A complex microbial inoculant for improving the quality of radix ophiopogonis and its application

Abstract

This invention discloses a compound microbial agent for improving the quality of Ophiopogon japonicus tubers and its application, belonging to the field of microbial technology. The compound microbial agent and accompanying Ophiopogon japonicus cultivation method provided by this invention can synergistically improve the yield, active ingredient content, and stress resistance of Ophiopogon japonicus tubers, while optimizing the rhizosphere soil microecology. The microbial agents used in this invention are all isolated from the rhizosphere soil of Ophiopogon japonicus, exhibiting good environmental compatibility and avoiding the residual risks associated with chemical fertilizers and regulators. The accompanying staged application method is simple to operate and low in cost, and can be widely applied to the standardized cultivation of Ophiopogon japonicus, contributing to the green and sustainable development of the traditional Chinese medicine industry.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a compound microbial agent for improving the quality of Ophiopogon japonicus tubers and its application. Background Technology

[0002] Ophiopogon japonicus Ophiopogon japonicus (Lf) Ker-Gawl. is a perennial herb belonging to the genus Ophiopogon of the Liliaceae family. Its dried tuberous root is a commonly used and valuable traditional Chinese medicine, Ophiopogon japonicus, which is rich in various active ingredients such as ruscosaponin and ophiopogonin D. It has important medicinal value in nourishing yin and promoting body fluid production, moistening the lungs and clearing the heart. At the same time, the yield and quality of Ophiopogon japonicus tuberous root directly determine its medicinal and economic value, which has attracted great attention from growers and the traditional Chinese medicine industry.

[0003] The current Ophiopogon japonicus (Liriope muscari) cultivation industry faces many pressing problems. On the one hand, Ophiopogon japonicus is quite sensitive to its growing environment, thriving in warm and humid climates. In low-temperature stress environments, it is prone to cell damage and growth stagnation, leading to hindered tuber enlargement and reduced yield. On the other hand, long-term continuous cropping and unreasonable fertilizer application patterns easily cause deterioration of the rhizosphere soil's physical and chemical properties, nutrient imbalance, and a reduction in beneficial microbial communities. This not only reduces soil fertility but also inhibits the absorption of nutrients by the roots, thus affecting the synthesis and accumulation of active ingredients in the tubers.

[0004] Furthermore, existing methods for improving the quality of Ophiopogon japonicus tubers have significant limitations. Traditional fertilization methods focus on supplementing macronutrients such as nitrogen, phosphorus, and potassium, making it difficult to precisely improve the rhizosphere microecological environment. While chemical regulators can promote growth in the short term, they may lead to unstable levels of active ingredients and pose a risk of residue. Microbial inoculants have become a research hotspot in the field of medicinal herb cultivation due to their advantages in improving soil, promoting crop growth, and enhancing quality. However, there are currently few types of inoculants specifically for Ophiopogon japonicus, and most inoculants have a single function, failing to simultaneously achieve multiple goals such as improving the low-temperature resistance of Ophiopogon japonicus, promoting the accumulation of active ingredients, and optimizing soil nutrients.

[0005] Therefore, screening and preparing compound microbial agents that can synergistically improve the yield, active ingredient content and stress resistance of Ophiopogon japonicus tubers, and establishing supporting efficient application techniques are of great significance for promoting the standardized and green cultivation of Ophiopogon japonicus and improving the economic benefits of the Chinese medicinal materials industry. Summary of the Invention

[0006] The purpose of this invention is to provide a compound microbial agent for improving the quality of Ophiopogon japonicus tubers and its application, so as to solve the problems existing in the prior art.

[0007] To achieve the above objectives, the present invention provides the following solution: One of the technical solutions of the present invention is a microbial inoculant, comprising Pantotheca cum Bacillus CTZT8 and / or Zürich Scutellaria cirrhosa CTZT22.

[0008] The second technical solution of the present invention is the application of the microbial agent in increasing the content of ruscosaponin in Ophiopogon japonicus tubers.

[0009] The third technical solution of the present invention is the application of the microbial agent in increasing the content of Ophiopogon japonicus saponin D in Ophiopogon japonicus tubers.

[0010] The fourth technical solution of the present invention is the application of the microbial agent in improving the low-temperature resistance of Ophiopogon japonicus.

[0011] The fifth technical solution of the present invention is a method for planting Ophiopogon japonicus, wherein the microbial agent is applied to the experimental field where Ophiopogon japonicus is growing during the growing season of Ophiopogon japonicus.

[0012] Based on the above technical solution, the present invention has the following technical effects: The compound microbial agent and the matching Ophiopogon japonicus planting method provided by this invention can synergistically improve the yield of Ophiopogon japonicus tubers, the content of active ingredients and stress resistance, and optimize the rhizosphere soil microecology. 1. Increase the content of active ingredients in Ophiopogon japonicus tuber roots The *Pantoinella asiatica* CTZT8, *Bacillus spp.* CTZT22, and the compound microbial agent (SynComs) described in this invention can all significantly increase the content of ruscosaponins in *Ophiopogon japonicus* tubers. Among them, *Pantoinella asiatica* CTZT8 showed the best increasing effect, with its content significantly higher than the control group and other microbial agent treatment groups. Simultaneously, *Pantoinella asiatica* CTZT8 and *Bacillus spp.* CTZT22 can effectively promote the synthesis of *Ophiopogon japonicus* saponin D, with a higher content than the control group, solving the problem of insufficient accumulation of active ingredients in *Ophiopogon japonicus* in traditional cultivation.

[0013] 2. Enhance the resistance of Ophiopogon japonicus to low-temperature stress Low-temperature stress leads to the accumulation of malondialdehyde (MDA) in Ophiopogon japonicus cells, causing cell damage. After application of the microbial agent of this invention, the MDA content of Ophiopogon japonicus plants was significantly lower than that of the control group. The peroxidase (POD), superoxide dismutase (SOD), and proline (Pro) contents in the microbial agent group were generally lower than those in the control group, indicating that low-temperature stress had a less stimulating effect on the microbial agent group. This demonstrates that the microbial agent of this invention can effectively alleviate the damage of low temperature to Ophiopogon japonicus and improve its growth adaptability in low-temperature environments.

[0014] 3. Promotes the growth of Ophiopogon japonicus and increases yield Apply during the tuber germination period of Ophiopogon japonicus Pantoea agglomeransApplying compound microbial agents during the rapid growth and shaping stages significantly increased the fresh weight and number of tubers of *Ophiopogon japonicus*. The SynComs treatment group showed the highest fresh weight and number of tubers during the germination stage, the SynComs treatment group had the most tubers during the rapid growth stage, and the CTZT8 treatment group had the highest fresh weight during the shaping stage. This application strategy provided sufficient nutrition and structural support for the tuber enlargement of *Ophiopogon japonicus*, effectively increasing its yield.

[0015] 4. Optimize the rhizosphere soil microecology of Ophiopogon japonicus The microbial agent of this invention can slow down the fluctuation rate of soil pH in the rhizosphere of Ophiopogon japonicus, maintaining the stability of the soil microenvironment. SynComs can promote the decomposition of soil organic matter in the early and middle stages of tuber development, and assist the roots of Ophiopogon japonicus in nutrient absorption in the later stages. The total nitrogen, total phosphorus, and total potassium contents of the soil in the CTZT8 and CTZT22 treatment groups continued to increase, and the available phosphorus content also steadily increased. The SynComs treatment group can promote soil nutrient dissolution in the early and middle stages of tuber growth, and stimulate efficient root absorption in the later stages, thereby improving soil fertility and nutrient supply capacity.

[0016] 5. Promote the development of green planting of Ophiopogon japonicus The microbial agents used in this invention are all isolated from the rhizosphere soil of Ophiopogon japonicus, exhibiting good environmental compatibility and avoiding the residue risks associated with chemical fertilizers and regulators. The accompanying staged application method is simple to operate and low in cost, and can be widely applied to the standardized cultivation of Ophiopogon japonicus, contributing to the green and sustainable development of the traditional Chinese medicine industry. Attached Figure Description

[0017] Figure 1 Analysis of the physicochemical properties of the rhizosphere soil of *Ophiopogon japonicus*. Where A represents pH, B represents organic matter content, C represents total nitrogen content, D represents available nitrogen content, E represents total phosphorus content, F represents available phosphorus content, G represents total potassium content, and H represents available potassium content.

[0018] Figure 2 Analysis of total flavonoids and soluble sugar content in Ophiopogon japonicus tubers. Where A represents total flavonoid content and B represents soluble sugar content.

[0019] Figure 3 The effect of inoculants on the response of Ophiopogon japonicus to low-temperature stress. In this study, A represents malondialdehyde (MDA) content, B represents peroxidase (POD) content, C represents superoxide dismutase (SOD) content, and D represents proline content.

[0020] Figure 4 The effect of microbial agents on the chlorophyll content of Ophiopogon japonicus leaves. Here, A represents total chlorophyll content, B represents chlorophyll a content, and C represents chlorophyll b content. Detailed Implementation

[0021] Unless otherwise specified, the technical solutions described in this invention are all conventional solutions in the field, and the reagents or raw materials used are all purchased from commercial channels or are publicly available unless otherwise specified.

[0022] This invention provides a microbial inoculant, comprising Pantotheca cum Bacillus CTZT8 and / or Zürich Scutellaria cirrhosa CTZT22.

[0023] In some specific implementation schemes, the Pantoea agglomerans CTZT8 was deposited on March 1, 2026, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 37823 and classification name: Pantoea agglomerans.

[0024] In some specific implementation schemes, the Zurich Siccibacter CTZT22 was deposited on March 1, 2026, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 37826 and classification name: Zurich Siccibacter turicensis.

[0025] In some specific implementations, the microbial agent is composed of Pantotheca cum Caulis CTZT8, Agrobacterium CTZT11, Microbacterium paraoxidans CTZT21, and Bacillus sicca Zuriensis CTZT22. The OD600 values ​​of the four bacterial solutions are adjusted to 1, and they are mixed in a volume ratio of 1:1:1:1. The Agrobacterium CTZT11 was deposited on March 1, 2026, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 37824 and classification name: Agrobacterium leguminum. The aforementioned *Microbacterium paraoxydans* CTZT21 was deposited on March 1, 2026, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCCNo. 37825 and classification name: *Microbacterium paraoxydans*.

[0026] This invention also provides the application of the microbial agent in increasing the content of ruscosaponin in Ophiopogon japonicus tubers.

[0027] This invention also provides the application of the microbial agent in increasing the content of ophiopogonin D in Ophiopogon japonicus tuber.

[0028] This invention also provides the application of the microbial agent in improving the low-temperature resistance of Ophiopogon japonicus.

[0029] This invention also provides a method for planting Ophiopogon japonicus, wherein the microbial agent is applied to the experimental field where Ophiopogon japonicus is growing during its growth period.

[0030] In some specific implementation plans, 4.5 liters of microbial inoculant are applied per square meter.

[0031] In some specific implementation schemes, the application method is as follows: applying Pantotheca acuminata CTZT8 during the germination period of Ophiopogon japonicus tubers, and applying the microbial agent during the rapid growth period and the tuber shaping period.

[0032] Example 1 1. Materials and Methods 1.1 Soil and Plants The rhizosphere soil samples used in this embodiment were collected from the rhizosphere soil of robust Ophiopogon japonicus plants located in the original ecological planting base in Santai County, Mianyang City, Sichuan Province, China (31°30'N, 104°95'E). The top 2cm of soil was removed, and the entire Ophiopogon japonicus plant was dug up with its roots intact. The rhizosphere soil was obtained by shaking the roots.

[0033] 1.2 Test culture medium and experimental methods 1.2.1 Isolation, purification, and culture The rhizosphere soil microorganisms of *Ophiopogon japonicus* samples were screened using the streak plate method. 5.0 g of sample was mixed with 45 ml of sterile water in a sterile Erlenmeyer flask and shaken on a shaker for 1 day. The resulting turbid soil solution was then diluted 10 times. 3 10 4 10 5 10 6 Prepare multiple times the amount of bacterial suspension. Streak the diluted bacterial suspensions at different concentrations onto LB agar plates, repeating the inoculation three times for each dilution. Incubate the plates at 28°C for 2-3 days. Select dominant bacterial groups with significant morphological differences and large numbers for purification. Store the purified strains in glycerol tubes at -80°C for later use.

[0034] 1.2.2 IAA Production Capability Assessment The Salkowski colorimetric method was used to identify the ability to produce IAA. Kings medium was prepared, and the strain was inoculated into the medium using a sterile toothpick. The culture was incubated at 180 rpm and 30°C for 2 days on a shaker. After centrifugation at 5000 rpm for 10 min, 500 μL of the supernatant was transferred to a 1.5 mL centrifuge tube, and an equal volume of Salkowski Reagent solution was added. The mixture was incubated in the dark for 30 min. If the liquid color changes from pale yellow to pink, magenta, or deep red, it indicates that the microorganism can produce IAA.

[0035] 1.2.3 Nitrogen fixation capacity assessment Ashby culture medium was used to identify nitrogen-fixing ability. Ashby medium was prepared, and purified dominant bacterial culture was inoculated onto the solid medium using a sterile toothpick. After inoculation, the medium was inverted and placed in a constant temperature incubator set to 28 °C for 3-7 days. The formation of a clear zone indicates that the strain has nitrogen-fixing ability.

[0036] 1.2.4 Identification of Phosphorus Solubility The organic phosphorus broth was used to identify the ability to solubilize organic phosphorus, while the PKO broth was used to identify the ability to solubilize inorganic phosphorus. Solid organic phosphorus and inorganic phosphorus broths were prepared separately. The test strains were inoculated onto the organic phosphorus broth plates and PKO broth plates using a spot inoculation method. The plates were then incubated upside down in a 28°C incubator for 3-7 days. The presence of a clear zone around the colony was observed; the formation of a clear zone indicated that the strain possessed phosphorus-solizing ability.

[0037] 1.2.5 Identification of Potassium Solubility The potassium-solubilizing ability of silicate bacteria was identified using culture media. A potassium-solubilizing medium was prepared. Using a sterile toothpick, the bacterial suspension to be tested was gently applied to multiple spots on the surface of the medium. The inoculated culture plate was then inverted and incubated at 28 °C for 3 days. The presence of transparent, oil-drop-like colonies on the silicate bacterial culture plate was observed. The appearance of these colonies indicates that the strain possesses potassium-solubilizing ability.

[0038] 1.2.6 Identification of Ferrophilic Production Capacity The ability of the strain to produce heptaphilin was identified by modifying the CAS medium. CAS medium was prepared, and the strain was inoculated into the CAS medium using a sterile toothpick. After incubation at 180 rpm and 28℃ for 2 days on a shaker, an appropriate amount of bacterial suspension was centrifuged at 10,000 rpm for 10 min. 3 mL of the supernatant was mixed with an equal volume of CAS test solution. The change in color of the mixture from blue to yellow indicates that the strain has the ability to secrete heptaphilin.

[0039] 1.2.7 Identification of ACC deaminase production capacity The ability to produce ACC deaminase was identified using DF and ADF media. ADF and DF media were prepared, and the strain was inoculated onto each medium using sterile toothpicks. If the growth in ADF medium was better than in DF medium, it indicated that the strain could grow using ACC as the sole nitrogen source, meaning that the strain could produce ACC deaminase.

[0040] 1.2.8 Detection of antagonistic effects The antagonistic relationship between growth-promoting bacteria was detected using the plate confrontation method. Selected growth-promoting strains were paired and inoculated onto LB agar plates, 3 cm apart. The plates were incubated at 28°C for 2 days. Observation was performed: if a blank appeared at the inoculation site with no other morphologically significantly different bacterial groups, the two bacterial groups were considered antagonistic; conversely, if the inoculated site contained the inoculated strain with no other morphologically significantly different bacterial groups, the two strains were considered not antagonistic.

[0041] 1.2.9 Strain Identification Bacterial species were identified using 16S rDNA sequencing. Total genomic DNA was extracted from the bacteria using a kit. The primer sequences were 27F: AGAGTTTGATCMTGGCTCAG; 1492R: GGTTACCTTGTTACGACTT. Using the extracted DNA as a template, a 1500 bp 16S rDNA fragment was amplified. After amplification, a clear and bright target band was observed by agarose gel electrophoresis. The PCR product was purified and sequenced. The sequencing results were compared with authoritative databases such as NCBI using BLAST to identify the bacterial species.

[0042] 1.3 Preparation of single-strain and compound-strain agents CTZT8 and CTZT22 are used as single microbial preparations, while CTZT8, CTZT11, CTZT21, and CTZT22 are used as a compound microbial preparation, SynComs. The ratio of CTZT8, CTZT11, CTZT21, and CTZT22 is as follows: the OD600 values ​​of the four bacterial solutions are adjusted to 1, and they are mixed in a volume ratio of 1:1:1:1.

[0043] From October 2024 to February 2025, microbial agents were applied once a month at fixed times in the experimental field in Santai County. The bacterial agents were propagated in 600 mL LB liquid medium until the OD600 value reached 1.0. After centrifugation, the supernatant was discarded, and the bacteria were resuspended in 4.5 L of pH 7.0 phosphate buffer (OD600 value approximately 0.13). The suspension was then evenly sprayed onto a 1×1 m experimental area (4.5 L / m²). 2 All other management measures remained consistent. Plant samples and rhizosphere soil samples were collected in November, January, and March for yield analysis.

[0044] 1.4 Detection of physicochemical properties of rhizosphere soil The pH of soil-water mixtures (1:2.5, w / v) was measured using a high-precision pH meter after 30 minutes of stirring (Zeeshan Ul Haq et al., 2025). Hydrolyzable nitrogen (AN) and total nitrogen (TN) in soil samples were determined using the alkaline hydrolysis-diffusion method and the Kjeldahl method, respectively (Restovich et al., 2019). Available phosphorus (AP) and total phosphorus (TP) were determined using the molybdenum-antimony colorimetric method (Restovich et al., 2019). Total potassium (TK) and available potassium (AK) were analyzed using a continuous flow chemistry analyzer following standardized leaching or digestion procedures (Ahmed et al., 2024). Soil organic matter was determined using the potassium dichromate method (Zhou et al., 2025).

[0045] 1.5 Determination of Ruscosaponin Content Accurately weigh three portions of the test sample, 1 g each, add 20 mL of 80% methanol to each portion, sonicate for 0.5 h, cool, and centrifuge an appropriate amount; after centrifugation, filter the supernatant through a 0.22 μm filter membrane, and use the filtrate as the test sample solution. The Ruscosaponin standard (CAS: 472-11-7, purity 98.5% HPLC) was dissolved in 80% methanol and diluted to 8.66, 17.32, 43.3, 86.6, and 173.2 ng / mL, respectively. The standard curve was determined to be Y = 263.295977X - 2072.085247, R² = 0.99968638.

[0046] 2. Experimental Results 2.1 Identification results of culturable bacteria in the rhizosphere soil of Ophiopogon japonicus Single colonies of microorganisms isolated from the rhizosphere soil of *Ophiopogon japonicus* were identified using 16S rDNA sequencing. The results are shown in Table 1. A total of 61 culturable bacterial species were isolated, including 10 fungi (A14, A15, A16, A32, A33, A34, A35, A36, A57, and A58) and 51 bacteria. The culturable bacteria mainly belonged to 5 genera, including *Bacillus*, *Pseudomonas*, *Arthrobacter*, *Bacillus*, and *Microbacterium*, as shown in Table 1.

[0047] Table 1. Identification results of culturable bacteria in the rhizosphere soil of Ophiopogon japonicus

[0048] 2.2 Results of the identification of the growth-promoting ability of rhizosphere microorganisms in Ophiopogon japonicus The growth-promoting abilities of 51 isolated rhizosphere bacteria were identified, and the results are shown in Table 2. Thirty of these strains possessed growth-promoting abilities, with A17 (CTZT8), A22 (CTZT11), A23, and A47 (CTZT22) exhibiting four different growth-promoting abilities, making them potential high-quality growth-promoting agents. This compound bacterial agent is composed of A17 (CTZT8), A22 (CTZT11), A47 (CTZT22), and A46 (CTZT21). A46 supplements the compound bacterial agent's ACC deaminase production function, while the other three strains enable the compound bacterial agent to possess potassium solubilization, nitrogen fixation, inorganic phosphorus solubilization, IAA secretion, and heparin production functions.

[0049] CTZT8 is Pantoea agglomerans Pantotheca, CTZT11 is Agrobacterium leguminum Agrobacterium, CTZT21 is Microbacterium paraoxydans Paraoxidizum microbacterium, CTZT22 is Siccibacter turicensis Zurich Scorching Bacillus.

[0050] Table 2. Results of the identification of the growth-promoting ability of rhizosphere microorganisms in Ophiopogon japonicus

[0051] 2.3 Determination of antagonism between growth-promoting bacteria Antagonism of growth-promoting bacteria was determined by the plate confrontation method, and the results are shown in Table 3. A4 is antagonistic to A8, A11, A12, A55, and A61; A12 is antagonistic to A10, A11, A47, and A48; A17 is antagonistic to A11 and A12; A18 is antagonistic to A50; A29 is antagonistic to A48, A49, A50, A55, and A56; and A53 is antagonistic to A55.

[0052] Table 3. Results of antagonistic assay of rhizosphere growth-promoting bacteria in Ophiopogon japonicus

[0053] 2.4 Yield analysis of single-strain and compound-strain agents The whole plant fresh weight and number of tubers were measured during the tuber germination period (November 2024), the rapid growth period (January 2025), and the tuber shaping period (March 2025). The results showed that SynComs had the highest whole plant fresh weight and number of tubers during the tuber germination period, SynComs had the most tubers during the rapid growth period, and CTZT8 had the highest whole plant fresh weight during the shaping period (Table 4).

[0054] Table 4 Fresh weight of whole plant and number of tubers of Ophiopogon japonicus

[0055] 2.5 Rhizosphere soil analysis after application of single and compound microbial agents Physicochemical indicators of the rhizosphere soil showed that the pH value first decreased and then increased. Compared with the control group, the rate of change in the microbial inoculation group was slower over time. This promoted the dissolution of soil nutrients while maintaining the relative stability of the microenvironment. Figure 1 The organic matter content in SynComs initially increased and then decreased sharply, indicating that the added strains mainly functioned in the decomposition of organic matter during the early and middle stages of tuber development, while assisting plant absorption in the later stages. Total nitrogen, total phosphorus, and total potassium in the CTZT8 and CTZT22 treatment areas increased continuously over time, while total nitrogen in the SynComs treatment area initially increased and then decreased, with no significant changes in total phosphorus and total potassium. Available phosphorus continuously increased in the CTZT8 and CTZT22 treatment areas, while available nitrogen continuously decreased in the CTZT22 and SynComs treatment areas. In the early and middle stages of tuber growth, the available nitrogen, phosphorus, and potassium contents of SynComs exceeded or equaled the control (CK), but were significantly lower than the CK in the later stages, indicating that SynComs first promoted the dissolution of soil nutrients and subsequently stimulated root absorption in Ophiopogon japonicus. In summary, CTZT8 showed strong root absorption promotion in the early stages of Ophiopogon japonicus tuber development, while SynComs showed strong absorption promotion in the middle and later stages.

[0056] 2.6 Analysis of Ruscosaponin Content The tuberous roots of Ophiopogon japonicus were harvested on March 19, 2025, and the active ingredient, ruscosaponin, was analyzed. The results showed that, compared with the control group, all fungal agents increased the content of ruscosaponin, and the data were statistically significant (P < 0.05). Their contents were ranked as follows: CTZT8 > CTZT22 > SynComs > CK (Table 5).

[0057] Table 5. Content of Ruscosaponins in Ophiopogon japonicus tuber roots

[0058] After detecting the content of Ophiopogon saponin D, it was found that CTZT8 and CTZT22 can promote the synthesis of Ophiopogon saponin D (Table 6).

[0059] Table 6. Content of Ophiopogon japonicus saponins D in Ophiopogon japonicus tubers

[0060] 2.7 Analysis of total flavonoids and soluble sugar content The results are as follows Figure 2 As shown, CTZT8 promotes flavonoid accumulation in tubers during the germination period, SynComs promotes flavonoid accumulation in tubers during the rapid growth period, and CTZT22 promotes flavonoid accumulation in tubers during the shaping period. CTZT8 and CTZT22 promote the accumulation of soluble sugars during the rapid growth period of tubers.

[0061] 2.8 Effects of microbial inoculants on the response of Ophiopogon japonicus to low-temperature Ophiopogon japonicus prefers a warm and humid growing environment (the suitable annual average temperature is 16.4~16.8℃). In Santai County, Mianyang City, the average temperature in November 2024 was 11~16℃, with a minimum of 4℃; the average temperature in January 2025 was 4~11℃, with a minimum of 0℃; and the average temperature in March 2025 was 8~16℃, with a minimum of 4℃. Therefore, Ophiopogon japonicus was subjected to varying degrees of low-temperature stress from November 2024 to March 2025.

[0062] Test results as follows Figure 3 As shown, the control group had the highest MDA content, which showed a continuous upward trend, while the bacterial agent group had a significantly lower MDA content than the control group, indicating that the bacterial agent reduced malondialdehyde accumulation and alleviated the cytotoxicity caused by low-temperature stress. The bacterial agent group had lower overall POD, SOD, and Pro contents than the control group, indicating that low-temperature stress did not cause strong stimulation to the bacterial agent group, and the bacterial agent group had stronger low-temperature resistance.

[0063] 2.9 Analysis of the relative content of steroidal saponins in the tuberous roots of Ophiopogon japonicus at different developmental stages 3-Hydroxy-spirostane-12-one (Hycosaponin): CTZT8 was significantly higher than CK in November and January, and higher than CK in the bacterial agent group in other stages, but the difference was not statistically significant.

[0064] Siberian Polygonin A: CTZT22 was lower than CK in November, CTZT8 was significantly higher than CK in January, and the bacterial agent group was higher than CK in other stages, but there was no significant difference.

[0065] Dioscorea triangularis saponins: CTZT8 was significantly higher than CK in November, and the relative content of the fungal agent group was greater than CK in other stages, but there was no significant difference.

[0066] Diosgenin-3-O-glucosyl(1→4)rhamnosyl(1→4)rhamnosyl(1→4)glucosinolate: The relative content of the inoculum group was greater than that of the control group. There was a significant difference between CTZT22 and CK in March, but no significant difference at other times.

[0067] Table 7. Relative content of hyosaccharin at different growth stages of Ophiopogon japonicus tuber.

[0068] Table 8. Relative content of Siberian polygalactosin A in Ophiopogon japonicus tubers at different growth stages

[0069] Table 9. Relative content of diosgenin in *Ophiopogon japonicus* tubers at different growth stages

[0070] Table 10. Relative contents of diosgenin-3-O-glucosyl (1→4) and rhamnosyl (1→4) glucosides at different growth stages of Ophiopogon japonicus tubers

[0071] 2.10 Chlorophyll Content Analysis like Figure 4 As shown, CTZT22 can promote the synthesis of total chlorophyll and chlorophyll a during the tuber germination period, while CTZT8 can promote the synthesis of total chlorophyll and chlorophyll a during the rapid growth period of tubers.

[0072] 2.11 Optimal Application Strategy of Microbial Agents Since the effects of each inoculant vary at different stages, we comprehensively analyzed the effects of three inoculants (CTZT8, CTZT22, and SynComs) on the whole plant fresh weight, number of tubers, soil nutrient absorption capacity, and ruscosaponin content at different developmental stages of Ophiopogon japonicus and designed an optimal application scheme. Specifically, applying CTZT8 during the tuber germination stage promotes root absorption of soil nutrients, providing a nutrient source for tuber germination and storing nutrients for tuber enlargement. Applying SynComs during the rapid growth period and the tuber shaping period stimulates an increase in the number of tubers, as well as soil nutrient dissolution and absorption. The increased number of tubers provides a structural basis for increased yield and also increases the root surface area, facilitating nutrient absorption. SynComs promotes soil nutrient dissolution, providing continuous impetus for efficient root absorption and continuously supplying raw materials for tuber enlargement.

[0073] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively describe all embodiments here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A microbial inoculant, characterized in that, This includes Pantotheca cumulus CTZT8 and / or Zürich xerobacterium CTZT22.

2. The microbial agent according to claim 1, characterized in that, The CTZT8 clump-forming bacteria was deposited on March 1, 2026, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 37823.

3. The microbial agent according to claim 2, characterized in that, The Zurich xerobacterium CTZT22 was deposited on March 1, 2026, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 37826.

4. The microbial agent according to claim 3, characterized in that, The microbial agent consists of Pantotheca cumulus CTZT8, Agrobacterium CTZT11, Microbacterium paraoxidans CTZT21, and Bacillus sicca CTZT22. The OD600 values ​​of the four bacterial solutions are adjusted to 1, and they are mixed in a volume ratio of 1:1:1:

1. The Agrobacterium CTZT11 was deposited on March 1, 2026, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 37824. The aforementioned *Paramicrobial paraoxidizum* CTZT21 was deposited on March 1, 2026, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 37825.

5. The application of the microbial inoculant as described in any one of claims 1-4 in increasing the content of ruscosaponin in Ophiopogon japonicus tubers.

6. The application of the microbial inoculant as described in any one of claims 1-4 in increasing the content of ophiopogon saponin D in Ophiopogon japonicus tubers.

7. The application of the microbial inoculant as described in any one of claims 1-4 in improving the low-temperature resistance of Ophiopogon japonicus.

8. A method for cultivating Ophiopogon japonicus, characterized in that, During the growing season of Ophiopogon japonicus, the microbial inoculant described in any one of claims 1-4 is applied to the experimental field where Ophiopogon japonicus is growing.

9. The method for planting Ophiopogon japonicus according to claim 8, characterized in that, Apply 4.5 liters of microbial inoculant per square meter.

10. The method for planting Ophiopogon japonicus according to claim 9, characterized in that, The application method is as follows: apply Pantotheca acuminata CTZT8 during the germination period of Ophiopogon japonicus tubers, and apply the microbial agent described in any one of claims 1-4 during the rapid growth period and the tuber shaping period.

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