strain had stable nitrogen fixation capacity.

[0044] 1.2 Quantitative determination: The nitrogenase activity of strain MX-56 was determined using a bacterial nitrogenase ELISA kit.

[0045] The results showed that strain MX-56 could grow on Ashby nitrogen-free medium (e.g., Figure 1 As shown in Table 1), the strain can be continuously transferred 5 times on solid Ashby nitrogen-free medium, demonstrating strong nitrogen-fixing ability. The nitrogenase activity of the strain is 214.25 IU / L (as shown in Table 1).

[0046] Table 1. Nitrogenase activity of endophytic bacteria MX-56 from Saposhnikovia divaricata.

[0047]

[0048] 2. Qualitative and quantitative determination of the strain's ability to solubilize inorganic phosphorus

[0049] 2.1 Qualitative analysis: Endophytic bacteria MX-56 were inoculated into NB liquid medium and cultured at 32℃ and 180 r / min for 36 h. The culture was then inoculated onto PKO solid medium using the filter paper disc method and cultured at 32℃ for 7 days. The phosphate-solubilizing effect of the strain was observed, i.e., whether a clear zone was formed around the colony.

[0050] 2.2 Quantitative Determination: Phosphorus standard solutions of varying concentrations were prepared, and the absorbance at 700 nm was measured using the molybdenum-antimony colorimetric method. A phosphorus standard curve was plotted based on the solution concentrations and absorbances, yielding the linear regression equation y = 0.2595x - 0.024, R0. 2 =0.9979, the seed culture of strain MX-56 was prepared and inoculated into PKO liquid medium at an inoculum of 1%, and cultured at 32℃ and 180 r / min for 7 days. The culture was centrifuged at 4℃ and 10000 r / min for 10 min, and the supernatant was collected and the phosphorus solubility of strain MX-56 was determined by molybdenum antimony colorimetric method.

[0051] The results showed that after inoculating strain MX-56 onto PKO medium and culturing for 7 days, transparent phosphate-lysing zones were observed on the medium (e.g., Figure 2 As shown in Table 2), the phosphorus soluble content was determined to be 250.05 mg / L by the molybdenum antimony colorimetric method.

[0052] Table 2. Determination of the inorganic phosphorus-dissolving capacity of the endophytic bacteria MX-56 from Saposhnikovia divaricata.

[0053]

[0054] 3. Qualitative determination of IAA production capacity

[0055] 3.1 Qualitative Analysis: After activation, strain MX-56 was inoculated into NB liquid medium containing L-tryptophan (100 mg / L) and cultured at 32°C with shaking at 180 rpm for 4 days. After centrifugation at 4°C for 10 min (10000 rpm), 1 mL of the supernatant was added to a porcelain spot plate, and an equal volume of Salkowski's reagent was added and mixed. The plate was then incubated at room temperature in the dark for 30 min, and the results were observed. A pink color indicates the bacterial strain can secrete IAA; the deeper the color, the stronger the IAA secretion ability. No color change indicates a negative result, meaning no IAA secretion. Quantitative analysis was performed after a positive result was obtained.

[0056] 3.2 Quantitative determination: Add 2 mL of bacterial culture medium and 2 mL of Salkowski reagent to a test tube, mix well, and let stand at 25℃ in the dark for 30 min. After the reaction, measure the absorbance of the reaction solution at 530 nm. Sterile NB liquid culture medium (containing tryptophan) was used as a control. IAA standard solutions with concentrations of 0, 10, 20, 30, 40, 50, 60, 70, and 80 μg / mL were prepared, and the linear regression equation was obtained as y = 0.0066x + 0.0955(R²). 2 =0.9981), calculate the IAA content in the culture medium based on the standard curve.

[0057] The results showed that when strain MX-56 was inoculated into NB liquid medium (containing 100 mg / L tryptophan) and cultured for 4 days, the addition of Salkowski's reagent resulted in a pale pink color in the endophytic bacteria MX-56 (as shown in the image). Figure 3 (As shown in Table 3). The IAA production of endophytic bacteria was calculated based on the IAA standard curve. The IAA content secreted by strain MX-56 was 39.09 mg / L.

[0058] Table 3. Determination of IAA production capacity of endophytic bacteria MX-56 from Saposhnikovia divaricata.

[0059]

[0060] 4. Qualitative and quantitative determination of iron-carrier production capacity:

[0061] 4.1 Qualitative analysis: Endophytic bacteria MX-56 were inoculated onto CAS solid medium and observed for the appearance of an orange halo around the colony. If an orange halo appeared around the colony, it indicated that the strain could produce siderophores. The diameter of the orange halo was measured and recorded.

[0062] 4.2 Quantitative analysis: The ability of the strain to produce siderophores was determined by the chromaine colorimetric method. The strain was inoculated into MKB liquid medium and cultured at 32℃ and 180 r / min for 48 h. After centrifugation at 4℃ for 10 min (10000 r / min), an equal volume of bacterial culture supernatant was mixed with CAS detection solution and reacted in the dark for 1 h. The absorbance As was measured at a wavelength of 680 nm. At the same time, the absorbance Ar of the reaction between the blank medium and an equal volume of CAS detection solution was measured, and the SU value was calculated.

[0063] Siderophore activity unit SU% = ((Ar-As) / Ar)*100

[0064] The isolated endophytic bacteria MX-56 were inoculated onto CAS medium and cultured for 7 days. An orange halo was observed around the colonies (e.g., ...). Figure 4 As shown in Table 4), it was initially determined that this strain could produce siderophores. After the quantitative reaction was completed, the absorbance value was measured at 630 nm, and the siderophore production capacity of endophytic bacteria MX-56 was 80.4% (as shown in Table 4).

[0065] Table 4. Determination of the siderophore production capacity of the endophytic bacteria MX-56 from Saposhnikovia divaricata.

[0066]

[0067] The results of this embodiment show that the strain MX-56 of the present invention has the functions of nitrogen fixation, inorganic phosphorus solubilization, IAA secretion, and siderophore production.

[0068] Example 3

[0069] This embodiment discloses the identification of Klebsiella michiganensis MX-56.

[0070] 1. Morphological and physiological biochemical identification

[0071] Morphological identification of the strain was performed according to Berger's Manual of Bacteriological Identification. Endophytic bacteria MX-56 were inoculated onto NB medium and incubated at 32°C for 1-2 days, with single colony morphology recorded and observed. Gram staining was also performed on the strain. Physiological and biochemical parameters of the endophytic bacteria were measured using microbial biochemical identification tubes.

[0072] Morphological identification results as follows Figure 5As shown: Strain MX-56 colonies are milky white in color, viscous in texture, opaque, with neat edges, a raised center, and are Gram-negative.

[0073] The physiological and biochemical identification results are shown in Table 1: strain MX-56 can decompose and utilize xylose, glucose and L-arabinose, but cannot utilize D-mannitol. The OF test is oxidized and does not produce hydrogen sulfide. The methyl red test, gelatin hydrolysis, starch hydrolysis and oxidase test are all negative, while the VP test and nitrate reduction test are positive.

[0074] Table 5. Physiological and biochemical identification results of the endophytic bacterium MX-56 from Saposhnikovia divaricata.

[0075]

[0076] In Table 1, "+" indicates a positive test result; "-" indicates a negative test result.

[0077] 2. Molecular biological identification

[0078] Strain MX-56 was inoculated into NB liquid culture medium and cultured at 32℃ and 180 r / min for 24 h. The bacterial cells were collected by centrifugation, and bacterial DNA was extracted using a bacterial genomic DNA extraction kit. Universal primers 27F (AGAGTTTGATCMTGGCTCAG) and 1492R (GGTTACCTTGTTACGACTT) were selected for amplification. The PCR reaction system was 50 μL: 2 μL genomic DNA template, 2 μL 10 mmol / L dNTPs, 2 μL 10×Buffer, 2 μL each of forward and reverse primers, 2 μL 2.5 U / μL Taq enzyme, and ddH2O to a final volume of 50 μL. 16S rRNA amplification conditions were: 94℃ for 5 min, 94℃ for 1 min, 58℃ for 30 s, 70℃ for 90 s, 35 cycles; 72℃ for 10 min. The PCR products were detected by 1% agarose gel electrophoresis and then sent to Sangon Biotech (Changchun) Co., Ltd. for sequencing.

[0079] The 16S rRNA gene sequence of endophytic strain MX-56 was assembled, with an effective length of 1440 bp. The sequence was uploaded to GenBank, accession number PQ864782. The 16S rRNA sequence of the strain was compared with the NCBI database using BLAST homology, and a phylogenetic tree was constructed (see attached). Figure 6 (As shown). The results showed that MX-56 clustered with Klebsiella michiganensis, with 100% homology. Based on the morphological characteristics, physiological and biochemical identification, and molecular biological analysis of the strain, MX-56 was preliminarily identified as Klebsiella michiganensis.

[0080] Example 4

[0081] This embodiment discloses an investigation into the colonization ability of the wind-resistant endophytic bacteria MX-56 of the present invention in soil.

[0082] Activated endophytic bacteria MX-56 were cultured at 32℃ and 180 rpm for 2 days to prepare a bacterial seed culture, which was then inoculated into NB medium (containing 20 μg / mL Rif) at a 1% inoculum and cultured under the same conditions for 4 days. When the medium became turbid, it was sequentially inoculated into NB medium containing Rif concentrations of 40, 60, 80, 100, 150, 200, and 300 μg / mL for gradient induction. The above-mentioned labeled strains were inoculated into NB liquid medium and cultured at 32℃ and 180 rpm with shaking for 2 days. The bacterial concentration of the culture was then diluted to approximately 10. 8 CFU / mL, using a pot cultivation method, the plants were established in the soil around the rhizosphere of *Saposhnikovia divaricata*. Two-year-old *Saposhnikovia divaricata* plants were transplanted into black PE seedling pots containing 1 kg of natural soil, one plant per pot. After establishment, 100 mL of the labeled bacterial suspension was evenly applied to the soil at the base of the stem using the root irrigation method. Soil samples were collected on days 4, 7, 10, 15, 20, and 30. Soil bacterial isolation procedures: The rhizosphere soil of *Saposhnikovia divaricata* was passed through a 20-mesh sieve. 10 g of soil sample was placed in a conical flask containing 90 mL of sterile water and incubated at 25℃ with shaking at 180 rpm for 30 min. After mixing, the sample was allowed to stand for 5 min. The soil suspension was then serially diluted to 10-10. -2 10 -3 10 -4 10 -5 200 μL of soil dilution was spread onto NA medium containing 300 μg / mLRif for each gradient and incubated at 32°C for 3 days. The bacterial count (CFU / g) in the soil was calculated after the incubation period.

[0083] Bacterial count = C / V*M

[0084] C represents the average number of colonies on the plate; V represents the volume of diluent used for plating; M represents the dilution factor.

[0085] The colonization rate of strain MX-56 in soil is shown in Table 6. Endophytic bacteria strain MX-56 exhibits strong colonization ability in soil. The bacterial count in soil initially decreased, then increased, and finally decreased again. The bacterial count of strain MX-56 in soil initially decreased rapidly, reaching its lowest point on day 7 at 2.03 × 10⁻⁶. 5 The CFU / g level gradually increased, reaching a peak on day 10, exceeding the minimum colonization value by 24.1 times, with a bacterial count of 5.10 × 10⁻⁶. 6 The bacterial count was initially set at CFU / g, then decreased, and remained at 2.67 × 10⁻⁶ on day 30. 5CFU / g.

[0086] Table 6. Colony numbers of the labeled strain MX-56 in soil.

[0087]

[0088] In Table 6, the different letters following the colonization numbers for different days indicate significant differences.

[0089] Example 5

[0090] This embodiment discloses an experimental study on the growth-promoting effect of Klebsiella michiganensis MX-56 on windbreak.

[0091] An outdoor pot experiment was conducted. Healthy, uniformly growing two-year-old *Saposhnikovia divaricata* plants were disinfected with a 500-fold dilution of carbendazim and then planted in seedling pots containing 1 kg of soil, one plant per pot, with each treatment replicated 15 times. After planting and thinning, the plants were irrigated with the bacterial solution using the root irrigation method, with a concentration of 1×10⁻⁶. 9 CFU / mL, each pot was watered with 100mL of bacterial solution, while the control group was watered with 100mL of sterile NB medium. The day of watering with bacterial solution was day 1 of the experiment, and the bacterial solution was replenished on day 30 after the start of the experiment to ensure the colonization of endophytic strains in the soil. No fertilization was performed during the experiment. The plant height, root length, root fresh weight and root dry weight of Saposhnikovia divaricata plants were measured after 60 days of potted experiment. The contents of Saposhnikovia divaricata's medicinal components, Saposhnikovia divaricata glycoside and 5-O-methylvisamidol glycoside, were determined by high performance liquid chromatography. The determination method was carried out according to the literature "Comprehensive Evaluation of Multi-Indicators of Saposhnikovia divaricata Medicinal Material Quality" (Ma Bingru, Cui Jingxuan, Wang Yuyi, et al. Journal of Zhejiang A&F University, 2024, 41(4):715-723).

[0092] The morphological diagram of Saposhnikovia divaricata after using strain MX-56 is shown below. Figure 7 As shown in Table 7, compared with the control group (CK), the plant height, root length, root diameter, aboveground fresh weight, root fresh weight, and root dry weight of Saposhnikovia divaricata inoculated with MX-56 increased significantly by 13.47%, 43.04%, 42.75%, 31.43%, 63.21%, and 77.12%, respectively. The 2020 edition of the Pharmacopoeia of the People's Republic of China stipulates that the total content of cimicifugoside and 5-O-methylvisamiloside in Saposhnikovia divaricata should not be less than 0.24%. The results of this study indicate that the total content of cimicifugoside and 5-O-methylvisamiloside in Saposhnikovia divaricata treated with the endophytic bacterium MX-56 was higher than the pharmacopoeia standard. Compared with CK, the total content of the two chromogens in Saposhnikovia divaricata plants treated with strain MX-56 increased by 30.23% (as shown in Table 8).

[0093] Table 7. Effects of the endophytic bacterium MX-56 on the growth characteristics and biomass of Saposhnikovia divaricata.

[0094]

[0095] Table 8. Effects of endophytic bacteria MX-56 on the contents of cimetidine and 5-O-methylvisamidol in Saposhnikovia divaricata.

[0096]

[0097] In summary, this invention screened MX-56, an endophytic bacterium with growth-promoting function, from the roots of two-year-old healthy Saposhnikovia divaricata, verified its colonization ability and its promoting effect on Saposhnikovia divaricata growth, and provided a theoretical basis for the development of microbial fertilizers.

[0098] This invention uses functional culture media to screen strains possessing nitrogen fixation, phosphorus solubilization, indoleacetic acid (IAA) production, and siderophore production characteristics. The taxonomic position of the strains is determined through morphological and physiological-biochemical characteristic analysis combined with 16S rRNA gene sequence molecular identification. Soil colonization ability and growth-promoting effects are measured using rifampicin resistance labeling and pot experiments. The results are as follows: the endophytic growth-promoting strain MX-56 possesses growth-promoting characteristics including nitrogen fixation, inorganic phosphorus solubilization, IAA secretion, and siderophore production. Phenotypic and molecular identification preliminarily identifies strain MX-56 as *Klebsiella michiganensis*. The growth-promoting strain MX-56 described in this invention exhibits a trend of initial decline, then increase, and then decline again in soil colonization, and can stably colonize in windbreak rhizosphere soil. Strain MX-56 exhibits a good growth-promoting effect on Saposhnikovia divaricata, significantly improving the morphological indicators and biomass of the plants. It also significantly affects root length, diameter, and weight, indicating that strain MX-56 can effectively promote root development. The total content of cimicifugoside and 5-O-methylvisamidolol in Saposhnikovia divaricata treated with strain MX-56 was significantly increased by 30.23% compared to the control group, meeting the content requirements stipulated in the Pharmacopoeia of the People's Republic of China. This invention provides high-quality strain resources and theoretical support for the development of microbial fertilizers and the green cultivation of Saposhnikovia divaricata and other Chinese medicinal herbs.

[0099] The above embodiments are merely one of the preferred embodiments of the present invention and should not be used to limit the scope of protection of the present invention. Any modifications or refinements made to the main design concept and spirit of the present invention that are not of substantial significance, but solve the same technical problem as the present invention, should be included within the scope of protection of the present invention.

Claims

1. A strain of Klebsiella michiganensis MX-56, characterized in that, It is deposited at the Guangdong Microbial Culture Collection Center, with accession number GDMCC No:66361, on May 19, 2025, at No. 100 Xianlie Middle Road, Guangzhou.

2. The application of the Klebsiella michiganensis MX-56 strain according to claim 1, characterized in that, Application in the preparation of windproof growth promoters.

3. The application according to claim 2, characterized in that, The wind-resistant growth promoter has at least one of the following functions: nitrogen fixation, dissolution of inorganic phosphorus, secretion of IAA, and iron carrier.

4. A wind-resistant growth promoter, characterized in that, Includes the strain of Klebsiella michiganensis MX-56 and its vector as described in claim 1.

5. A method for promoting the growth of *Pteris vittata* using *Klebsiella michiganensis* MX-56 as described in claim 1, characterized in that, The MX-56 bacterial solution was irrigated using the root irrigation method.

6. The method according to claim 5, characterized in that, The bacterial concentration was 0.5 × 10⁻⁶. 9 ~10×10 9 CFU / mL.

7. The method according to claim 5, characterized in that, The bacterial concentration is 1×10 9 CFU / mL.

8. The method according to claim 5, characterized in that, After planting and thinning, the bacterial solution is irrigated by root irrigation.

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